1. A method of producing aflibercept MiniTrap from a clarified harvest of
a cell cultured in a chemically defined medium (CDM) and expresses
aflibercept, comprising: (a) binding aflibercept from said clarified
harvest to a first capture chromatography; (b) eluting said aflibercept
of step (a) and subjecting said aflibercept to enzymatic cleavage to
remove its Fc domain thereby forming MiniTrap; (c) subjecting (b) to a
second capture chromatography, wherein said second capture chromatography
step is subjected to one or more washes, and wherein a first flowthrough
fraction comprises MiniTrap and has a first color; (d) subjecting said
first flowthrough fraction of step (c) to anion exchange chromatography
(AEX); and (e) washing said AEX column of step (d), wherein said MiniTrap
is collected in a second flowthrough fraction and has a second color, and
wherein said first color is a more intense yellow brown color than said
second color.
2. The method of claim 1, wherein said both first and second capture
chromatography comprises Protein A resin.
3. The method of claim 1, wherein said first color has a b* value ranging
from about 1.5 to about 15.0 when protein concentration is normalized to
5.0 g/L.
4. The method of claim 1, wherein said second color has a b* value
ranging from about 0.5 to about 5.0 when protein concentration is
normalized to 5.0 g/L.
5. The method of claim 1, wherein said cell is selected from a group
consisting of CHO, NS0, Sp2/0, embryonic kidney cells and BHK.
6. The method of claim 1, wherein said clarified harvest comprises one or
more aflibercept variants, wherein said variants have at least one
oxidized amino acid residue.
7. The method of claim 6, wherein said oxidized amino acid residue is
selected from group consisting of methionine, tryptophan, histidine,
phenylalanine, tyrosine and a combination thereof.
8. The method of claim 7, wherein said oxidized amino acid residue is
histidine.
9. The method of claim 7, wherein said oxidized amino acid residue is
tryptophan.
10. The method of claim 6, wherein said aflibercept variant is selected
from an amino acid residue on a polypeptide having an amino acid sequence
as set forth in the group consisting of: SEQ ID NO.: 17, SEQ ID NO.: 18,
SEQ ID NO.: 19, SEQ ID NO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, SEQ ID
NO.: 23, SEQ ID NO.: 62, SEQ ID NO.: 63, SEQ ID NO.: 64, SEQ ID NO.: 65,
SEQ ID NO.: 66, SEQ ID NO.: 67, SEQ ID NO.: 68, SEQ ID NO.: 69, SEQ ID
NO.: 70, SEQ ID NO.: 71 and combinations thereof.
11. The method of claim 1, wherein said AEX column comprises an anionic
exchange substituent including diethylaminoethyl (DEAE), quaternary
aminoethyl (QAE) and quaternary amine (Q) groups.
12. The method of claim 1, further comprising after clarified harvest,
subjecting aflibercept to one or more further chromatographic steps
selected from the group consisting of: cation exchange chromatography,
hydrophobic interactive chromatography, size exclusion chromatography and
a combination thereof.
13. A method of producing aflibercept MiniTrap from a clarified harvest
of a cell cultured in a chemically defined medium (CDM), comprising: (a)
binding aflibercept from said clarified harvest to a first capture
chromatography; (b) eluting said aflibercept of step (a) and subjecting
said aflibercept to enzymatic cleavage to remove its Fc domain thereby
forming MiniTrap; (c) subjecting (b) to a second capture chromatography,
wherein said second capture chromatography step is subjected to one or
more washes, and wherein a first flowthrough fraction comprises MiniTrap,
wherein said MiniTrap has one or more acidic species; (d) subjecting said
first flowthrough fraction of step (c) to anion exchange chromatography
(AEX); and (e) washing said AEX column of step (d) and collected in a
second flowthrough fraction, wherein the percent of acidic species of
MiniTrap in said affinity eluate of step (b) is greater than the percent
of acidic species of MiniTrap in said AEX second flowthrough fraction
when concentration of protein in said eluate and AEX flowthrough fraction
are normalized, wherein said acidic species of MiniTrap correspond to the
peaks that elute earlier than the main peak in a strong cation exchange
chromatography (CEX) chromatogram of aflibercept, and wherein a
chromatogram is generated using a first mobile phase of 20 mM
2-(N-morpholino)ethanesulfonic acid (MES), pH 5.7 and a second mobile
phase of 40 mM sodium phosphate, 100 mM sodium chloride pH 9.0 (Mobile
phase B), and wherein a chromatogram is generated using detection at 280
nm.
14. The method of claim 13, wherein said both first and second capture
chromatography comprises Protein A resin.
15. The method of claim 13, wherein said AEX flowthrough fractions
comprises less than 20% total acidic species of MiniTrap.
16. The method of claim 13, wherein said acidic species of MiniTrap
comprises at least one oxidized amino acid residue selected from group
consisting of methionine, tryptophan, histidine, phenylalanine, tyrosine
and a combination thereof.
17. The method of claim 13, wherein the pH of both said equilibration and
wash buffers for said AEX column are from about 8.30 to about 8.60.
18. The method of claim 13, wherein the conductivity of both said
equilibration and wash buffers for said AEX column can be from about 1.50
to about 3.0 mS/cm.
19. The method of claim 13, wherein the enzymatic cleavage to remove the
Fc domain from aflibercept to generate MiniTrap uses proteolytic
digestion employing a protease or an enzymatically active variant
thereof.
20. The method of claim 19, wherein said protease is an
immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS).
21. The method of claim 13, further comprising after binding aflibercept
from said clarified harvest, subjecting aflibercept to one or more
further chromatographic steps selected from the group consisting of:
cation exchange chromatography (CEX), hydrophobic interactive
chromatography, size exclusion chromatography and a combination thereof.
22. A method of producing MiniTrap from a clarified harvest of a cell
cultured in a chemically defined medium (CDM), comprising: (a) binding
aflibercept from said clarified harvest to a Protein A resin; (b) eluting
said aflibercept of step (a) and subjecting said aflibercept to enzymatic
cleavage to remove its Fc domain thereby forming MiniTrap; (c) subjecting
(b) to a second capture chromatography, wherein said second capture
chromatography step is subjected to one or more washes, and wherein a
first flowthrough fraction comprises MiniTrap, wherein said MiniTrap has
one or more oxidized species of MiniTrap; (d) subjecting said first
flowthrough fraction of step (c) to anion exchange chromatography (AEX);
and (e) washing said AEX column of step (d) to obtain a second
flowthrough fraction, wherein the percent of oxidized species of MiniTrap
in said affinity eluate of step (b) is greater than the percent of
oxidized species in said AEX second flowthrough fraction when the
concentration protein in said eluate and flowthrough fraction are
normalized, and wherein said oxidized species of MiniTrap is measured by
subjecting said affinity eluate and said flowthrough fractions to
digestion, followed by their analysis using reverse-phase
ultra-performance chromatography (UPLC), detection at wavelengths of 280
nm, 320 nm and 350 nm and mass spectrometry analysis using a first mobile
phase of 0.1% formic acid in water and a second mobile phase of 0.1%
formic acid in acetonitrile.
23. The method of claim 22, wherein said oxidized amino acid residue is
selected from group consisting of methionine, tryptophan, histidine,
phenylalanine, tyrosine and a combination thereof.
24. The method of claim 22, wherein said oxidized amino acid residue is
selected from an amino acid residue on a polypeptide having an amino acid
sequence as set forth in the group consisting of: SEQ ID NO.: 17, SEQ ID
NO.: 18, SEQ ID NO.: 19, SEQ ID NO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22,
SEQ ID NO.: 23, SEQ ID NO.: 64, SEQ ID NO.: 65, SEQ ID NO.: 66, SEQ ID
NO.: 67, SEQ ID NO.: 69, SEQ ID NO.: 70, SEQ ID NO.: 71 and combinations
thereof.
25. A method of producing aflibercept MiniTrap from a clarified harvest
of a cell cultured in a chemically defined medium (CDM), comprising: (a)
binding aflibercept from said clarified harvest to a first capture
chromatography, wherein said first capture chromatography is Protein A
resin; (b) eluting said aflibercept of step (a) and subjecting said
aflibercept to enzymatic cleavage to remove its Fc domain thereby forming
MiniTrap; (c) subjecting (b) to a second capture chromatography, wherein
said second capture chromatography step is subjected to one or more
washes, and wherein a first flowthrough fraction comprises MiniTrap,
wherein said flowthrough fraction has b* value of more than 0.5 when
protein concentration is normalized to 5.0 g/L; (d) subjecting said first
flowthrough fraction of step (c) to anion exchange chromatography (AEX);
and (e) washing said AEX column of step (d), wherein said MiniTrap is
collected in a second flowthrough fraction and has a b* value, and
wherein said b* value is lower than the b* value in (c) when protein
concentration is normalized to 5.0 g/L.
26. The method of claim 25, wherein the pH of both said equilibration and
wash buffers for the AEX column can be from about 7.0 to about 8.6.
27. The method of claim 25, wherein said enzymatic cleavage to remove the
Fc domain from aflibercept to generate MiniTrap uses an
immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS).
28. The method claim of 27, wherein said IdeS include a polypeptide
having an amino acid sequence as set forth in the group consisting of SEQ
ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6,
SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.:
11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, SEQ
ID NO.: 16 and combinations thereof.
29. The method of claim 25, wherein said clarified harvest comprises one
or more aflibercept variants, wherein said variants have at least one
oxidized amino acid residue selected from group consisting of methionine,
tryptophan, histidine, phenylalanine, tyrosine and a combination thereof.
30. The method of claim 25, further comprising after binding aflibercept
from said clarified harvest, subjecting aflibercept to one or more
further chromatographic steps selected from the group consisting of:
cation exchange chromatography, hydrophobic interactive chromatography,
size exclusion chromatography and a combination thereof.
Description
SEQUENCE LISTING
[0001] The instant application contains a Sequence Listing which has been
filed electronically in ASCII format and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Aug. 13, 2020, is
named 070816-02400_SL.txt and is 130,880 bytes in size.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application claims priority to and the benefit of U.S.
Provisional Patent Application No. 63/065,012, filed on Aug. 13, 2020,
and U.S. Provisional Patent Application No. 62/944,635, filed on Dec. 6,
2019, the content of which is incorporated herein by reference in its
entirety.
FIELD
[0003] The present invention generally pertains to anti-VEGF compositions
and methods for producing the same.
BACKGROUND
[0004] Protein-based biopharmaceutical compositions have emerged as
important products for research, the treatment of ophthalmological
diseases, cancer, autoimmune disease, infection as well as other diseases
and disorders. Biopharmaceuticals represent one of the fastest growing
product segments of the pharmaceutical industry.
[0005] A class of cell-derived dimeric mitogens with selectivity for
vascular endothelial cells has been identified and designated vascular
endothelial cell growth factor (VEGF).
[0006] Persistent angiogenesis may cause or exacerbate certain diseases
such as psoriasis, rheumatoid arthritis, hemangiomas, angiofibromas,
diabetic retinopathy and neovascular glaucoma. An inhibitor of VEGF
activity would be useful as a treatment for such diseases and other
VEGF-induced pathological angiogenesis and vascular permeability
conditions, such as tumor vascularization. The angiopoietins and members
of the vascular endothelial growth factor (VEGF) family are the only
growth factors thought to be largely specific for vascular endothelial
cells.
[0007] Several eye disorders are associated with pathological
angiogenesis. For example, the development of age-related macular
degeneration (AMD) is associated with a process called choroidal
neovascularization (CNV). Leakage from the CNV causes macular edema and
collection of fluid beneath the macula resulting in vision loss. Diabetic
macular edema (DME) is another eye disorder with an angiogenic component.
DME is the most prevalent cause of moderate vision loss in patients with
diabetes and is a common complication of diabetic retinopathy, a disease
affecting the blood vessels of the retina. Clinically significant DME
occurs when fluid leaks into the center of the macula, the
light-sensitive part of the retina responsible for sharp, direct vision.
Fluid in the macula can cause severe vision loss or blindness.
[0008] Various VEGF inhibitors, such as the VEGF trap Eylea (aflibercept),
have been approved to treat such eye disorders.
SUMMARY
[0009] The present invention relates to anti-VEGF proteins including the
VEGF trap protein aflibercept, which is a fusion protein. The instant
invention also pertains to a new anti-VEGF protein, the aflibercept
MiniTrap or VEGF MiniTrap (collectively referred to as MiniTrap unless
otherwise noted). Disclosed herein are methods of making these anti-VEGF
proteins, including production modalities that provide efficient and
effective means to produce the proteins of interest. In one aspect, the
instant invention is directed towards the use of chemically defined media
(CDM) to produce anti-VEGF proteins. In a particular aspect, the CDMs of
interest are those that, when used, produce a protein sample wherein the
sample has a yellow-brown color and may comprise oxidized species. Still
further in the present application, protein variants of aflibercept and
VEGF MiniTrap are disclosed together with attendant production methods.
Production of Aflibercept
[0010] The present disclosure describes the production of aflibercept
using a cell culture medium. In one embodiment, the cell culture medium
is a chemically defined medium ("CDM"). CDM is often used because it is a
protein-free, chemically-defined formula using no animal-derived
components and there is certainty as to the composition of the medium. In
another embodiment, the cell culture medium is a soy hydrolysate medium.
[0011] In one embodiment, a method of producing a recombinant protein
comprises: (a) providing a host cell genetically engineered to express a
recombinant protein of interest; (b) culturing the host cell in a CDM
under suitable conditions in which the cell expresses the recombinant
protein of interest; and (c) harvesting a preparation of the recombinant
protein of interest produced by the cell. In one aspect, the recombinant
protein of interest is an anti-VEGF protein. In a particular aspect, the
anti-VEGF protein is selected from the group consisting of aflibercept
and recombinant MiniTrap (examples of which are disclosed in U.S. Pat.
No. 7,279,159), an aflibercept scFv and other anti-VEGF proteins. In a
preferred aspect, the recombinant protein of interest is aflibercept.
[0012] In one aspect of the present embodiment, aflibercept is expressed
in a suitable host cell. Non-limiting examples of such host-cells
include, but are not limited to, CHO, CHO K1, EESYR.RTM., NICE.RTM., NS0,
Sp2/0, embryonic kidney cells and BHK.
[0013] Suitable CDMs include Dulbecco's Modified Eagle's (DME) medium,
Ham's Nutrient Mixture, Excell medium, and IS CHO-CD medium. Other CDMs
known to those skilled in the art are also contemplated to be within the
scope of the present invention. In a particular aspect, a suitable CDM is
CDM1B (Regeneron) or Excell Advanced Medium (SAFC).
[0014] In one embodiment, a clarified harvest sample from a CDM culture
comprising aflibercept is subjected to a capture chromatography
procedure. In one aspect, the capture step is an affinity chromatography
procedure using, for example, Protein A. In a further aspect, the eluate
of the affinity procedure exhibits a certain color, for example, the
eluate can exhibit a yellow-brown color. As described in more detail
infra, color can be assessed using (i) the European Color Standard "BY"
in which a qualitative visual inspection is made or (ii) a colorimetric
assay, CIE L*, a*, b* (or CIELAB), which is more quantitative than the BY
system. However, in either case, color assessment between multiple
samples should be normalized against protein concentration in order to
assure a meaningful assessment. For example, referring to Example 9
below, the Protein A eluate has a "b*" value of around 2.52 which
corresponds to a BY value of approximately BY5 (when measured at a
concentration of 5 g/L protein in the protein A eluate). If the color of
the Protein A eluate is to be compared to another sample, then the
comparison should be made against the same protein concentration. The b*
value in the CIELAB color space is used to expresses coloration of the
samples and covers blue (-) to yellow (+). The higher the b* value of a
sample is compared to another indicates a more intense yellow-brown
coloration in the sample compared to the other.
[0015] In one embodiment, aflibercept is produced from a host cell
genetically engineered to express aflibercept using CDM. In one aspect,
other species or variants of aflibercept are also produced. These
variants include aflibercept isoforms that comprise one or more oxidized
amino acid residues collectively referred to as oxo-variants. A clarified
harvest sample produced using CDM comprising aflibercept as well as its
oxo-variants can be subjected to a capture chromatography procedure. In
one aspect, the capture step is an affinity chromatography procedure
using, for example, a Protein A column. When a sample extracted from an
affinity eluate, which may or may not manifest a yellow-brown color, is
analyzed using, for example, liquid chromatography--mass
spectrophotometry (LC-MS), one or more oxidized variants of aflibercept
may be detected. Certain amino acid residues of a modified aflibercept
are shown to be oxidized including, but not limited to, histidine and/or
tryptophan residues. In one aspect, the variants can include oxidation of
one or more methionine residues as well as other residues, see infra.
[0016] In another aspect, the variants can include oxidation of one or
more tryptophan residues to form N-formylkynurenine. In a further aspect,
the variants can include oxidation of one or more tryptophan residues to
form mono-hydroxyl tryptophan. In a particular aspect, the protein
variants can include oxidation of one or more tryptophan residues to form
di-hydroxyl tryptophan. In a particular aspect, the protein variants can
include oxidation of one or more tryptophan residues to form tri-hydroxyl
tryptophan.
[0017] In another aspect, the variants can include one or more
modifications selected from the group consisting of: deamidation of, for
example, one or more asparagines; one or more aspartic acids converted to
iso-aspartate and/or Asn; oxidation of one or more methionines; oxidation
of one or more tryptophans to N-formylkynurenin; oxidation of one or more
tryptophans to mono-hydroxyl tryptophan; oxidation of one or more
tryptophans to di-hydroxyl tryptophan; oxidation of one or more
tryptophans to tri-hydroxyl tryptophan; Arg 3-deoxyglucosonation of one
or more arginines; removal of C-terminal glycine; and presence of one or
more non-glycosylated glycosites.
[0018] In another embodiment, the invention is directed to methods for
producing aflibercept. In one aspect, a clarified harvest sample
comprising aflibercept and its variants are subjected to a capture step
such as Protein A affinity chromatography. Subsequent to the affinity
step, an affinity eluate can be subjected to ion exchange chromatography.
The ion exchange chromatography can be either cation or anion exchange
chromatography. Also contemplated to be within the scope of the present
embodiment is mixed-mode or multimodal chromatography as well as other
chromatographic procedures which are discussed further below. In a
particular aspect, the ion exchange chromatography is anion exchange
chromatography (AEX). Suitable conditions for employing AEX include, but
are not limited to, Tris hydrochloride at a pH of about 8.3 to about 8.6.
Following equilibration using, for example, Tris hydrochloride at a pH of
about 8.3 to about 8.6, the AEX column is loaded with sample. Following
the loading of the column, the column can be washed one or multiple times
using, for example, the equilibrating buffer. In a particular aspect, the
conditions used can facilitate the differential chromatographic behavior
of aflibercept and its oxidized variants such that a fraction comprising
aflibercept absent significant amounts of oxo-variants can be collected
in a flowthrough fraction while a significant portion of oxo-variants are
retained on the solid-phase of the AEX column and can be obtained upon
stripping the column--see Example 2 below, FIG. 11. Referring to FIG. 11
and Example 2, changes in oxo-variants can be observed between the
different production steps. For example, this change can be illustrated
by data in the "Tryptophan Oxidation Level (%)" section, specifically,
the "W138(+16)" column. There it can be observed that the oxo-variants
(specifically, oxo-tryptophan) went from about 0.131% in a load sample to
about 0.070% in a flowthrough sample following AEX chromatography (AEX
separation 2), indicating that there was a reduction in oxo-variant of
aflibercept using AEX.
[0019] Use of ion exchange can be used to mitigate or minimize color. In
one aspect of the present embodiment, a clarified harvest sample is
subjected to capture chromatography, for example, using Protein A
affinity chromatography. The affinity column is eluted and has a first
color with a particular BY and/or b* value assigned thereto. This Protein
A eluate is then subjected to ion exchange chromatography such as anion
exchange chromatography (AEX). The ion exchange column is washed and the
flowthrough is collected and has a second color having a particular BY
and/or b* value assigned thereto. In a particular aspect, the color value
(either "BY" or "b*") of the first color differs from the second color.
In a further aspect, the first color of the Protein A eluate has a more
yellow-brown color as compared to the second color of the AEX flowthrough
as reflected by the respective BY and/or b* value. Typically, there is a
reduction in yellow-brown color of the second color following AEX when
compared to the first color of the Protein A eluate. For example, the use
of anion exchange reduced the yellow-brown color observed in a Protein A
eluate sample from a b* value of about 3.06 (first color) to about 0.96
(second color) following AEX--see Example 2, Table 2-3 below.
[0020] In one aspect of the embodiment, the pH of both the equilibration
and wash buffers for the AEX column can be from about 8.30 to about 8.60.
In another aspect, the conductivity of both the equilibration and wash
buffers for the AEX column can be from about 1.50 to about 3.00 mS/cm.
[0021] In one aspect of the embodiment, the equilibration and wash buffers
can be about 50 mM Tris hydrochloride. In one aspect, the strip buffer
comprises 2M sodium chloride or 1N sodium hydroxide or both (see Table
2-2).
[0022] The present embodiment can include the addition of one or more
steps, in no particular order, such as hydrophobic interaction
chromatography (HIC), affinity chromatography, multimodal chromatography,
viral inactivation (e.g., using low pH), viral filtration, and/or
ultra/diafiltration as well as other well-known chromatographic steps.
[0023] In one embodiment, the anti-VEGF protein is glycosylated at one or
more asparagines as follows: G0-GlcNAc glycosylation; G1-GlcNAc
glycosylation; G1S-GlcNAc glycosylation; G0 glycosylation; G1
glycosylation; G1S glycosylation; G2 glycosylation; G2S glycosylation;
G2S2 glycosylation; G0F glycosylation; G2F2S glycosylation; G2F2S2
glycosylation; G1F glycosylation; G1FS glycosylation; G2F glycosylation;
G2FS glycosylation; G2FS2 glycosylation; G3FS glycosylation; G3FS3
glycosylation; G0-2GlcNAc glycosylation; Man4 glycosylation; Man4_A1G1
glycosylation; Man4_A1G1S1 glycosylation; Man5 glycosylation; Man5_A1G1
glycosylation; Man5_A1G1S1 glycosylation; Man6 glycosylation;
Man6_G0+Phosphate glycosylation; Man6+Phosphate glycosylation; and/or
Man7 glycosylation. In one aspect, the anti-VEGF protein can be
aflibercept, anti-VEGF antibody or VEGF MiniTrap.
[0024] In one aspect, glycosylation profile of a composition of an
anti-VEGF protein is as follows: about 40% to about 50% total fucosylated
glycans, about 30% to about 55% total sialylated glycans, about 6% to
about 15% mannose-5, and about 60% to about 79% galactosylated glycans
(see Example 6). In one aspect, the anti-VEGF protein has Man5
glycosylation at about 32.4% of asparagine 123 residues and/or about
27.1% of asparagine 196 residues.
[0025] In one embodiment, the process can further comprise formulating a
drug substance using a pharmaceutically acceptable excipient. In one
aspect of the embodiment, the pharmaceutically acceptable excipient can
be selected from the following: water, buffering agents, sugar, salt,
surfactant, amino acid, polyol, chelating agent, emulsifier and
preservative. Other well-known excipients to the skilled artisan are
within the purview of this embodiment.
[0026] In one aspect of the embodiment, the formulation can be suitable
for administration to a human subject. In particular, administration can
be affected by intravitreal injection. In one aspect, the formulation can
have about 40 to about 200 mg/mL of the protein of interest.
[0027] The formulation can be used as a method of treating or preventing
angiogenic eye disorders which can include: age-related macular
degeneration (e.g., wet or dry), macular edema, macular edema following
retinal vein occlusion, retinal vein occlusion (RVO), central retinal
vein occlusion (CRVO), branch retinal vein occlusion (BRVO), diabetic
macular edema (DME), choroidal neovascularization (CNV), iris
neovascularization, neovascular glaucoma, post-surgical fibrosis in
glaucoma, proliferative vitreoretinopathy (PVR), optic disc
neovascularization, corneal neovascularization, retinal
neovascularization, vitreal neovascularization, pannus, pterygium,
vascular retinopathy, diabetic retinopathy in a subject with diabetic
macular edema; or diabetic retinopathies (e.g., non-proliferative
diabetic retinopathy (e.g., characterized by a Diabetic Retinopathy
Severity Scale (DRSS) level of about 47 or 53 or proliferative diabetic
retinopathy; e.g., in a subject that does not suffer from DME).
Production of VEGF MiniTrap
[0028] The present disclosure describes the production of a modified
version of aflibercept wherein the Fc portion is removed or absent and is
referred to as aflibercept MiniTrap or VEGF MiniTrap. This MiniTrap can
be produced in cell culture medium including a chemically defined medium
(CDM) or soy hydrolysate medium.
[0029] In one embodiment, the MiniTrap is produced using CDM. In one
aspect of MiniTrap production, full length aflibercept is produced using
a suitable host and under suitable conditions and is further processed
whereby the Fc portion is enzymatically removed thus yielding MiniTrap.
Alternatively, a gene encoding MiniTrap (e.g., a nucleotide sequence
encoding aflibercept absent its Fc portion) can be produced under
suitable conditions using a suitable host cell.
[0030] In one embodiment, a method for manufacturing MiniTrap includes
producing a full-length aflibercept fusion protein followed by cleavage
of the Fc region. In one aspect, the method involves producing a
recombinant protein, namely a full-length aflibercept fusion protein
(see, U.S. Pat. No. 7,279,159, the entire teaching of which is
incorporated herein by reference), comprising: (a) providing a host cell
genetically engineered to express full length aflibercept; (b) culturing
the host cell in CDM under suitable conditions in which the cell
expresses the full length aflibercept; (c) harvesting a preparation of
the full length aflibercept produced by the cell; and (d) subjecting the
full length aflibercept to enzymatic cleavage specific for removing the
Fc portion of the fusion protein. In another aspect, a nucleotide
sequence encoding aflibercept minus its Fc portion is expressed from a
suitable host cell under suitable conditions well known to those skilled
in the art (see U.S. Pat. No. 7,279,159).
[0031] In one aspect of the present embodiment, the aflibercept is
expressed in a suitable host cell. Non-limiting examples of such
host-cells include, but are not limited to, CHO, CHO K1, EESYR.RTM.,
NICE.RTM., NS0, Sp2/0, embryonic kidney cells and BHK.
[0032] Suitable CDMs include Dulbecco's Modified Eagle's (DME) medium,
Ham's Nutrient Mixture, EX-CELL medium (SAFC), and IS CHO-CD medium
(Irvine). Other CDMs known to those skilled in the art are also
contemplated to be within the scope of the present invention. In a
particular aspect, a suitable CDM is CDM1B (Regeneron) or Excell medium
(SAFC).
[0033] In one aspect, during the production of MiniTrap, a sample
comprising a protein of interest (i.e., aflibercept fusion protein and/or
MiniTrap) along with its variants (including oxo-variants) can exhibit
certain color properties--a yellow-brown color. For example, an eluate
sample from an affinity chromatography step can exhibit a certain
yellow-brown color measured using the BY and/or b* system (see Examples 2
and 9 below). Exemplary sources for a "sample" may include an affinity
chromatography, such as Protein A, eluate; the sample may be obtained
from a flowthrough fraction of ion exchange chromatography procedure; it
may also be obtained from the strip of an ion exchange column--there are
other sources during a production process well known to those skilled in
the art from which a sample may be analyzed. As mentioned above and
described further below, color can be assessed using (i) the European
Color Standard "BY" in which a qualitative visual inspection is made or
(ii) a colorimetric assay, CIELAB, which is more quantitative than the BY
system. However, in either case, color assessment between multiple
samples should be normalized, for example, using protein concentration,
in order to assure a meaningful assessment between samples.
[0034] In one aspect of the present embodiment, a full-length aflibercept
fusion protein can be subjected to enzymatic processing ("cleavage
activity") in order to generate a VEGF MiniTrap, for example, using
proteolytic digestion employing a protease or enzymatically active
variant thereof. In one aspect of this embodiment, the protease can be an
immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS). In
another aspect, the protease can be thrombin trypsin, endoproteinase
Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, outer membrane
protease T (OmpT), IdeS, chymotrypsin, pepsin, thermolysin, papain,
pronase, or protease from Aspergillus saitoi. In one aspect, the protease
can be a cysteine protease. In a particular aspect of the embodiment, the
protease can be IdeS. In another aspect, the protease can be a variant of
IdeS. Non-limiting examples of variants of IdeS are described infra and
include a polypeptide having an amino acid sequence as set forth in the
group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID
NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ
ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.:
14, SEQ ID NO.: 15 and SEQ ID NO.: 16. In one aspect, the protease can be
immobilized on agarose or another suitable matrix.
[0035] In one aspect, a protein of interest (together with its variants)
is produced using CDM. In a particular aspect, the protein of interest
includes aflibercept or MiniTrap. The variants comprise one or more
oxidized amino acid residues, collectively oxo-variants. Examples of
oxidized residues include, but are not limited to, one or more histidine
and/or tryptophan residues, other oxidized residues using LC-MS have also
been detected and described below such as oxidized methionine. Subsequent
chromatography such as AEX can be used to isolate these oxo-variants from
the protein of interest in a given sample and are described herein.
[0036] In one aspect, the variants can include oxidation of one or more
tryptophan residues to form N-formylkynurenines. In a further aspect, the
variants can include oxidation of one or more tryptophan residues to form
mono-hydroxyl tryptophan. In a particular aspect, the protein variants
can include oxidation of one or more tryptophan residues to form
di-hydroxyl tryptophan. In a particular aspect, the protein variants can
include oxidation of one or more tryptophan residues to form tri-hydroxyl
tryptophan.
[0037] In another aspect, the oxo-variants can include one or more
modifications selected from the group consisting of: deamidation of one
or more asparagine residues; one or more aspartic acids converted to
iso-aspartate and/or Asn; oxidation of one or more methionine residues;
oxidation of one or more tryptophan residues to form N-formylkynurenin;
oxidation of one or more tryptophan residues to form mono-hydroxyl
tryptophan; oxidation of one or more tryptophan residues to form
di-hydroxyl tryptophan; oxidation of one or more tryptophan residues to
form tri-hydroxyl tryptophan; Arg 3-deoxyglucosonation of one or more
arginine residues; removal of C-terminal Glycine; and presence of one or
more non-glycosylated glycosites.
[0038] In one embodiment, the method of manufacturing a MiniTrap protein
comprises (a) capturing a full-length aflibercept fusion protein on a
first chromatographic platform and (b) cleaving the aflibercept thereby
forming a MiniTrap protein, i.e., aflibercept absent its Fc domain. In
one aspect, the first chromatographic support comprises an affinity
chromatography media, an ion-exchange chromatography media, or a
hydrophobic interaction chromatography media. In a particular aspect, the
first chromatographic platform comprises an affinity chromatography
platform such as a Protein A. In a further aspect, the protein of capture
step (a) is eluted from the first chromatography platform prior to
cleavage step (b). In a still further aspect, a second capture step
follows cleavage step (b). In a particular aspect, this second capture
step can be facilitated by affinity chromatography such as Protein A
affinity chromatography. The flowthrough of this second capture step
(comprising MiniTrap) has a first color, for example, a yellow-brown
color and measured having a particular BY and/or b* value--see, e.g.,
Example 9 below. Additionally, LC-MS analysis of this second capture
flowthrough may demonstrate the presence of oxo-variants wherein one or
more residues of MiniTrap are oxidized (see Example 9 below).
[0039] In a further aspect, the second capture flowthrough can be
subjected to ion exchange chromatography such as AEX. This AEX column can
be washed using a suitable buffer and an AEX flowthrough fraction can be
collected comprising essentially MiniTrap. This AEX flowthrough fraction
can have a second color that is of a yellow-brown coloration having a
particular BY and/or b* value. In a further aspect, the first color
(flowthrough from second capture step) and second color (flowthrough of
the ion exchange procedure) have different colors as measured either by
the BY and/or b* system. In one aspect, the second color demonstrates a
diminished yellow-brown color when compared to the first color using
either a BY and/or b* value following AEX.
[0040] In another embodiment, the cleavage activity of step (b) can be
performed using a chromatographic column wherein the cleavage activity,
for example, an enzyme activity, is affixed or immobilized to a column
matrix. The column used in step (b) can comprise one or more of the
proteases already alluded to and more fully described below.
[0041] In one embodiment, the ion-exchange chromatography procedure can
comprise an anion-exchange (AEX) chromatography media. In another aspect,
the ion-exchange chromatography media comprises a cation exchange (CEX)
chromatography media. Suitable conditions for employing AEX include, but
are not limited to, Tris hydrochloride at a pH of about 8.3 to about 8.6.
Following equilibration using, for example, Tris hydrochloride at a pH of
about 8.3 to about 8.6, the AEX column is loaded with sample. Following
the loading of the column, the column can be washed one or multiple times
using, for example, the equilibrating buffer. In a particular aspect, the
conditions used can facilitate the differential chromatographic behavior
of MiniTrap and its oxo-variants using AEX such that the MiniTrap is
substantially in the flowthrough fraction while the oxo-variants are
substantially retained on the AEX column and can be collected by
stripping the column (see Example 9 below).
[0042] In one example, samples from different stages of production were
analyzed for color and presence of oxo-variants. Referring to Example 9,
the affinity flowthrough pool (flowthrough from a second Protein A
affinity step) had a first b* value of about 1.58 (see Table 9-3). This
second affinity flowthrough was subjected to AEX. The AEX flowthrough had
a second b* value of about 0.50, indicating a significant reduction in
yellow-brown color following the use of AEX. Stripping of the AEX column
yielded a strip sample and a third b* value of about 6.10 was observed,
indicating that this strip sample has a more yellow-brown color when
compared to either the load or flowthrough.
[0043] Referring again to Example 9, oxo-variant analysis was also
performed. Samples analyzed were the affinity flowthrough pool (second
Protein A affinity eluate), AEX flowthrough, and AEX strip. Referring to
Table 9-5 and Table 9-6, changes in oxo-variants can be observed between
the different production steps. For example, this change can be
illustrated by data in the "Tryptophan Oxidation Level (%)" section,
specifically, the "W58(+16)" column. There it can be observed that the
oxo-variants (specifically, oxo-tryptophan) went from about 0.055% in a
load sample to about 0.038% in a flowthrough sample following AEX
chromatography, indicating that there was a reduction in oxo-variant
following AEX. The AEX strip was analyzed and the percent oxo-tryptophan
species was found to be about 0.089%. When this strip value was compared
to the load (as well as the flowthrough), it appeared that a significant
portion of this oxo-variant was retained on the AEX column.
[0044] The present embodiment can include the addition of one or more
steps, in no particular order, such as hydrophobic interaction
chromatography, affinity chromatography, multimodal chromatography, viral
inactivation (e.g., using low pH), viral filtration, and/or
ultra/diafiltration.
[0045] One embodiment of the present invention is directed to a method for
regenerating a chromatography column comprising a resin. In one aspect of
the embodiment, the resin has an immobilized hydrolyzing agent. In yet
another aspect of the embodiment, the resin comprises an immobilized
protease enzyme. In still another aspect of the embodiment, the resin is
a FabRICATOR.RTM. resin or a mutant of the resin. In one aspect of the
embodiment, the method of regenerating a column comprising a resin
improves reaction efficiency of the resin.
[0046] In one aspect of the embodiment, a method of regenerating a column
comprising a resin includes incubating the column resin with acetic acid.
In one aspect, the concentration of acetic acid used is from about 0.1 M
to about 2 M. In one aspect, the concentration of acetic acid is about
0.5 M. In one aspect, the resin is incubated for at least about 10
minutes. In another aspect, the resin is incubated for at least about 30
minutes. In yet another aspect of this embodiment, the resin is incubated
for at least about 50 minutes. In yet another aspect of this embodiment,
the resin is incubated for at least about 100 minutes. In yet another
aspect of this embodiment, the resin is incubated for at least about 200
minutes. In yet another aspect of this embodiment, the resin is incubated
for at least about 300 minutes.
[0047] Optionally, the column resin is further incubated with guanidine
hydrochloride (Gu-HCl). In one aspect, Gu-HCl absent acetic acid is used
to regenerate the column resin. The concentration of Gu-HCl employed is
from about 1 N to about 10 N. In another aspect, the concentration of
Gu-HCl is about 6 N. In a further aspect, the column resin can be
incubated for at least about 10 minutes with the regenerative agents
(acetic acid, Gu-HCl). In yet another aspect, the resin is incubated for
at least about 30 minutes. In still another aspect, the resin is
incubated for at least about 50 minutes. In yet another aspect, said
resin is incubated for at least about 100 minutes.
[0048] In one embodiment, the column comprising a resin is stored in
ethanol. In one aspect, the column is stored in ethanol, wherein the
ethanol percentage is from about 5% v/v to about 20% v/v. In a particular
aspect, the column is stored using 20% v/v ethanol.
[0049] In one embodiment, the process can further comprise formulating the
VEGF MiniTrap using a pharmaceutically acceptable excipient. In a one
aspect, the pharmaceutically acceptable excipient can be selected from
the following: water, buffering agents, sugar, salt, surfactant, amino
acid, polyol, chelating agent, emulsifier and preservative. Other
well-known excipients to the skilled artisan are within the purview of
this embodiment.
[0050] The formulation of the present invention is suitable for
administration to a human subject. In one aspect of the present
embodiment, administration can be effected by intravitreal injection. In
one aspect, the formulation can have about 40 to about 200 mg/mL of the
protein of interest. In a particular aspect, the protein of interest is
either aflibercept or aflibercept MiniTrap.
[0051] The formulation can be used in a method of treating or preventing
angiogenic eye disorders which can include: age-related macular
degeneration (e.g., wet or dry), macular edema, macular edema following
retinal vein occlusion, retinal vein occlusion (RVO), central retinal
vein occlusion (CRVO), branch retinal vein occlusion (BRVO), diabetic
macular edema (DME), choroidal neovascularization (CNV), iris
neovascularization, neovascular glaucoma, post-surgical fibrosis in
glaucoma, proliferative vitreoretinopathy (PVR), optic disc
neovascularization, corneal neovascularization, retinal
neovascularization, vitreal neovascularization, pannus, pterygium,
vascular retinopathy, diabetic retinopathy in a subject with diabetic
macular edema; or diabetic retinopathies (e.g., non-proliferative
diabetic retinopathy (e.g., characterized by a Diabetic Retinopathy
Severity Scale (DRSS) level of about 47 or 53 or proliferative diabetic
retinopathy; e.g., in a subject that does not suffer from DME).
Variants of IdeS
[0052] The present disclosure describes the use of IdeS (FabRICATOR) (SEQ
ID NO.: 1) or other polypeptides which are IdeS variants (SEQ ID NO.: 2
to 16) to produce a VEGF MiniTrap. IdeS (SEQ ID NO.: 1) includes
asparagine residues at position 87, 130, 182 and/or 274 (shown as "N*"
bolded and italicized in SEQ ID NO.: 1 below). The asparagine at these
positions may be mutated to an amino acid other than asparagine to form
IdeS variants (and the mutated amino acid(s) are shown as italicized and
underscored amino acid(s)):
[0053] In one embodiment, the polypeptide has an isolated amino acid
sequence comprising at least 70% sequence identity over a full length of
an isolated amino acid sequence as set forth in the group consisting of
SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.:
6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID
NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15
and SEQ ID NO.: 16. In one aspect, the isolated amino acid sequence has
at least about 80% sequence identity over a full length of the isolated
amino acid sequence. In another aspect, the isolated amino acid sequence
has at least about 90% sequence identity over a full length of the
isolated an amino acid sequence. In another aspect, the isolated amino
acid sequence has about 100% sequence identity over a full length of the
isolated an amino acid sequence. In one aspect, the polypeptide can be
capable of cleaving a target protein into fragments. In a particular
aspect, the target protein is an IgG. In another aspect, the target
protein is a fusion protein. In yet another aspect, the fragments can
comprise a Fab fragment and/or an Fc fragment.
[0054] The SEQ ID 1, NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5,
SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.:
10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ
ID NO.: 15 and SEQ ID NO.: 16.
[0055] The present disclosure also includes an isolated nucleic acid
molecule encoding a polypeptide having an isolated amino acid sequence
comprising at least 70% sequence identity over a full length of the
isolated amino acid sequence as set forth in the group consisting of SEQ
ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6,
SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.:
11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and
SEQ ID NO.: 16. In one aspect, the isolated amino acid sequence has at
least about 80% sequence identity over a full length of the isolated
amino acid sequence. In another aspect, the isolated amino acid sequence
has at least about 90% sequence identity over a full length of the
isolated amino acid sequence. In another aspect, the isolated amino acid
sequence has about 100% sequence identity over a full length of the
isolated amino acid sequence. In one aspect, the polypeptide can be
capable of cleaving a target protein into fragments. In a particular
aspect, the target protein is an IgG. In another particular aspect, the
target protein is a fusion protein. In yet another particular aspect, the
fragments can comprise a Fab fragment and/or an Fc fragment.
[0056] The present disclosure also includes a vector which comprises a
nucleic acid encoding a polypeptide having an isolated amino acid
sequence comprising at least 70% sequence identity over a full length of
the isolated amino acid sequence as set forth in the group consisting of
SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.:
6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID
NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15
and SEQ ID NO.: 16. In one aspect, the nucleic acid molecule is
operatively linked to an expression control sequence capable of directing
its expression in a host cell. In one aspect, the vector can be a
plasmid. In one aspect, the isolated amino acid sequence has at least
about 80% sequence identity over a full length of the isolated amino acid
sequence. In another aspect, the isolated amino acid sequence has at
least about 90% sequence identity over a full length of the isolated
amino acid sequence. In another aspect, the isolated amino acid sequence
has about 100% sequence identity over a full length of the isolated amino
acid sequence. In one aspect, the polypeptide can be capable of cleaving
a target protein into fragments. In a particular aspect, the target
protein is an IgG. In another aspect, the target protein is a fusion
protein. In yet another aspect, the fragments can comprise a Fab fragment
and/or an Fc fragment.
[0057] In one embodiment, the isolated amino acid can comprise a parental
amino acid sequence defined by SEQ ID NO.: 1 with an asparagine residue
at position 87, 130, 182 and/or 274 mutated to an amino acid other than
asparagine. In one aspect, the mutation can confer an increased chemical
stability at alkaline pH-values compared to the parental amino acid
sequence. In another aspect, the mutation can confer an increase in
chemical stability by 50% at alkaline pH-values compared to the parental
amino acid sequence. In one aspect, the amino acid can be selected from
aspartic acid, leucine, and arginine. In a particular aspect, the
asparagine residue at position 87 is mutated to aspartic acid residue. In
another aspect, the asparagine residue at position 130 is mutated to
arginine residue. In a yet another aspect, the asparagine residue at
position 182 is mutated to a leucine residue. In a yet another aspect,
the asparagine residue at position 274 is mutated to aspartic acid
residue. In a yet another aspect, the asparagine residues at positions 87
and 130 are mutated. In a yet another aspect, the asparagine residues at
positions 87 and 182 are mutated. In a yet another aspect, the asparagine
residues at positions 87 and 274 are mutated. In a yet another aspect,
the asparagine residues at positions 130 and 182 are mutated. In a yet
another aspect, the asparagine residues at positions 130 and 274 are
mutated. In a yet another aspect, the asparagine residues at positions
182 and 274 are mutated. In a yet another aspect, the asparagine residues
at positions 87, 130 and 182 are mutated. In a yet another aspect, the
asparagine residues at positions 87, 182 and 274 are mutated. In a yet
another aspect, the asparagine residues at positions 130, 182 and 274 are
mutated. In a yet another aspect, the asparagine residues at positions
87, 130, 182 and 274 are mutated.
[0058] In a related embodiment, the disclosure includes an isolated
nucleic acid molecule encoding a polypeptide having an isolated amino
acid sequence comprising a parental amino acid sequence defined by SEQ ID
NO.: 1 with asparagine residues at positions 87, 130, 182 and/or 274
mutated to an amino acid other than asparagine--see above. The mutation
can confer an increased chemical stability at alkaline pH-values compared
to the parental amino acid sequence.
[0059] In a further related embodiment, the disclosure includes a vector,
which comprises a nucleic acid molecule encoding a polypeptide having an
isolated amino acid sequence comprising a parental amino acid sequence
defined by SEQ ID NO.: 1 with asparagine residue at position 87, 130, 182
and/or 274 mutated to an amino acid other than asparagine--see above. The
mutation can confer an increased chemical stability at alkaline pH-values
compared to the parental amino acid sequence. In one aspect, the nucleic
acid molecule is operatively linked to an expression control sequence
capable of directing its expression in a host cell. In one aspect, the
vector can be a plasmid.
Affinity-based Production
[0060] The present disclosure also provides methods for reducing host-cell
proteins as well as other undesirable proteins and nucleic acids during
production of an anti-VEGF protein using affinity chromatography.
[0061] In one embodiment, a method of producing a recombinant protein
comprises: (a) providing a host cell genetically engineered to express a
recombinant protein of interest; (b) culturing the host cell under
suitable conditions in which the cell expresses the recombinant protein
of interest; and (c) harvesting a preparation of the recombinant protein
of interest produced by the cell. In one aspect, the recombinant protein
of interest is an anti-VEGF protein. In a particular aspect, the
anti-VEGF protein is selected from the group consisting of aflibercept,
MiniTrap, recombinant MiniTrap (an example of which is disclosed in U.S.
Pat. No. 7,279,159), a scFv and other anti-VEGF proteins.
[0062] In one aspect of the present embodiment, the recombinant protein of
interest is expressed in a suitable host cell. Non-limiting examples of a
suitable host-cells include, but are not limited to, CHO, CHO K1,
EESYR.RTM., NICE.RTM., NS0, Sp2/0, embryonic kidney cells and BHK.
[0063] In one aspect of the present embodiment, the recombinant protein of
interest is cultured in a CDM. A suitable CDM includes Dulbecco's
Modified Eagle's (DME) medium, Ham's Nutrient Mixture, Excell medium, IS
CHO-CD medium, and CDM1B. Other CDMs known to those skilled in the art
are also contemplated to be within the scope of the present invention.
[0064] The production preparation can comprise at least one contaminant
including one or more host-cell proteins in addition to the recombinant
protein of interest. The at least one contaminant can be derived from
cell-substrate, cell culture or a downstream process.
[0065] In one embodiment, the invention is directed to methods for
producing an anti-VEGF protein from a biological sample using affinity
chromatography. In a particular aspect, methods disclosed herein can be
used to separate, at least in part, the anti-VEGF protein from one or
more host-cell proteins and nucleic acids (e.g., DNA) formed during the
culture production process of an anti-VEGF protein.
[0066] In one aspect, the method can comprise subjecting a biological
sample comprising the anti-VEGF protein and along with accompanying
contaminants to an affinity chromatography under suitable conditions. In
a particular aspect, the affinity chromatography can comprise material
capable of selectively or specifically binding to the anti-VEGF protein
("capture"). Non-limiting examples of such chromatographic material
include: Protein A, Protein G, chromatographic material comprising, for
example, protein capable of binding to the anti-VEGF protein, and
chromatographic material comprising an Fc binding protein. In a specific
aspect, the protein capable of binding to or interacting with the
anti-VEGF protein can be an antibody, fusion protein or a fragment
thereof. Non-limiting examples of such material capable of selectively or
specifically binding to the anti-VEGF protein are described in Example 7.
[0067] In one aspect of the present embodiment, the method can comprise
subjecting a biological sample comprising an anti-VEGF protein and one or
more host-cell proteins/contaminants to affinity chromatography under
suitable conditions, wherein the affinity chromatography stationary phase
comprises a protein capable of selectively or specifically binding to the
anti-VEGF protein. In a particular aspect, the protein can be an
antibody, a fusion protein, a scFv or an antibody fragment. In a specific
aspect, the protein can be VEGF.sub.165, VEGF.sub.121, VEGF forms from
other species, such as, rabbit. For example, as exemplified in Table 7-1
and Table 7-10, using VEGF.sub.165 as the protein capable of selectively
or specifically binding to or interacting with the anti-VEGF protein led
to a successful production of MT5 (an anti-VEGF protein), aflibercept and
an anti-VEGF scFv fragment. In another specific aspect, the protein can
be one or more of the proteins having amino acid sequence as shown in SEQ
ID NO.: 73-80. Table 7-1 also discloses successful production of MT5
using the proteins having amino acid sequence as shown in SEQ ID NO.:
73-80 as the protein capable of selectively or specifically binding to
the anti-VEGF protein (MT5).
[0068] In one aspect of the present embodiment, the method can comprise
subjecting a biological sample comprising the anti-VEGF protein and one
or more host-cell proteins/contaminants to affinity chromatography under
suitable conditions, wherein the affinity chromatography stationary phase
comprises a protein capable of selectively or specifically binding to or
interacting with the anti-VEGF protein, wherein the anti-VEGF protein can
be selected from aflibercept, VEGF MiniTrap, or an anti-VEGF antibody. In
a particular aspect, the VEGF MiniTrap can be obtained from VEGF receptor
components, further; it can be formed by a recombinant expression of the
VEGF MiniTrap in a host-cell. Performing the method can reduce the amount
of the one or more host-cell proteins in the sample. For example, FIG.
35A and FIG. 35B show a significant reduction in total host-cell proteins
in the sample comprising MT5 (an anti-VEGF protein) on using five
different affinity chromatography columns comprising (i) VEGF.sub.165
(SEQ ID NO.: 72); (ii) mAb1 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ
ID NO.: 73 is a heavy chain and SEQ ID NO.: 74 is a light chain); (iii)
mAb2 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 75 is a heavy
chain and SEQ ID NO.: 76 is a light chain); (iv) mAb3 (a mouse
anti-VEGFR1 mAb mouse IgG1 where SEQ ID NO.: 77 is a heavy chain and SEQ
ID NO.: 78 is a light chain) and (v) mAb4 (a mouse anti-VEGFR1 mAb mouse
IgG1 where SEQ ID NO.: 79 is a heavy chain and SEQ ID NO.: 80 is a light
chain) as different proteins capable of selectively or specifically
binding to MT5. As seen in FIG. 35A and FIG. 35B, the eluates from each
of the affinity-based production processes reduced the host cell proteins
from above 7000 ppm to about 25 ppm, and to about 55 ppm respectively.
[0069] Suitable conditions for employing affinity chromatography can
include, but are not limited to, equilibration of an affinity
chromatography column using an equilibration buffer. Following
equilibration using, for example, Tris hydrochloride at a pH of about 8.3
to about 8.6, the affinity chromatography column is loaded with a
biological sample. Following loading of the column, the column can be
washed one or multiple times using, for example, the equilibrating buffer
such as Dulbecco's Phosphate-Buffered Saline (DPBS). Other washes
including washes employing different buffers can be used before eluting
the column. Column elution can be affected by the buffer type and pH and
conductivity, other elution conditions well known to those skilled in the
art can be applied. Following elution using one or more types of elution
buffers, for example, glycine at a pH of about 2.0 to about 3.0, the
eluted fractions can be neutralized with the addition of a neutralizing
buffer for example, 1 M Tris at pH 7.5.
[0070] In one aspect of the embodiment, the pH of both the wash and
equilibration buffer can be from about 7.0 to about 8.6. In one aspect of
the embodiment, the wash buffer can be DPBS. In one aspect, the elution
buffer can comprise 100 mM glycine buffer with pH of about 2.5. In
another aspect, the elution buffer can be a buffer with a pH of about 2.0
to about 3.0. In one aspect, the neutralizing buffer can comprise 1 M
tris with pH of about 7.5.
[0071] In one aspect of the present embodiment, the method can further
comprise washing the column with a wash buffer. In one aspect of the
present embodiment, the method can further comprise eluting the column
with an elution buffer to obtain elution fractions. In a particular
aspect, the amount of host-cell proteins in the eluted fractions is
significantly reduced as compared to the amount of host-cell proteins in
the biological sample, for example, by about 70%, about 80%, 90%, about
95%, about 98%, or about 99%.
[0072] The present embodiment can include the addition of one or more
steps, in no particular order, such as hydrophobic interaction
chromatography, affinity-based chromatography, multimodal chromatography,
viral inactivation (e.g., using low pH), viral filtration, and/or
ultra/diafiltration.
[0073] In one aspect, glycosylation profile of a composition of an
anti-VEGF protein is as follows: about 40% to about 50% total fucosylated
glycans, about 30% to about 55% total sialylated glycans, about 6% to
about 15% mannose-5, and about 60% to about 79% galactosylated glycans.
[0074] In one aspect of this embodiment, the anti-VEGF protein has Man5
glycosylation at about 32.4% of asparagine 123 residues and/or about
27.1% of asparagine 196 residues. In a specific embodiment, the anti-VEGF
protein can be aflibercept, anti-VEGF antibody or VEGF MiniTrap.
[0075] In one embodiment, the method can further comprise formulating a
drug substance using a pharmaceutically acceptable excipient. In a one
aspect, the pharmaceutically acceptable excipient can be selected from
the following: water, buffering agents, sugar, salt, surfactant, amino
acid, polyol, chelating agent, emulsifier and preservative. Other
well-known excipients to the skilled artisan are within the purview of
this embodiment.
[0076] In one aspect of the embodiment, the formulation can be suitable
for administration to a human subject. In one aspect of the present
embodiment, administration can be effected by intravitreal injection. In
one aspect, the formulation can have about 40 to about 200 mg/mL of the
protein of interest. In a particular aspect, the protein of interest can
be aflibercept, anti-VEGF antibody or VEGF MiniTrap.
[0077] The formulation can be used in a method of treating or preventing
angiogenic eye disorders which can include: age-related macular
degeneration (e.g., wet or dry), macular edema, macular edema following
retinal vein occlusion, retinal vein occlusion (RVO), central retinal
vein occlusion (CRVO), branch retinal vein occlusion (BRVO), diabetic
macular edema (DME), choroidal neovascularization (CNV), iris
neovascularization, neovascular glaucoma, post-surgical fibrosis in
glaucoma, proliferative vitreoretinopathy (PVR), optic disc
neovascularization, corneal neovascularization, retinal
neovascularization, vitreal neovascularization, pannus, pterygium,
vascular retinopathy, diabetic retinopathy in a subject with diabetic
macular edema; or diabetic retinopathies (e.g., non-proliferative
diabetic retinopathy (e.g., characterized by a Diabetic Retinopathy
Severity Scale (DRSS) level of about 47 or 53) or proliferative diabetic
retinopathy; e.g., in a subject that does not suffer from DME).
Synthesis of Oxo-Species
[0078] One embodiment of the present invention is directed to one or more
methods for synthesizing oxidized protein species using light. In one
aspect of the present embodiment, the protein of interest is an anti-VEGF
protein. In a particular aspect, the anti-VEGF protein is aflibercept. In
another aspect, the anti-VEGF protein is a VEGF MiniTrap including
recombinant VEGF MiniTrap. In yet another aspect of the present
embodiment, the anti-VEGF protein is a single-chain variable fragment
(scFv).
[0079] In one aspect of the present embodiment, a sample comprises a
protein of interest, for example, aflibercept fusion protein with minimal
or no oxo-variants. The sample is photo-stressed to synthesize oxidized
species of aflibercept. In a particular aspect, the sample is
photo-stressed by using cool-white light. In another particular aspect,
the sample is photo-stressed by using ultraviolet light.
[0080] In a specific aspect of the embodiment, a sample comprising
aflibercept or another anti-VEGF protein is exposed to cool-white light
for about 30 hours to about 300 hours resulting in about 1.5 to about
50-fold increase in modified oligopeptide. These peptides are
enzymatically digested and analyzed comprising one or more from the group
consisting of:
DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), EIGLLTC*EATVNGH*LYK (SEQ ID NO.:
18), QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), TELNVGIDFNWEYPSSKH*QHK
(SEQ ID NO.: 20), TNYLTH*R (SEQ ID NO.: 21), SDTGRPFVEMYSEIPEIIH*MTEGR
(SEQ ID NO.: 22), VH*EKDK (SEQ ID NO.: 23), SDTGRPFVEM*YSEIPEIIHMTEGR
(SEQ ID NO.: 64), SDTGRPFVEMYSEIPEIIHM*TEGR (SEQ ID NO.: 65), TQSGSEM*K
(SEQ ID NO.: 66), SDQGLYTC*AASSGLM*TK (SEQ ID NO.: 67),
IIW*DSR/RIIW*DSR/IIW*DSRK (SEQ ID NO.: 28), TELNVGIDFNW*EYPSSK (SEQ ID
NO.: 29), GFIISNATY*K (SEQ ID NO.: 69), KF*PLDTLIPDGK (SEQ ID NO.: 70)
F*LSTLTIDGVTR (SEQ ID NO.: 32), wherein H* is a histidine is oxidized to
2-oxo-histidine, wherein C* is a cysteine is carboxymethylated, wherein
M* is a oxidized methionine, wherein W* is a oxidized tryptophan, wherein
Y* is a oxidized tyrosine, and wherein F* is a oxidized phenylalanine.
The digestion can be performed by proteases alluded to before, for
example, trypsin. The oligopeptides can be analyzed using mass
spectrometry.
[0081] In a specific aspect of the embodiment, a sample comprising
aflibercept or other anti-VEGF protein is exposed to ultraviolet light
for about 4 hours to about 40 hours resulting in about 1.5 to about
25-fold increase in modified oligopeptide products (obtained on
performing digestion) wherein the sample comprises one or more modified
oligopeptides selected from the group consisting of:
DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), EIGLLTC*EATVNGH*LYK (SEQ ID NO.:
18), QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), TELNVGIDFNWEYPSSKH*QHK
(SEQ ID NO.: 20), TNYLTH*R (SEQ ID NO.: 21), SDTGRPFVEMYSEIPEIIH*MTEGR
(SEQ ID NO.: 22), VH*EKDK (SEQ ID NO.: 23), SDTGRPFVEM*YSEIPEIIHMTEGR
(SEQ ID NO.: 64), SDTGRPFVEMYSEIPEIIHM*TEGR (SEQ ID NO.: 65), TQSGSEM*K
(SEQ ID NO.: 66), SDQGLYTC*AASSGLM*TK (SEQ ID NO.: 67),
IIW*DSR/RIIW*DSR/IIW*DSRK (SEQ ID NO.: 28), TELNVGIDFNW*EYPSSK (SEQ ID
NO.: 29), GFIISNATY*K (SEQ ID NO.: 69), KF*PLDTLIPDGK (SEQ ID NO.: 70)
F*LSTLTIDGVTR (SEQ ID NO.: 32), wherein H* is a histidine is oxidized to
2-oxo-histidine, wherein C* is a cysteine is carboxymethylated, wherein
M* is a oxidized methionine, wherein W* is a oxidized tryptophan, wherein
Y* is a oxidized tyrosine, and wherein F* is a oxidized phenylalanine.
The digestion can be performed by proteases alluded to before, for
example, trypsin. The oligopeptides can be analyzed using mass
spectrometry.
Methods to Minimize Yellow-Brown Color
[0082] The present disclosure provides methods for reducing yellow-brown
coloration during production of aflibercept, MiniTrap or the like
produced in a CDM.
[0083] In one embodiment, the method comprises culturing a host cell in a
CDM under suitable conditions, wherein the host cell expresses a
recombinant protein of interest and then harvesting a preparation
comprising the recombinant protein of interest. In one aspect, the
recombinant protein of interest is an anti-VEGF protein. In a particular
aspect, the anti-VEGF protein is selected from the group consisting of
aflibercept, MiniTrap, recombinant MiniTrap (examples of which are
disclosed in U.S. Pat. No. 7,279,159, which is incorporated herein by
reference in its entirety), a scFv and other anti-VEGF proteins. In one
aspect, the method can produce a preparation of the recombinant protein
of interest, wherein the color of the preparation is characterized using
the European BY method or the CIELAB method (b*). Additionally, the
presence of oxo-variants can be analyzed using, for example, LC-MS.
[0084] In one aspect of the present embodiment, mitigation conditions
include increasing or decreasing cumulative concentrations of one or more
media components, for example, amino acids, metals or anti-oxidants,
including, salts and precursors, corresponding to a reduction in color
and protein variants of aflibercept and VEGF MiniTrap. Non-limiting
examples of amino acids include alanine, arginine, asparagine, aspartic
acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, proline, serine, threonine,
tryptophan, tyrosine, and valine. In a particular aspect, lowering of
cysteine can be effective in lowering the yellow-brown color of a
preparation. Cysteine concentration can also affect oxo-variants.
[0085] In one embodiment, the method comprises culturing a host cell in a
CDM under suitable conditions, wherein the host cell expresses a
recombinant protein of interest, such as aflibercept, and harvesting a
preparation of the protein of interest produced by the cell, wherein the
suitable conditions are obtained, in part, by lowering the cumulative
concentration of cysteine in the CDM to less than or equal to about 10
mM. Examples of suitable media include, but are not limited to, CDM1B,
Excell or the like. As used herein, the term "cumulative amount" refers
to the total amount of a particular component added to a bioreactor over
the course of the cell culture to form the CDM, including amounts added
at the beginning of the culture (CDM at day 0) and subsequently added
amounts of the component. Amounts of a component added to a seed-train
culture or inoculum prior to the bioreactor production (i.e., prior to
the CDM at day 0) are also included when calculating the cumulative
amount of the component. A cumulative amount is unaffected by the loss of
a component over time during the culture (for example, through metabolism
or chemical degradation). Thus, two cultures with the same cumulative
amounts of a component may nonetheless have different absolute levels,
for example, if the component is added to the two cultures at different
times (e.g., if in one culture all of the component is added at the
outset, and in another culture the component is added over time). A
cumulative amount is also unaffected by in situ synthesis of a component
over time during the culture (for example, via metabolism or chemical
conversion). Thus, two cultures with the same cumulative amounts of a
given component may nonetheless have different absolute levels, for
example, if the component is synthesized in situ in one of the two
cultures by way of a bioconversion process. A cumulative amount may be
expressed in units such as, for example, grams or moles of the component.
[0086] As used herein, the term "cumulative concentration" refers to the
cumulative amount of a component divided by the volume of liquid in the
bioreactor at the beginning of the production batch, including the
contribution to the starting volume from any inoculum used in the
culture. For example, if a bioreactor contains 2 liters of cell culture
medium at the beginning of the production batch, and one gram of
component X is added at days 0, 1, 2, and 3, then the cumulative
concentration after day 3 is 2 g/L (i.e., 4 grams divided by 2 liters).
If, on day 4, an additional one liter of liquid not containing component
X were added to the bioreactor, the cumulative concentration would remain
2 g/L. If, on day 5, some quantity of liquid were lost from the
bioreactor (for example, through evaporation), the cumulative
concentration would remain 2 g/L. A cumulative concentration may be
expressed in units such as, for example, grams per liter or moles per
liter.
[0087] In an aspect of this embodiment, the method comprises culturing a
host cell in a CDM under suitable conditions, wherein the host cell
expresses a recombinant protein of interest, harvesting a preparation of
the protein produced by the cell, wherein the suitable conditions are
obtained by lowering the ratio of cumulative cysteine concentration from
about 1:10 to 1:29 to a cumulative total amino acid concentration from
about 1:50 to about 1:30.
[0088] In one embodiment, the method comprises (i) culturing a host cell
in a CDM under suitable conditions, wherein the host cell expresses a
recombinant protein of interest, such as aflibercept, and (ii) harvesting
a preparation of the recombinant protein of interest produced by the
cell, wherein the suitable conditions are obtained by lowering the
cumulative concentration of iron in the CDM to less than about 55.0
.mu.M. In an aspect of this embodiment, the preparation obtained by this
method shows lesser yellow-brown color than the preparation obtained by a
method wherein the cumulative concentration of iron in the CDM to more
than about 55.0 .mu.M.
[0089] In one embodiment, the method comprises culturing a host cell in a
CDM under suitable conditions, wherein the host cell expresses a
recombinant protein of interest, such as aflibercept. The method further
comprises harvesting a preparation of the recombinant protein of interest
produced by the cell, wherein the suitable conditions are obtained by
lowering the cumulative concentration of copper in the CDM to less than
or equal to about 0.8 .mu.M. In an aspect of this embodiment, the
preparation obtained by this method shows lesser yellow-brown color than
the preparation obtained by a method wherein the cumulative concentration
of copper in the CDM to more than about 0.8 .mu.M.
[0090] In one embodiment, the method comprises culturing a host cell in a
CDM under suitable conditions, wherein the host cell expresses a
recombinant protein of interest, such as aflibercept, and harvesting a
preparation of the recombinant protein of interest produced by the cell,
wherein the suitable conditions are obtained by lowering the cumulative
concentration of nickel in the CDM to less than or equal to about 0.40
.mu.M. In an aspect of this embodiment, the preparation obtained by this
method shows lesser yellow-brown color than the preparation obtained by a
method wherein the cumulative concentration of nickel in the CDM to more
than about 0.40 .mu.M.
[0091] In one embodiment, the method comprises culturing a host cell in a
CDM under suitable conditions, wherein the host cell expresses a
recombinant protein of interest, such as aflibercept. The method further
comprises harvesting a preparation of the recombinant protein of interest
produced by the cell, wherein the suitable conditions are obtained by
lowering the cumulative concentration of zinc in the CDM to less than or
equal to about 56 .mu.M. In an aspect of this embodiment, the preparation
obtained by this method shows lesser yellow-brown color than the
preparation obtained by a method wherein the cumulative concentration of
zinc in the CDM to more than about 56 .mu.M.
[0092] In one embodiment, the method comprises culturing a host cell in a
CDM under suitable conditions, wherein the host cell expresses a
recombinant protein of interest, such as aflibercept. The method further
comprises harvesting a preparation of the recombinant protein of interest
produced by the cell, wherein the suitable conditions are obtained by
presence of anti-oxidants in the CDM in a cumulative concentration of
about 0.001 mM to about 10 mM for a single antioxidant and no more than
about 30 mM cumulative concentration if multiple antioxidants are added
in said CDM. In an aspect of this embodiment, the preparation obtained by
this method shows lesser yellow-brown color than the preparation obtained
by a method wherein the method without anti-oxidants in the CDM in a
cumulative concentration of about less than about 0.01 mM or above about
100 mM. Non-limiting examples of the anti-oxidant can be taurine,
hypotaurine, glycine, thioctic acid, glutathione, choline chloride,
hydrocortisone, Vitamin C, Vitamin E, chelating agents, catalase,
S-carboxymethyl-L-cysteine, and combinations thereof. Non-limiting
examples of chelating agents include aurintricarboxylic acid (ATA),
deferoxamine (DFO), EDTA and citrate.
[0093] In one embodiment, the method comprises culturing a host cell in a
CDM under suitable conditions, wherein the host cell expresses a
recombinant protein of interest, such as aflibercept. The method further
comprises harvesting a preparation of the recombinant protein of interest
produced by the cell, wherein the suitable conditions include a CDM with
a: cumulative concentration of iron in said CDM that is less than about
55 .mu.M, cumulative concentration of copper in said CDM that is less
than or equal to about 0.8 .mu.M, cumulative concentration of nickel in
said CDM that is less than or equal to about 0.40 .mu.M, cumulative
concentration of zinc in said CDM that is less than or equal to about 56
.mu.M, cumulative concentration of cysteine in said CDM that is less than
10 mM; and/or an anti-oxidant in said CDM in a concentration of about
0.001 mM to about 10 mM for a single antioxidant, and no more than about
30 mM cumulative concentration if multiple antioxidants are added in said
CDM.
[0094] In one aspect of the present embodiment, the preparation obtained
from using suitable conditions results in a reduction in protein variants
of aflibercept and VEGF MiniTrap to a desired amount of protein variants
of aflibercept and VEGF MiniTrap (referred to as a "target value" of
protein variants of aflibercept and VEGF MiniTrap). In further aspect of
this embodiment, the preparation obtained from using suitable conditions
results in a reduction in color of the preparations to a desired b* value
or BY value (referred to as a "target b* value" "target BY value"
respectively) when the preparation of protein, including variants of
aflibercept and VEGF MiniTrap are normalized to a concentration of 5 g/L
or 10 g/L. In a further aspect of the present embodiment, the target b*
value (or target BY value) and/or target value of variants can be
obtained in a preparation where the titer increases or does not
significantly decrease.
[0095] These and other aspects of the invention will be better appreciated
and understood when considered in conjunction with the following
description and the accompanying drawings. The following description,
while indicating various embodiments and numerous specific details
thereof, is given by way of illustration and not of limitation. Many
substitutions, modifications, additions, or rearrangements may be made
within the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0096] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office upon
request and payment of the necessary fee.
[0097] FIG. 1 depicts a VEGF MiniTrap generated using an exemplary
embodiment, including VEGFR1 (SEQ ID NO.: 34), VEGFR2 (SEQ ID NO.: 36,
Hinge domain fragment (SEQ ID NO.: 60) and the cleaved off Fc fragment
from aflibercept (SEQ ID NO.: 55).
[0098] FIG. 2 depicts a proposed mechanism for histidine oxidation to
2-oxo-histidine (14 Da).
[0099] FIG. 3 depicts a proposed mechanism for histidine oxidation to
2-oxo-histidine (16 Da).
[0100] FIG. 4 depicts proposed mechanism for oxidation of tryptophan to
N-formylkynurenine and kynurenine.
[0101] FIG. 5 depicts an exemplary embodiment for production of
aflibercept.
[0102] FIG. 6 depicts an exemplary embodiment for production of VEGF
MiniTrap.
[0103] FIG. 7 depicts an exemplary embodiment for production of
aflibercept.
[0104] FIG. 8 depicts an exemplary embodiment for production of VEGF
MiniTrap.
[0105] FIG. 9 depicts a chart of calculated BY standards versus b* value
calculated as an exemplary embodiment.
[0106] FIG. 10 depicts results of an experiment performed to evaluate the
percentage of 2-oxo-histidines and tryptophan oxidation (where
underscoring represents oxidation of the residue) in oligopeptides from
protease-digested AEX load and flowthrough, including fragments of
reduced and alkylated aflibercept (SEQ ID NO.: 55).
[0107] FIG. 11 depicts the relative abundance of the peptides identified
from the peptide mapping analysis performed using oligopeptides from
protease-digested AEX load and flowthrough (where underscoring represents
oxidation of the residue in the peptide sequence), including fragments of
aflibercept (SEQ ID NO.: 55).
[0108] FIG. 12A depicts a full-view of the chromatogram chart of
absorbance versus time (minutes) for MT4 and MT1 at 350 nm.
[0109] FIG. 12B depicts an expanded-view of the chromatogram chart of
absorbance versus time (16-30 minutes) for MT4 and MT1 at 350 nm,
including SEQ ID NO.: 21 and 28.
[0110] FIG. 12C depicts an expanded-view of the chromatogram chart of
absorbance versus time (30-75 minutes) for MT4 and MT1 at 350 nm,
including SEQ ID NO.: 17, 18, 19, 20.
[0111] FIG. 13 depicts results of an experiment performed to evaluate the
percentage of 2-oxo-histidines (and tryptophan dioxidation) in
oligopeptides from protease digested MT1 which has been processed by AEX
chromatography and oligopeptides from protease digested MT1 which has
been stripped from AEX chromatography, including SEQ ID NO.: 17, 18, 19,
20, 21, 28.
[0112] FIG. 14 depicts results of an experiment performed to compare the
acidic species present in different production lots of MT1 and the acidic
acid fractions obtained on performing a strong cation exchange (CEX)
chromatography, including SEQ ID NO.: 17, 18, 19, 20, 21, 28.
[0113] FIG. 15 depicts an exemplary method for the enrichment of the
acidic species and other variants present in cell culture harvest samples
using strong cation exchange chromatography.
[0114] FIG. 16 depicts the fractions from performing strong cation
exchange chromatography according to an exemplary embodiment.
[0115] FIG. 17 depicts strong cation exchange chromatograms performed
according to an exemplary embodiment for the MT1 production (prior to any
production procedure, .ltoreq.BY3) subjected to CEX and for enriched
variants of desialylated MiniTrap (dsMT1) using a dual salt-pH gradient.
[0116] FIG. 18A depicts a 3D chromatogram for unfractionated parental
control carried out by strong cation exchange chromatography according to
an exemplary embodiment.
[0117] FIG. 18B depicts a 3D chromatogram for MT1, fraction 1 representing
some of the tailing feature for the experiment carried out by strong
cation exchange chromatography according to an exemplary embodiment.
[0118] FIG. 18C depicts a 3D chromatogram for MT1, fraction 2 feature
carried out by strong cation exchange chromatography according to an
exemplary embodiment.
[0119] FIG. 18D depicts a 3D chromatogram for MT1, fraction 3 feature
carried out by strong cation exchange chromatography according to an
exemplary embodiment.
[0120] FIG. 18E depicts a 3D chromatogram for MT1, fraction 4 feature
carried out by strong cation exchange chromatography according to an
exemplary embodiment.
[0121] FIG. 18F depicts a 3D chromatogram for MT1, fraction 5 feature
carried out by strong cation exchange chromatography according to an
exemplary embodiment.
[0122] FIG. 18G depicts a 3D chromatogram for MT1, fraction 6 feature
carried out by strong cation exchange chromatography according to an
exemplary embodiment.
[0123] FIG. 1811 depicts a 3D chromatogram for MT1, fraction 7 feature
carried out by strong cation exchange chromatography according to an
exemplary embodiment.
[0124] FIG. 19 depicts imaged capillary isoelectric focusing (icIEF)
electropherograms performed according to an exemplary embodiment for the
MT1 production.
[0125] FIG. 20 depicts results of a study correlating the exposure of MT1
cool white light or UVA light with the appearance of oxidized amino acid
residues, including SEQ ID NO.: 17, 18, 19, 20, 21, 28, 29, and 83.
[0126] FIG. 21 depicts the 3D SEC-PDA (size exclusion chromatography
coupled to photodiode array detection) chromatograms on CWL-stressed MT1
with absorbance at .about.350 nm (see, e.g., circle highlighting
.about.350 nm) according to an exemplary embodiment where A shows the
chromatogram at T=0, B shows the chromatogram at 0.5.times.ICH, 0.0 shows
the chromatogram at 2.0.times.ICH, and D images of MT1 in vials
(normalized to 80 mg/mL) stressed by CWL for different time intervals.
[0127] FIG. 22 depicts the 3D SEC-PDA chromatograms on UVA-stressed MT1
with absorbance at .about.350 nm (see, e.g., circle highlighting
.about.350 nm) according to an exemplary embodiment where A shows the
chromatogram at T=0, B shows the chromatogram at 0.5.times.ICH, 0.0 shows
the chromatogram at 2.0.times.ICH, and D images of MT1 in vials
(normalized to 80 mg/mL) stressed by UVA for different time intervals.
[0128] FIG. 23 A depicts A320/280 absorbance ratios quantitated from
SEC-PDA chromatograms for the samples stressed using CWL (top panel) and
B a chart of A320/280 absorbance ratios for size variants in the samples
stressed using CWL (bottom panel), wherein the samples are stressed
according to an exemplary embodiment.
[0129] FIG. 24 A depicts A320/280 absorbance ratios quantitated from
SEC-PDA chromatograms for the samples stressed using UVA (top panel) and
B a chart of A320/280 absorbance ratios for size variants in the samples
stressed using UVA (bottom panel), wherein the samples are stressed
according to an exemplary embodiment.
[0130] FIG. 25 A depicts a scaled estimate of the effect that incubation
of various components with aflibercept have on the generation of color
(CIE L*, a*, b* predicted b value); and B actual against predicted b
value plot.
[0131] FIG. 26 depicts the effect of CDMs comprising low cysteine and low
metals on the titer of aflibercept (A), viable cell concentration (B),
viability (C), ammonia (D), and osmolality (E).
[0132] FIG. 27 is a chart showing prediction profile of the color of the
harvest (seen as Day 13 b* values) on increasing/decreasing
concentrations of metals and cysteine according to an exemplary
embodiment.
[0133] FIG. 28 (A-B) depicts the effect of incubation of various
components with aflibercept in spent CDM on the generation of color (CIE
L*, a*, b* predicted b value) (A); and by a plot of scaled predicted
impacts on b value (B).
[0134] FIG. 28C depicts the scaled estimated effects of incubation of
various components with aflibercept in CDM on the generation of color
(CIE L*, a*, b* predicted b value) in a shake flask culture.
[0135] FIG. 28D depicts the effect of incubation of hypotaurine and
deferoxamine mesylate salt (DFO) with aflibercept in spent CDM on the
generation of color (CIE L*, a*, b* predicted "b" value).
[0136] FIG. 28E depicts the effect of incubation of various components
individually with aflibercept from shake flask culture on the generation
of color (CIE L*, a*, b* predicted "b" value).
[0137] FIG. 29 is a chart showing the effect of addition of uridine,
manganese, galactose and dexamethasone in CDMs on the titer of the
production of aflibercept produced.
[0138] FIG. 30 is a chart showing the effect of addition of uridine,
manganese, galactose and dexamethasone in CDMs on the viability of cells
expressing aflibercept, wherein the aflibercept is produced.
[0139] FIG. 31 is a chart showing the effect of addition of uridine,
manganese, galactose and dexamethasone in CDMs on the viable cell count
of cells expressing aflibercept, wherein the aflibercept is produced.
[0140] FIG. 32 is a chart showing a standard curve of absorbance versus
host cell protein concentrations (ng/mL) prepared using standard host
cell protein solutions from Cygnus 3G (F550).
[0141] FIG. 33 is an image of SDS-PAGE analysis performed using
non-reducing SDS-PAGE sample buffer.
[0142] FIG. 34 is an image of SDS-PAGE analysis performed using reducing
SDS-PAGE sample buffer.
[0143] FIG. 35A is a chart of total host cell protein detected in loading
solution, eluted fractions from affinity chromatography columns 1-3
comprising VEGF.sub.165, mAb1 and mAb2, respectively.
[0144] FIG. 35B is a chart of total host cell proteins detected in loading
solution, eluted fractions from affinity chromatography columns 1, 2, 4
and 5 comprising VEGF.sub.165, mAb1, mAb3 and mAb4, respectively.
[0145] FIG. 36 depicts the SEC profiles of VEGF MiniTrap A before and B
after performing affinity chromatography production.
[0146] FIG. 37 depicts a cartoon representation of the kinetic study of
VEGF MiniTrap to VEGF.sub.165, wherein the VEGF MiniTrap constructs
studied were from before and after performing affinity chromatography
production according to some exemplary embodiments.
[0147] FIG. 38 depicts SPR sensorgrams from the kinetic study of VEGF
MiniTrap to VEGF.sub.165, wherein the VEGF MiniTrap constructs studied
were from before and after performing affinity chromatography production
according to some exemplary embodiments.
[0148] FIG. 39 is a chart of total host cell protein detected in loading
solution, eluted fractions from affinity chromatography columns used
repeatedly for columns comprising VEGF.sub.165, mAb1 and mAb2.
[0149] FIG. 40 depicts the structure of VEGF MiniTrap MT1 (SEQ ID NO.: 46)
according to an exemplary embodiment.
[0150] FIG. 41 depicts the structure of VEGF MiniTrap MT6 (SEQ ID NO.: 51)
according to an exemplary embodiment.
[0151] FIG. 42 depicts Total Ion Chromatograms (TIC) of relative
absorbance versus time (minutes) for native SEC-MS analysis of MT1, MT5
and MT6 and a zoomed view of low molecular weight region from the TICs.
[0152] FIG. 43 depicts a deconvoluted mass spectra of the main peak for
MT1 and MT5 to confirm the MiniTrap dimer identity with elucidation for
some PTMs.
[0153] FIG. 44 depicts a deconvoluted mass spectra of the main peak for
MT6 confirm the single chain MiniTrap identity with elucidation for some
PTMs.
[0154] FIG. 45A depicts a chart of relative absorbance versus time
(minutes) for low molecular weight impurities in MT1.
[0155] FIG. 45B depicts mass spectra for the low molecular weight
impurities in MT1.
[0156] FIG. 46 depicts relative absorbance versus time (minutes) for MT1
which shows absence of the FabRICATOR enzyme which was used to cleave
aflibercept into MT1.
[0157] FIG. 47 depicts relative absorbance versus time (minutes) for low
molecular weight impurities in MT5.
[0158] FIG. 48 depicts relative absorbance versus time (minutes) for low
molecular weight impurities in MT6.
[0159] FIG. 49A depicts a chart of absorbance versus time (minutes)
obtained on performing HILIC-UV/MS for VEGF MiniTrap MT6, wherein the
chart shows the elution of main peak at 21 minutes and O-glycans at
around 21.5 minutes.
[0160] FIG. 49B depicts a mass spectrum obtained on performing HILIC-UV/MS
for VEGF MiniTrap MT6 showing the main peak at 47985.8 Da.
[0161] FIG. 49C depicts a mass spectra of 0-glycans of VEGF MiniTrap MT6
obtained on performing HILIC-UV/MS.
[0162] FIG. 50 is an image of VEGF MiniTrap dimer wherein the disulfide
bridge in the hinge region (SEQ ID NO.: 83) of the VEGF MiniTrap can be
parallel or crossed.
[0163] FIG. 51 depicts relative abundance of distribution of glycans
observed at Asn36, including SEQ ID NO.: 100, among MT1, MT5 and MT6.
[0164] FIG. 52 depicts relative abundance of distribution of glycans
observed at Asn68, including SEQ ID NO.: 101, among MT1, MT5 and MT6.
[0165] FIG. 53 depicts relative abundance of distribution of glycans
observed at Asn123, including SEQ ID NO.: 102, among MT1, MT5 and MT6.
[0166] FIG. 54 depicts relative abundance of distribution of glycans
observed at Asn196, including SEQ ID NO.: 103, among MT1, MT5 and MT6.
[0167] FIG. 55 depicts the released N-linked glycan analysis by
hydrophilic interaction chromatography (HILIC) coupled to florescence
detection and mass spectrometry analysis (full scale and stacked).
[0168] FIG. 56 depicts a HILIC-FLR chromatograms for MT1, MT5 and MT6.
[0169] FIG. 57 depicts the released N-linked glycan analysis by HILIC
coupled to fluorescence detection and mass spectrometry analysis (full
scale, stacked and normalized).
[0170] FIG. 58A is a table of detailed glycan identification and
quantification from VEGF MiniTrap samples MT1, MT5 and MT6.
[0171] FIG. 58B is a table of detailed glycan identification and
quantification from VEGF MiniTrap samples MT1, MT5 and MT6.
[0172] FIG. 58C is a table of detailed glycan identification and
quantification from VEGF MiniTrap samples MT1, MT5 and MT6.
[0173] FIG. 59 depicts an exemplary production procedure for manufacturing
MiniTrap according to an exemplary embodiment.
DETAILED DESCRIPTION
[0174] Angiogenesis, the growth of new blood vessels from preexisting
vasculature, is a highly orchestrated process that is critical for proper
embryonic and postnatal vascular development. Abnormal or pathological
angiogenesis is a hallmark of cancer and several retinal diseases where
the upregulation of proangiogenic factors, such as vascular endothelial
growth factor (VEGF) leads to increases in endothelial proliferation,
changes in vasculature morphology, and increased vascular permeability.
Elevated levels of VEGF have been found in the vitreous fluid and retinal
vasculature of patients with various ocular diseases. Blocking VEGF
activity has also become the therapy of choice for treating DME, wet AMD,
CNV, retinal vein occlusions, and other ocular diseases where abnormal
angiogenesis is the underlying etiology.
[0175] As used herein, aflibercept is one such anti-VEGF protein
comprising all-human amino acid sequence comprising the second Ig domain
of human VEGFR1 and the third Ig domain of human VEGFR2 expressed as an
inline fusion with a (Fc) of human IgG1. Aflibercept binds all forms of
VEGF-A (VEGF) but in addition binds P1GF and VEGF-B. Several other
homodimeric VEGF MiniTraps have been generated as enzymatically cleaved
products from aflibercept or recombinantly expressed directly from host
cell lines. One such example of a VEGF MiniTrap is shown in FIG. 1. In
this figure, a terminal lysine is depicted (k), some culture processes
remove this terminal lysine while others do not. FIG. 1 illustrates a
process whereby the terminal lysine remains. In general, aflibercept
encompasses both the presence and absence of the terminal lysine.
[0176] As demonstrated herein, the present invention, in part, discloses
the production of anti-VEGF proteins (Example 1) using a CDM. Analysis of
solutions comprising aflibercept produced using certain CDMs demonstrated
a certain color property, such as an intense yellow-brown color. The
intensity of the solution's color depended upon the CDM used. Not all
CDMs examined produced a sample with a distinct yellow-brown color after
the solutions were normalized to a concentration of 5 g/L.
[0177] A color, such as yellow-brown, in an injectable therapeutic drug
solution, can be an undesirable feature. It may be an important parameter
employed for determining if a drug product satisfies a predetermined
level of purity and quality for a particular therapeutic. A color such as
yellow-brown observed along the manufacturing route of a biologic can be
caused by chemical modifications of that biologic, degradation products
of formulation excipients, or degradation products formed through the
reaction of the biologic and formulation excipients. However, such
information can be valuable for understanding the cause of the color
change. It can also assist in designing short-term as well as long-term
storage conditions to prevent modifications facilitating such a color
change.
[0178] The inventors observed that use of AEX during the production of an
anti-VEGF protein solution minimized yellow-brown coloration.
Additionally, the inventors discovered that the yellow-brown coloration
can be decreased by modifying the cell culture used to produce a
recombinant protein, such as aflibercept or a modified aflibercept like
MiniTrap.
[0179] The present invention encompasses anti-VEGF proteins and their
production using CDM. Additionally, the present invention is based on the
identification and optimization of upstream and downstream process
technologies for protein production.
[0180] As demonstrated herein, some of the Examples set forth below
describe the production of anti-VEGF proteins (Example 1), production of
oxidized species of anti-VEGF proteins (Example 4), methods to reduce
oxidized species of anti-VEGF proteins by optimizing culture medium
(Example 5) and by optimizing production methods (Example 2).
[0181] A number of recent patent applications and granted patents purport
to describe various aflibercept species and methods of producing the
same, but none describe or suggest the anti-VEGF compositions and methods
for producing the same described herein. See, e.g., U.S. application Ser.
No. 16/566,847 to Coherus Biosciences Inc., U.S. Pat. No. 10,646,546 to
Sam Chun Dang Pharm. Co., Ltd., U.S. Pat. No. 10,576,128 to Formycon AG,
International Application No. PCT/US2020/015659 to Amgen Inc., and U.S.
Pat. Nos. 8,956,830; 9,217,168; 9,487,810; 9,663,810; 9,926,583; and U.S.
Pat. No. 10,144,944 to Momenta Pharmaceuticals, Inc.
I. Explanation of Selected Terms
[0182] Unless described otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Methods and materials
similar or equivalent to those described herein known to the skilled
artisan can be used in the practice of particular embodiments described
herein. All publications mentioned are hereby incorporated by reference
in their entirety.
[0183] The term "a" should be understood to mean "at least one" and the
terms "about" and "approximately" should be understood to permit standard
variation as would be understood by those of ordinary skill in the art
and where ranges are provided, endpoints are included.
[0184] As used herein, the term "angiogenic eye disorder" means any
disease of the eye, which is caused by or associated with the growth or
proliferation of blood vessels or by blood vessel leakage.
[0185] As used herein, the term "chemically defined medium" or "chemically
defined media" (both abbreviated "CDM") refers to a synthetic growth
medium in which the identity and concentration of all the ingredients are
defined. Chemically defined media do not contain bacterial, yeast,
animal, or plant extracts, animal serum or plasma although individual
plant or animal-derived components (e.g., proteins, polypeptides, etc.)
may be added. Chemically defined media may contain inorganic salts such
as phosphates, sulfates, and the like needed to support growth. The
carbon source is defined, and is usually a sugar such as glucose,
lactose, galactose, and the like, or other compounds such as glycerol,
lactate, acetate, and the like. While certain chemically defined culture
media also use phosphate salts as a buffer, other buffers may be employed
such as sodium bicarbonate, HEPES, citrate, triethanolamine, and the
like. Examples of commercially available chemically defined media
include, but are not limited to, various Dulbecco's Modified Eagle's
(DME) media (Sigma-Aldrich Co; SAFC Biosciences, Inc.), Ham's Nutrient
Mixture (Sigma-Aldrich Co; SAFC Biosciences, Inc.), various EX-CELLs
mediums (Sigma-Aldrich Co; SAFC Biosciences, Inc.), various IS CHO-CD
mediums (FUJIFILM Irvine Scientific), combinations thereof, and the like.
Methods of preparing chemically defined culture media are known in the
art, for example, in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO
2007/077217, and U.S. Patent Application Publication Nos. 2008/0009040
and 2007/0212770, the entire teachings of which are herein incorporated
by reference.
[0186] As used herein, the term "cumulative amount" refers to the total
amount of a particular component added to a bioreactor over the course of
the cell culture to form the CDM, including amounts added at the
beginning of the culture (CDM at day 0) and subsequently added amounts of
the component. Amounts of a component added to a seed-train culture or
inoculum prior to the bioreactor production (i.e., prior to the CDM at
day 0) are also included when calculating the cumulative amount of the
component. A cumulative amount is unaffected by the loss of a component
over time during the culture (for example, through metabolism or chemical
degradation). Thus, two cultures with the same cumulative amounts of a
component may nonetheless have different absolute levels, for example, if
the component is added to the two cultures at different times (e.g., if
in one culture all of the component is added at the outset, and in
another culture the component is added over time). A cumulative amount is
also unaffected by in situ synthesis of a component over time during the
culture (for example, via metabolism or chemical conversion). Thus, two
cultures with the same cumulative amounts of a given component may
nonetheless have different absolute levels, for example, if the component
is synthesized in situ in one of the two cultures by way of a
bioconversion process. A cumulative amount may be expressed in units such
as, for example, grams or moles of the component.
[0187] As used herein, the term "cumulative concentration" refers to the
cumulative amount of a component divided by the volume of liquid in the
bioreactor at the beginning of the production batch, including the
contribution to the starting volume from any inoculum used in the
culture. For example, if a bioreactor contains 2 liters of cell culture
medium at the beginning of the production batch, and one gram of
component X is added at days 0, 1, 2, and 3, then the cumulative
concentration after day 3 is 2 g/L (i.e., 4 grams divided by 2 liters).
If, on day 4, an additional one liter of liquid not containing component
X were added to the bioreactor, the cumulative concentration would remain
2 g/L. If, on day 5, some quantity of liquid were lost from the
bioreactor (for example, through evaporation), the cumulative
concentration would remain 2 g/L. A cumulative concentration may be
expressed in units such as, for example, grams per liter or moles per
liter.
[0188] As used herein, the term "formulation" refers to a protein of
interest that is formulated together with one or more pharmaceutically
acceptable vehicles. In one aspect, the protein of interest is
aflibercept and/or MiniTrap. In some exemplary embodiments, the amount of
protein of interest in the formulation can range from about 0.01 mg/mL to
about 600 mg/mL. In some specific embodiments, the amount of the protein
of interest in the formulation can be about 0.01 mg/mL, about 0.02 mg/mL,
about 0.03 mg/mL, about 0.04 mg/mL, about 0.05 mg/mL, about 0.06 mg/mL,
about 0.07 mg/mL, about 0.08 mg/mL, about 0.09 mg/mL, about 0.1 mg/mL,
about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about
0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, about 1
mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about
6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL,
about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35
mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL,
about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 5 mg/mL, about 80
mg/mL, about 85 mg/mL, about 90 mg/mL, about 100 mg/mL, about 110 mg/mL,
about 120 mg/mL, about 130 mg/mL, about 140 mg/mL, about 150 mg/mL, about
160 mg/mL, about 170 mg/mL, about 180 mg/mL, about 190 mg/mL, about 200
mg/mL, about 225 mg/mL, about 250 mg/mL, about 275 mg/mL, about 300
mg/mL, about 325 mg/mL, about 350 mg/mL, about 375 mg/mL, about 400
mg/mL, about 425 mg/mL, about 450 mg/mL, about 475 mg/mL, about 500
mg/mL, about 525 mg/mL, about 550 mg/mL, about 575 mg/mL, or about 600
mg/mL. In some exemplary embodiments, pH of the composition can be
greater than about 5.0. In one exemplary embodiment, the pH can be
greater than about 5.0, greater than about 5.5, greater than about 6,
greater than about 6.5, greater than about 7, greater than about 7.5,
greater than about 8, or greater than about 8.5.
[0189] As used herein, the term "database" refers to a bioinformatics
tool, which provides for the possibility of searching the uninterpreted
MS-MS spectra against all possible sequences in the database(s).
Non-limiting examples of such tools are Mascot
(http://www.matrixscience.com), Spectrum Mill
(http://www.chem.agilent.com), PLGS (http://www.waters.com), PEAKS
(http://www.bioinformaticssolutions.com), Proteinpilot
(http://download.appliedbiosystems.com//proteinpilot), Phenyx
(http://www.phenyx-ms.com), Sorcerer (http://www.sagenresearch.com),
OMSSA (http://www.pubchem.ncbi.nlm.nih.gov/omssa/), X!Tandem
(http://www.thegpm.org/TANDEM/), Protein Prospector
(http://www.http://prospector.ucsf.edu/prospector/mshome.htm), Byonic
(https://www.proteinmetrics.com/products/byonic) or Sequest
(http://fields.scripps.edu/sequest).
[0190] As used herein, the term "ultrafiltration" or "UF" can include a
membrane filtration process similar to reverse osmosis, using hydrostatic
pressure to force water through a semi-permeable membrane.
Ultrafiltration is described in detail in: LEOS J. ZEMAN & ANDREW L.
ZYDNEY, MICROFILTRATION AND ULTRAFILTRATION: PRINCIPLES AND APPLICATIONS
(1996), the entire teaching of which is herein incorporated. Filters with
a pore size of smaller than 0.1 .mu.m can be used for ultrafiltration. By
employing filters having such small pore size, the volume of the sample
can be reduced through permeation of the sample buffer through the filter
while proteins are retained behind the filter.
[0191] As used herein, "diafiltration" or "DF" can include a method of
using ultrafilters to remove and exchange salts, sugars, and non-aqueous
solvents, to separate free from bound species, to remove low
molecular-weight material, and/or to cause the rapid change of ionic
and/or pH environments. Microsolutes are removed most efficiently by
adding solvent to a solution being ultrafiltered at a rate approximately
equal to the ultrafiltration rate. This washes microspecies from the
solution at a constant volume. In certain exemplary embodiments of the
present invention, a diafiltration step can be employed to exchange
various buffers used in connection with the instant invention, for
example, prior to chromatography or other production steps, as well as to
remove impurities from the protein preparation. As used herein, the term
"downstream process technology" refers to one or more techniques used
after the upstream process technologies to produce a protein. Downstream
process technology includes, for example, production of a protein
product, using, for example, affinity chromatography, including Protein A
affinity chromatography as well as affinity chromatography that uses a
solid phase having a well-defined molecule like VEGF that can interact
with its cognate like a VEGF receptor (VEGF R), ion exchange
chromatography, such as anion or cation exchange chromatography,
hydrophobic interaction chromatography, or displacement chromatography.
[0192] The phrase "recombinant host cell" (or simply "host cell") includes
a cell into which a recombinant expression vector coding for a protein of
interest has been introduced. It should be understood that such a term is
intended to refer not only to a particular subject cell but to a progeny
of such a cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences, such
progeny may not, in fact, be identical to the parent cell, but are still
be included within the scope of the term "host cell" as used herein. In
an embodiment, host cells include prokaryotic and eukaryotic cells
selected from any of the kingdoms of life. In one aspect, eukaryotic
cells include protist, fungal, plant and animal cells. In a further
aspect, host-cells include eukaryotic cells such as plant and/or animal
cells. The cells can be mammalian cells, fish cells, insect cells,
amphibian cells or avian cells. In a particular aspect, the host cell is
a mammalian cell. A wide variety of mammalian cell lines suitable for
growth in culture are available from the American Type Culture Collection
(Manassas, Va.) and other depositories as well as commercial vendors.
Cells that can be used in the processes of the invention include, but not
limited to, MK2.7 cells, PER-C6 cells, Chinese hamster ovary cells (CHO),
such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec.
Genet., 12:555-556; Kolkekar et al., 1997, Biochemistry, 36: 10901-10909;
and WO 01/92337 A2), dihydrofolate reductase negative CHO cells
(CHO/-DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA,
77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney
cells (CV1, ATCC CCL-70); monkey kidney CV1 cells transformed by SV40
(COS cells, COS-7, ATCC CRL-1651); HEK 293 cells, and Sp2/0 cells, 5L8
hybridoma cells, Daudi cells, EL4 cells, HeLa cells, HL-60 cells, K562
cells, Jurkat cells, THP-1 cells, Sp2/0 cells, primary epithelial cells
(e.g., keratinocytes, cervical epithelial cells, bronchial epithelial
cells, tracheal epithelial cells, kidney epithelial cells and retinal
epithelial cells) and established cell lines and their strains (e.g.,
human embryonic kidney cells (e.g., 293 cells, or 293 cells subcloned for
growth in suspension culture, Graham et al., 1977, J. Gen. Virol.,
36:59); baby hamster kidney cells (BHK, ATCC CCL-10); mouse sertoli cells
(TM4, Mather, 1980, Biol. Reprod., 23:243-251); human cervical carcinoma
cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human
lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065);
mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver
cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NY Acad.
Sci., 383:44-68); MCR 5 cells; FS4 cells; PER-C6 retinal cells, MDBK
(NBL-1) cells, 911 cells, CRFK cells, MDCK cells, BeWo cells, Chang
cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB
cells, LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI 2650 cells,
SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells,
LLC-MK.sub.2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells,
Y-1 cells, LLC-PK.sub.1 cells, PK(15) cells, GH.sub.1 cells, GH.sub.3
cells, L2 cells, LLC-RC 256 cells, MH.sub.1C.sub.1 cells, XC cells, MDOK
cells, VSW cells, and TH-I, B1 cells, or derivatives thereof), fibroblast
cells from any tissue or organ (including but not limited to heart,
liver, kidney, colon, intestines, esophagus, stomach, neural tissue
(brain, spinal cord), lung, vascular tissue (artery, vein, capillary),
lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow, and blood),
spleen, and fibroblast and fibroblast-like cell lines (e.g., TRG-2 cells,
IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey
cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit
529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells,
Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells,
WI-26 cells, MiCl.sub.1 cells, CV-1 cells, COS-1 cells, COS-3 cells,
COS-7 cells, African green monkey kidney cells (VERO-76, ATCC CRL-1587;
VERO, ATCC CCL-81); DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2
cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells,
C3H/IOTI/2 cells, HSDM.sub.1C.sub.3 cells, KLN205 cells, McCoy cells,
Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells,
L-MTK (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells,
Swiss/3T3 cells, Indian muntac cells, SIRC cells, C.sub.H cells, and
Jensen cells, or derivatives thereof) or any other cell type known to one
skilled in the art.
[0193] As used herein, the term "host-cell proteins" (HCP) includes
protein derived from a host cell and can be unrelated to the desired
protein of interest. Host-cell proteins can be a process-related impurity
which can be derived from the manufacturing process and can include three
major categories: cell substrate-derived, cell culture-derived and
downstream derived. Cell substrate-derived impurities include, but are
not limited to, proteins derived from a host organism and nucleic acid
(host cell genomic, vector, or total DNA). Cell culture-derived
impurities include, but are not limited to, inducers, antibiotics, serum,
and other media components. Downstream-derived impurities include, but
are not limited to, enzymes, chemical and biochemical processing reagents
(e.g., cyanogen bromide, guanidine, oxidizing and reducing agents),
inorganic salts (e.g., heavy metals, arsenic, nonmetallic ion), solvents,
carriers, ligands (e.g., monoclonal antibodies), and other leachables.
[0194] In some exemplary embodiments, the host-cell protein can have a pI
in the range of about 4.5 to about 9.0. In an exemplary embodiment, the
pI can be about 4.5, about 5.0, about 5.5, about 5.6, about 5.7, about
5.8, about 5.9, about 6.0, about 6.1 about 6.2, about 6.3, about 6.4,
about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about
7.1 about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7,
about 7.8, about 7.9, about 8.0, about 8.1 about 8.2, about 8.3, about
8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.
[0195] As used herein, the term "hydrolyzing agent" refers to any one or
combination of a large number of different agents that can perform
digestion of a protein. Non-limiting examples of hydrolyzing agents that
can carry out enzymatic digestion include protease from Aspergillus
saitoi, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N,
chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC
endoproteinase (Lys-C), endoproteinase Asp-N(Asp-N), endoproteinase
Arg-C(Arg-C), endoproteinase Glu-C(Glu-C) or outer membrane protein T
(OmpT), immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS),
thermolysin, papain, pronase, V8 protease or biologically active
fragments or homologs thereof or combinations thereof. Non-limiting
examples of hydrolyzing agents that can carry out non-enzymatic digestion
include the use of high temperature, microwave, ultrasound, high
pressure, infrared, solvents (Non-limiting examples are ethanol and
acetonitrile), immobilized enzyme digestion (IMER), magnetic particle
immobilized enzymes, and on-chip immobilized enzymes. For a recent review
discussing the available techniques for protein digestion, see Switzar et
al., "Protein Digestion: An Overview of the Available Techniques and
Recent Developments" (Linda Switzar, Martin Giera & Wilfried M. A.
Niessen, Protein Digestion: An Overview of the Available Techniques and
Recent Developments, 12 JOURNAL OF PROTEOME RESEARCH 1067-1077 (2013),
the entire teachings of which are herein incorporated). One or a
combination of hydrolyzing agents can cleave peptide bonds in a protein
or polypeptide, in a sequence-specific manner, generating a predictable
collection of shorter peptides. The ratio of hydrolyzing agent to protein
and the time required for digestion can be appropriately selected to
obtain optimal digestion of the protein. When the enzyme to substrate
ratio is unsuitably high, the correspondingly high digestion rate will
not allow sufficient time for the peptides to be analyzed by mass
spectrometer, and sequence coverage will be compromised. On the other
hand, a low E/S ratio would need long digestion and thus long data
acquisition time. The enzyme to substrate ratio can range from about
1:0.5 to about 1:200. As used herein, the term "digestion" refers to
hydrolysis of one or more peptide bonds of a protein. There are several
approaches to carrying out digestion of a protein in a biological sample
using an appropriate hydrolyzing agent, for example, enzymatic digestion
or non-enzymatic digestion. One of the widely accepted methods for
digestion of proteins in a sample involves the use of proteases. Many
proteases are available and each of them have their own characteristics
in terms of specificity, efficiency, and optimum digestion conditions.
Proteases refer to both endopeptidases and exopeptidases, as classified
based on the ability of the protease to cleave at non-terminal or
terminal amino acids within a peptide. Alternatively, proteases also
refer to the six distinct classes--aspartic, glutamic, and
metalloproteases, cysteine, serine, and threonine proteases, as
classified based on the mechanism of catalysis. The terms "protease" and
"peptidase" are used interchangeably to refer to enzymes, which hydrolyze
peptide bonds.
[0196] The term "in association with" indicates that components, an
anti-VEGF composition of the present invention, along with another agent
such as anti-ANG2, can be formulated into a single composition for
simultaneous delivery, or formulated separately into two or more
compositions (e.g., a kit including each component). Components
administered in association with each another can be administered to a
subject at a different time than when the other component is
administered; for example, each administration may be given
non-simultaneously (e.g., separately or sequentially) at intervals over a
given period of time. Separate components administered in association
with each another may also be administered essentially simultaneously
(e.g., at precisely the same time or separated by a non-clinically
significant time period) during the same administration session.
Moreover, the separate components administered in association with each
other may be administered to a subject by the same or by a different
route, for example, a composition of aflibercept along with another agent
such as anti-ANG2, wherein the composition of aflibercept comprises about
15% or less of its variants.
[0197] As used herein, the term "liquid chromatography" refers to a
process in which a biological/chemical mixture carried by a liquid can be
separated into components as a result of differential distribution of the
components as they flow through (or into) a stationary liquid or solid
phase. Non-limiting examples of liquid chromatography include reverse
phase liquid chromatography, ion-exchange chromatography, size exclusion
chromatography, affinity chromatography, mixed-mode chromatography,
hydrophobic chromatography or mixed-mode chromatography.
[0198] As used herein, "affinity chromatography" can include separations
including any method by which two substances are separated based upon
their affinity to a chromatographic material. It can comprise subjecting
the substances to a column comprising a suitable affinity chromatographic
media. Non-limiting examples of such chromatographic media include, but
are not limited to, Protein A resin, Protein G resin, affinity supports
comprising an antigen against which a binding molecule (e.g., antibody)
was produced, protein capable of binding to a protein of interest and
affinity supports comprising an Fc binding protein. In one aspect, an
affinity column can be equilibrated with a suitable buffer prior to
sample loading. An example of a suitable buffer can be a Tris/NaCl
buffer, pH around 7.0 to 8.0. A skilled artisan can develop a suitable
buffer without undue burden. Following this equilibration, a sample can
be loaded onto the column. Following the loading of the column, the
column can be washed one or multiple times using, for example, the
equilibrating buffer. Other washes including washes employing different
buffers can be used before eluting the column. The affinity column can
then be eluted using an appropriate elution buffer. An example of a
suitable elution buffer can be an acetic acid/NaCl buffer, pH around 2.0
to 3.5. Again, the skilled artisan can develop an appropriate elution
buffer without undue burden. The eluate can be monitored using techniques
well known to those skilled in the art, including UV, for example, the
absorbance at 280 nm can be employed especially if the sample of interest
comprises aromatic rings (e.g., proteins having aromatic amino acids like
tryptophan).
[0199] As used herein, "ion exchange chromatography" can refer to
separations including any method by which two substances are separated
based on differences in their respective ionic charges, either on the
molecule of interest and/or chromatographic material as a whole or
locally on specific regions of the molecule of interest and/or
chromatographic material, and thus can employ either cationic exchange
material or anionic exchange material. Ion exchange chromatography
separates molecules based on differences between the local charges of the
molecules of interest and the local charges of the chromatographic
material. A packed ion-exchange chromatography column or an ion-exchange
membrane device can be operated in a bind-elute mode, a flowthrough mode,
or a hybrid mode. After washing the column or the membrane device with an
equilibration buffer or another buffer, product recovery can be achieved
by increasing the ionic strength (i.e., conductivity) of the elution
buffer to compete with the solute for the charged sites of the ion
exchange matrix. Changing the pH and thereby altering the charge of the
solute can be another way to achieve elution of the solute. The change in
conductivity or pH may be gradual (gradient elution) or stepwise (step
elution). Anionic or cationic substituents may be attached to matrices in
order to form anionic or cationic supports for chromatography.
Non-limiting examples of anionic exchange substituents include
diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary
amine (Q) groups. Cationic substituents include carboxymethyl (CM),
sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S).
Cellulose ion exchange medias or support can include DE23.TM., DE32.TM.
DE52.TM., CM-23.TM., CM-32.TM., and CM-52.TM. are available from Whatman
Ltd. Maidstone, Kent, U.K. SEPHADEX.RTM.-based and -locross-linked ion
exchangers are also known. For example, DEAE-, QAE-, CM-, and
SP-SEPHADEX.RTM. and DEAE-, Q-, CM- and S-SEPHAROSE.RTM. and
SEPHAROSE.RTM. Fast Flow, and Capto.TM. S are all available from GE
Healthcare. Further, both DEAE and CM derivitized ethylene
glycol-methacrylate copolymer such as TOYOPEARL.TM. DEAE-650S or M and
TOYOPEARL.TM. CM-650S or M are available from Toso Haas Co.,
Philadelphia, Pa., or Nuvia S and UNOSphere.TM. S from BioRad, Hercules,
Calif., Eshmuno.RTM. S from EMD Millipore, MA.
[0200] As used herein, the term "hydrophobic interaction chromatography
resin" can include a solid phase, which can be covalently modified with
phenyl, octyl, butyl or the like. It can use the properties of
hydrophobicity to separate molecules from one another. In this type of
chromatography, hydrophobic groups such as, phenyl, octyl, hexyl or butyl
can form the stationary-phase of a column. Molecules such as proteins,
peptides and the like pass through a HIC (hydrophobic interactive
chromatography) column that possess one or more hydrophobic regions on
their surface or have hydrophobic pockets and are able to interact with
hydrophobic groups comprising a HIC's stationary phase. Examples of HIC
resins or support include Phenyl sepharose FF, Capto Phenyl (GE
Healthcare, Uppsala, Sweden), Phenyl 650-M (Tosoh Bioscience, Tokyo,
Japan) and Sartobind Phenyl (Sartorius corporation, New York, USA).
[0201] As used herein, the term "Mixed Mode Chromatography" or "multimodal
chromatography" (both "MMC") includes a chromatographic method in which
solutes interact with a stationary phase through more than one
interaction mode or mechanism. MMC can be used as an alternative or
complementary tool to traditional reversed-phased (RP), ion exchange
(IEX) and normal phase chromatography (NP). Unlike RP, NP and IEX
chromatography, in which hydrophobic interaction, hydrophilic interaction
and ionic interaction respectively are the dominant interaction modes,
mixed-mode chromatography can employ a combination of two or more of
these interaction modes. Mixed mode chromatography media can provide
unique selectivity that cannot be reproduced by single mode
chromatography. Mixed mode chromatography can also provide potential cost
savings, longer column lifetimes and operation flexibility compared to
affinity-based methods. In some exemplary embodiments, mixed mode
chromatography media can be comprised of mixed mode ligands coupled to an
organic or inorganic support, sometimes denoted a base matrix, directly
or via a spacer. The support may be in the form of particles, such as
essentially spherical particles, a monolith, filter, membrane, surface,
capillaries, etc. In some exemplary embodiments, the support can be
prepared from a native polymer such as cross-linked carbohydrate
material, such as agarose, agPV, cellulose, dextran, chitosan, konjac,
carrageenan, gellan, alginate, etc. To obtain high adsorption capacities,
the support can be porous and ligands are then coupled to the external
surfaces as well as to the pore surfaces. Such native polymer supports
can be prepared according to standard methods, such as inverse suspension
gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964), the
entire teachings of which are herein incorporated). Alternatively, the
support can be prepared from a synthetic polymer such as cross-linked
synthetic polymers, for example, styrene or styrene derivatives,
divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl
esters, vinyl amides and the like. Such synthetic polymers can be
produced according to standard methods, for example, "Styrene based
polymer supports developed by suspension polymerization" (R Arshady:
Chimica e L'Industria 70(9), 70-75 (1988), the entire teachings of which
are herein incorporated). Porous native or synthetic polymer supports are
also available from commercial sources, such as such as GE Healthcare,
Uppsala, Sweden.
[0202] As used herein, the term "mass spectrometer" includes a device
capable of identifying specific molecular species and measuring their
accurate masses. The term is meant to include any molecular detector into
which a polypeptide or peptide may be characterized. A mass spectrometer
can include three major parts: the ion source, the mass analyzer, and the
detector. The role of the ion source is to create gas phase ions. Analyte
atoms, molecules, or clusters can be transferred into gas phase and
ionized either concurrently (as in electrospray ionization) or through
separate processes. The choice of ion source depends on the application.
In some exemplary embodiments, the mass spectrometer can be a tandem mass
spectrometer. As used herein, the term "tandem mass spectrometry"
includes a technique where structural information on sample molecules is
obtained by using multiple stages of mass selection and mass separation.
A prerequisite is that the sample molecules be transformed into a gas
phase and ionized so that fragments are formed in a predictable and
controllable fashion after the first mass selection step. Multistage
MS/MS, or MS.sup.n, can be performed by first selecting and isolating a
precursor ion (MS.sup.2), fragmenting it, isolating a primary fragment
ion (MS.sup.3), fragmenting it, isolating a secondary fragment
(MS.sup.4), and so on, as long as one can obtain meaningful information,
or the fragment ion signal is detectable. Tandem MS has been successfully
performed with a wide variety of analyzer combinations. What analyzers to
combine for a certain application can be determined by many different
factors, such as sensitivity, selectivity, and speed, but also size,
cost, and availability. The two major categories of tandem MS methods are
tandem-in-space and tandem-in-time, but there are also hybrids where
tandem-in-time analyzers are coupled in space or with tandem-in-space
analyzers. A tandem-in-space mass spectrometer comprises an ion source, a
precursor ion activation device, and at least two non-trapping mass
analyzers. Specific m/z separation functions can be designed so that in
one section of the instrument ions are selected, dissociated in an
intermediate region, and the product ions are then transmitted to another
analyzer for m/z separation and data acquisition. In tandem-in-time, mass
spectrometer ions produced in the ion source can be trapped, isolated,
fragmented, and m/z separated in the same physical device. The peptides
identified by the mass spectrometer can be used as surrogate
representatives of the intact protein and their post translational
modifications. They can be used for protein characterization by
correlating experimental and theoretical MS/MS data, the latter generated
from possible peptides in a protein sequence database. The
characterization includes, but is not limited, to sequencing amino acids
of the protein fragments, determining protein sequencing, determining
protein de novo sequencing, locating post-translational modifications, or
identifying post translational modifications, or comparability analysis,
or combinations thereof.
[0203] As used herein, "Mini-Trap" or "MiniTrap" or "MiniTrap binding
molecule" is capable of binding to a VEGF molecule. Such MiniTraps can
include (i) chimeric polypeptides as well as (ii) multimeric (e.g.,
dimeric) molecules comprising two or more polypeptides which are bound
non-covalently, for example, by one or more disulfide bridges. MiniTraps
can be produced through chemical modification, enzymatic activity, or
recombinantly manufactured.
[0204] As used herein, "VEGF MiniTrap" or "VEGF MiniTrap binding molecule"
can be a molecule or complex of molecules that binds to VEGF and has one
or more sets of VEGF receptor Ig-like domains (or variants thereof)
(e.g., VEGFR1 Ig domain 2 and/or VEGFR2 Ig domain 3 and/or 4) and a
modified or absent multimerizing component (MC), for example, wherein the
MC is a modified immunoglobulin Fc. The modification may be the result of
proteolytic digestion of a VEGF trap (e.g., aflibercept or conbercept) or
direct expression of the resulting polypeptide chains with the shortened
MC sequence. (See the molecular structure depicted in FIG. 1.) FIG. 1 is
a depiction of a VEGF MiniTrap molecule, which is the product of
proteolysis of aflibercept with Streptococcus pyogenes IdeS. The
homodimeric molecule is depicted having an Ig hinge domain fragment
connected by two parallel disulfide bonds. The VEGFR1 domain, the VEGFR2
domain and the hinge domain fragment (MC) is indicated. The point in
aflibercept where IdeS cleavage occurs is indicated with a "//". The
cleaved off Fc fragment from aflibercept is also indicated. A single such
chimeric polypeptide, which is not dimerized, may also be a VEGF MiniTrap
if it has VEGF binding activity. The term "VEGF MiniTrap" includes a
single polypeptide comprising a first set of one or more VEGF receptor Ig
domains (or variants thereof), lacking an MC, but fused with a linker
(e.g., a peptide linker) to one or more further sets of one or more VEGF
receptor Ig domains (or variants thereof). The VEGF binding domains in a
VEGF MiniTrap of the present invention may be identical or different from
another (see WO2005/00895, the entire teachings of which are herein
incorporated).
[0205] For example, in an embodiment of the invention, the unmodified
immunoglobulin Fc domain comprises the amino acid sequence or amino acids
1-226 thereof:
TABLE-US-00002
DKTHTCPX.sub.1CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKX.sub.2TPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.: 33;
wherein X.sub.1 is L or P and X.sub.2 is A or T)
[0206] Inhibition of VEGF includes, for example, antagonism of VEGF
binding to VEGF receptor, for example, by competition with VEGF receptor
for VEGF (e.g., VEGF.sub.110, VEGF.sub.121 and/or VEGF.sub.165) binding.
Such inhibition may result in inhibition of VEGF-mediated activation of
VEGFR, for example, inhibition of luciferase expression in a cell line
(e.g., HEK293) expressing chimeric VEGF Receptor (e.g., a homodimer
thereof) having VEGFR extracellular domains fused to IL18R.alpha. and/or
IL18R.beta. intracellular domains on the cell surface and also having an
NFkB-luciferase-IRES-eGFP reporter gene, for example, the cell line
HEK293/D9/Flt-IL18R.alpha./Flt-IL18R.beta. as set forth herein.
[0207] The VEGF receptor Ig domain components of the VEGF MiniTraps of the
present invention can include:
[0208] (i) one or more of the immunoglobulin-like (Ig) domain 2 of VEGFR1
(Flt1) (R1D2),
[0209] (ii) one or more of the Ig domain 3 of VEGFR2 (Flk1 or KDR)
(F1k1D3) (R2D3),
[0210] (iii) one or more of the Ig domain 4 of VEGFR2 (Flk1 or KDR)
(F1k1D4) (R2D4) and/or
[0211] (iv) one or more of the Ig domain 3 of VEGFR3 (Flt4) (F1t1D3 or
R3D3).
[0212] Immunoglobulin-like domains of VEGF receptors may be referred to
herein as VEGFR "Ig" domains. VEGFR Ig domains which are referenced
herein, for example, R1D2 (which may be referred to herein as
VEGFR1(d2)), R2D3 (which may be referred to herein as VEGFR2(d3)), R2D4
(which may be referred to herein as VEGFR2(d4)) and R3D3 (which may be
referred to herein as VEGFR3(d3)) are intended to encompass not only the
complete wild-type Ig domain, but also variants thereof which
substantially retain the functional characteristics of the wild-type
domain, for example, retain the ability to form a functioning VEGF
binding domain when incorporated into a VEGF MiniTrap. It will be readily
apparent to one of skilled in the art that numerous variants of the above
Ig domains, which will retain substantially the same functional
characteristics as the wild-type domain, can be obtained.
[0213] The present invention provides a VEGF MiniTrap polypeptide
comprising the following domain structure: [0214]
((R1D2)-(R2D3)).sub.a-linker-((R1D2)-(R2D3)).sub.b; [0215]
((R1D2)-(R2D3)-(R2D4)).sub.c-linker-((R1D2)-(R2D3)-(R2D4)).sub.d; [0216]
((R1D2)-(R2D3)).sub.e-(MC).sub.g; [0217]
((R1D2)-(R2D3)-(R2D4)).sub.f-(MC).sub.g; wherein, [0218] R1D2 is the VEGF
receptor 1 (VEGFR1) Ig domain 2 (D2); [0219] R2D3 is the VEGFR2 Ig domain
3; [0220] R2D4 is the VEGFR2 Ig domain 4; [0221] MC is a multimerizing
component (e.g., an IgG hinge domain or fragment thereof, for example
from IgG1); [0222] linker is a peptide comprising about 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15 or 16 amino acids, for example, (GGGS).sub.g; and,
[0224] In an embodiment of the invention, R1D2 comprises the amino acid
sequence: SDTGRPFVEMYSEIPEIIHMTEGRELVWCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG
FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIID (SEQ ID NO.: 34). In one aspect,
the R1D2 lacks the N-terminal SDT.
[0225] In an embodiment of the invention, R1D2 comprises the amino acid
sequence:
TABLE-US-00003
(SEQ ID NO.: 35)
PFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKR
IIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT.
[0226] In an embodiment of the invention, R2D3 comprises the amino acid
sequence:
TABLE-US-00004
(SEQ ID NO.: 36)
VVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNR
DLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVH
EK.
[0227] In an embodiment of the invention, R2D4 comprises the amino acid
sequence:
TABLE-US-00005
(SEQ ID NO.: 37)
PFVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTI
KAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPGPG.
[0228] In an embodiment of the invention, R2D4 comprises the amino acid
sequence:
TABLE-US-00006
(SEQ ID NO.: 38)
FVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIK
AGHVLTIMEVSERDTGNYTVILTNPIKSEKQSHVVSLVVYVP.
[0229] In an embodiment of the invention, a multimerizing component (MC)
for use in a VEGF MiniTrap is a peptide, for example, a modified Fc
immunoglobulin (e.g., from an IgG1) which is capable of binding to
another multimerizing component. In one aspect, an MC is a modified Fc
immunoglobulin that includes the immunoglobulin hinge region. For
example, in an embodiment of the invention, an MC is a peptide comprising
one or more (e.g., 1, 2, 3, 4, 5 or 6) cysteines that are able to form
one or more cysteine bridges with cysteines in another MC, for example,
DKTHTCPPC (SEQ ID NO.: 39), DKTHTCPPCPPC (SEQ ID NO.: 40),
DKTHTCPPCPPCPPC (SEQ ID NO.: 41), DKTHTC(PPC).sub.h, wherein h is 1, 2,
3, 4, or 5, DKTHTCPPCPAPELLG (SEQ ID NO.: 60), DKTHTCPLCPAPELLG (SEQ ID
NO.: 43), DKTHTC (SEQ ID NO.: 44) or DKTHTCPLCPAP (SEQ ID NO.: 45).
[0230] The present invention also provides a VEGF MiniTrap polypeptide
comprising the following domain structure:
(i) (R1D2).sub.a-(R2D3).sub.b-(MC).sub.c; or (ii)
(R1D2).sub.a-(R2D3).sub.b-(R2D4).sub.c-(MC).sub.d; which may be
homodimerized with a second of said polypeptides, for example, by binding
between the MCs of each polypeptide, wherein (i) said R1D2 domains
coordinate; (ii) said R2D3 domains coordinate; and/or (iii) said R2D4
domains coordinate, to form a dimeric VEGF binding domain.
[0231] In an embodiment of the invention, the VEGF MiniTrap polypeptide
comprises the amino acid sequence:
TABLE-US-00007
(SEQ ID NO.: 46; MC underscored)
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPN.sub.36ITVTLKKFPLDTL
IPDGKRIIWDSRKGFIISN.sub.68ATYKEIGLLTCEATVNGHLYKTNYLTHRQT
NTIIDVVLSPSHGIELSVGEKLVLN.sub.123CTARTELNVGIDFNWEYPSSKHQ
HKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK
N.sub.196STFVRVHEKDKTHTCPPCPAPELLG;
(SEQ ID NO.: 47; MC underscored)
GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPD
GKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTI
IDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKL
VNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTF
VRVHENLSVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIP
LESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYV
PPGPGDKTHTCPLCPAPELLG;
(SEQ ID NO.: 48; MC underscored)
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPN.sub.36ITVTLKKFPLDT
LIPDGKRIIWDSRKGFIISN.sub.68ATYKEIGLLTCEATVNGHLYKTNYLTHR
QTNTIIDVVLSPSHGIELSVGEKLVLN.sub.123CTARTELNVGIDFNWEYPSS
KHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLM
TKKN.sub.396STFVRVHEKDKTHTCPPC;
(SEQ ID NO.: 49; MC underscored)
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPN.sub.36ITVTLKKFPLDT
LIPDGKRIIWDSRKGFIISN.sub.68ATYKEIGLLTCEATVNGHLYKTNYLTHR
QTNTIIDVVLSPSHGIELSVGEKLVLN.sub.123CTARTELNVGIDFNWEYPSS
KHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLM
TKKN.sub.196STFVRVHEKDKTHTCPPCPPC;
(SEQ ID NO.: 50; MC underscored)
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPN.sub.36ITVTLKKFPLDT
LIPDGKRIIWDSRKGFIISN.sub.68ATYKEIGLLTCEATVNGHLYKTNYLTHR
QTNTIIDVVLSPSHGIELSVGEKLVLN.sub.123CTARTELNVGIDFNWEYPSS
KHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLM
TKKN.sub.196STFVRVHEKDKTHTCPPCPPCPPC;
or
(SEQ ID NO.: 106)
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTL
IPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT
NTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQH
KKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKN
STFVRVHEKDKTHTC-(PPC)x (MC underscored;
wherein x is 1, 2, 3, 4 or 5). As discussed, such polypeptides may be
multimerized (e.g., dimerized (e.g., homodimerized)) wherein binding
between the polypeptides is mediated via the multimerizing components.
[0232] In an embodiment of the invention, the VEGFR1 Ig-like domain 2 of
the monomeric VEGF MiniTraps of the present invention, have N-linked
glycosylation at N36 and/or N68; and/or an intrachain disulfide bridge
between C30 and C79; and/or, the VEGFR2 Ig-like domain 3 of the monomeric
VEGF MiniTraps of the present invention, have N-linked glycosylation at
N123 and/or N196; and/or an intrachain disulfide bridge between C124 and
C185.
[0233] In an embodiment of the invention, the VEGF MiniTrap comprises the
structure: [0234]
(R1D2).sub.1-(R2D3).sub.1-(G4S).sub.3-(R1D2).sub.1-(R2D3).sub.1; [0235]
(R1D2).sub.1-(R2D3).sub.1-(G4S).sub.6-(R1D2).sub.1-(R2D3).sub.1; [0236]
(R1D2).sub.1-(R2D3).sub.1-(G.sub.4S).sub.9-(R1D2).sub.1-(R2D3).sub.1; or
[0237] (R1D2).sub.1-(R2D3).sub.1-(G4S).sub.12-(R1D2).sub.1-(R2D3).sub.1.
G4S is -Gly-Gly-Gly-Gly-Ser-
[0238] In an embodiment of the invention, the VEGF MiniTrap comprises the
amino acid sequence:
As discussed herein, these polypeptides may comprise a secondary
structure wherein like VEGFR Ig domains associate to form an intra-chain
VEGF binding domain (e.g., FIG. 2). In an embodiment of the invention,
two or more of such polypeptides multimerize (e.g., dimerize (e.g.,
homodimerize)) wherein the VEGFR Ig domains of each chain associate with
like Ig domains of another chain to form an inter-chain VEGF binding
domain.
[0239] In a certain embodiment of the invention, a VEGF MiniTrap of the
present invention lacks any significant modification of the amino acid
residues of a VEGF MiniTrap polypeptide (e.g., directed chemical
modification such as PEGylation or iodoacetamidation, for example at the
N- and/or C-terminus).
[0240] In an embodiment of the invention, the polypeptide comprises a
secondary structure wherein like VEGFR Ig domains in a single chimeric
polypeptide (e.g.,
(R1D2).sub.a-(R2D3).sub.b-linker-(R1D2).sub.c-(R2D3).sub.d; or
(R1D2).sub.a-(R2D3).sub.b-(R2D4).sub.c-linker-(R1D2).sub.d-(R2D3).sub.e-
(R2D4).sub.f) or in separate chimeric polypeptides (e.g., homodimers)
coordinate to form a VEGF binding domain. For example, wherein
(i) said R1D2 domains coordinate; (ii) said R2D3 domains coordinate;
and/or (iii) said R2D4 domains coordinate, to form a VEGF binding domain.
FIG. 2 is a description of a single chain VEGF MiniTrap depicting such
domain coordination. The VEGFR1, VEGFR2 and linker domains are indicated.
The linker shown is (G4S).sub.6. The present invention includes single
chain VEGF MiniTraps with a (G4S).sub.3; (G4S).sub.9 or (G4S).sub.12
linker.
[0241] In addition, the present invention also provides a complex
comprising a VEGF MiniTrap as discussed herein complexed with a VEGF
polypeptide or a fragment thereof or fusion thereof. In an embodiment of
the invention, the VEGF (e.g., VEGF.sub.165) is homodimerized and/or the
VEGF MiniTrap is homodimerized in a 2:2 complex (2 VEGFs:2 MiniTraps)
and/or VEGF MiniTrap is homodimerized in a 1:1 complex. Complexes can
include homodimerized VEGF molecules bound to homodimerized VEGF MiniTrap
polypeptides. In an embodiment of the invention, the complex is in vitro
(e.g., immobilized to a solid substrate) or is in the body of a subject.
The present invention also includes a composition of complexes of a VEGF
dimer (e.g., VEGF.sub.165) complexed with a VEGF MiniTrap.
[0242] As used herein, the term "protein" or "protein of interest" can
include any amino acid polymer having covalently linked amide bonds.
Examples of protein of interest include, but are not limited to,
aflibercept and MiniTrap. Proteins comprise one or more amino acid
polymer chains, generally known in the art as "polypeptides."
"Polypeptide" refers to a polymer composed of amino acid residues,
related naturally occurring structural variants, and synthetic
non-naturally occurring analogs thereof linked via peptide bonds, related
naturally occurring structural variants, and synthetic non-naturally
occurring analogs thereof. "Synthetic peptides or polypeptides" refers to
a non-naturally occurring peptide or polypeptide. Synthetic peptides or
polypeptides can be synthesized, for example, using an automated
polypeptide synthesizer. Various solid phase peptide synthesis methods
are known to those of skill in the art. A protein may comprise one or
multiple polypeptides to form a single functioning biomolecule. Another
exemplary aspect, a protein can include antibody fragments, nanobodies,
recombinant antibody chimeras, cytokines, chemokines, peptide hormones,
and the like. Protein of interest can include any of bio-therapeutic
proteins, recombinant proteins used in research or therapy, trap proteins
and other chimeric receptor Fc-fusion proteins, chimeric proteins,
antibodies, monoclonal antibodies, polyclonal antibodies, human
antibodies, and bispecific antibodies. In a particular aspect, the
protein of interest is an anti-VEGF fusion protein (e.g., aflibercept or
MiniTrap). Proteins may be produced using recombinant cell-based
production systems, such as the insect bacculovirus system, yeast systems
(e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHO
derivatives like CHO-K1 cells). For a recent review discussing
biotherapeutic proteins and their production, see Ghaderi et al.,
"Production platforms for biotherapeutic glycoproteins. Occurrence,
impact, and challenges of non-human sialylation," (Darius Ghaderi et al.,
Production platforms for biotherapeutic glycoproteins. Occurrence,
impact, and challenges of non-human sialylation, 28 BIOTECHNOLOGY AND
GENETIC ENGINEERING REVIEWS 147-176 (2012), the entire teachings of which
are herein incorporated). In some exemplary embodiments, proteins
comprise modifications, adducts, and other covalently linked
moieties--these modifications, adducts and moieties include, for example,
avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine,
galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and
other monosaccharides), PEG, polyhistidine, FLAGtag, maltose binding
protein (MBP), chitin binding protein (CBP), glutathione-S-transferase
(GST) myc-epitope, fluorescent labels and other dyes, and the like.
Proteins can be classified on the basis of compositions and solubility
and can thus include simple proteins, such as, globular proteins and
fibrous proteins; conjugated proteins, such as, nucleoproteins,
glycoproteins, mucoproteins, chromoproteins, phosphoproteins,
metalloproteins, and lipoproteins; and derived proteins, such as, primary
derived proteins and secondary derived proteins.
[0243] In some exemplary embodiments, the protein of interest can be a
recombinant protein, an antibody, a bispecific antibody, a multispecific
antibody, antibody fragment, monoclonal antibody, fusion protein, scFv
and combinations thereof.
[0244] As used herein, the term "recombinant protein" refers to a protein
produced as the result of the transcription and translation of a gene
carried on a recombinant expression vector that has been introduced into
a suitable host cell. In certain exemplary embodiments, the recombinant
protein can be a fusion protein. In a particular aspect, the recombinant
protein is an anti-VEGF fusion protein (e.g., aflibercept or MiniTrap).
In certain exemplary embodiments, the recombinant protein can be an
antibody, for example, a chimeric, humanized, or fully human antibody. In
certain exemplary embodiments, the recombinant protein can be an antibody
of an isotype selected from group consisting of: IgG, IgM, IgA1, IgA2,
IgD, or IgE. In certain exemplary embodiments the antibody molecule is a
full-length antibody (e.g., an IgG1) or alternatively the antibody can be
a fragment (e.g., an Fc fragment or a Fab fragment).
[0245] The term "antibody," as used herein includes immunoglobulin
molecules comprising four polypeptide chains, two heavy (H) chains and
two light (L) chains inter-connected by disulfide bonds, as well as
multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain
variable region (abbreviated herein as HCVR or VH) and a heavy chain
constant region. The heavy chain constant region comprises three domains,
CH1, CH2 and CH3. Each light chain comprises a light chain variable
region (abbreviated herein as LCVR or VL) and a light chain constant
region. The light chain constant region comprises one domain (CL1). The
VH and VL regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions (CDRs),
interspersed with regions that are more conserved, termed framework
regions (FR). Each VH and VL is composed of three CDRs and four FRs,
arranged from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In different embodiments of the
invention, the FRs of the anti-big-ET-1 antibody (or antigen-binding
portion thereof) may be identical to the human germline sequences or may
be naturally or artificially modified. An amino acid consensus sequence
may be defined based on a side-by-side analysis of two or more CDRs. The
term "antibody," as used herein, also includes antigen-binding fragments
of full antibody molecules. The terms "antigen-binding portion" of an
antibody, "antigen-binding fragment" of an antibody, and the like, as
used herein, include any naturally occurring, enzymatically obtainable,
synthetic, or genetically engineered polypeptide or glycoprotein that
specifically binds an antigen to form a complex. Antigen-binding
fragments of an antibody may be derived, for example, from full antibody
molecules using any suitable standard techniques such as proteolytic
digestion or recombinant genetic engineering techniques involving the
manipulation and expression of DNA encoding antibody variable and
optionally constant domains. Such DNA is known and/or is readily
available from, for example, commercial sources, DNA libraries
(including, e.g., phage-antibody libraries), or can be synthesized. The
DNA may be sequenced and manipulated chemically or by using molecular
biology techniques, for example, to arrange one or more variable and/or
constant domains into a suitable configuration, or to introduce codons,
create cysteine residues, modify, add or delete amino acids, etc.
[0246] As used herein, an "antibody fragment" includes a portion of an
intact antibody, such as, for example, the antigen-binding or variable
region of an antibody. Examples of antibody fragments include, but are
not limited to, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a
scFv fragment, a Fv fragment, a dsFv diabody, a dAb fragment, a Fd'
fragment, a Fd fragment, and an isolated complementarity determining
region (CDR) region, as well as triabodies, tetrabodies, linear
antibodies, single-chain antibody molecules, and multi specific
antibodies formed from antibody fragments. Fv fragments are the
combination of the variable regions of the immunoglobulin heavy and light
chains, and ScFv proteins are recombinant single chain polypeptide
molecules in which immunoglobulin light and heavy chain variable regions
are connected by a peptide linker. In some exemplary embodiments, an
antibody fragment comprises a sufficient amino acid sequence of the
parent antibody of which it is a fragment that it binds to the same
antigen as does the parent antibody; in some exemplary embodiments, a
fragment binds to the antigen with a comparable affinity to that of the
parent antibody and/or competes with the parent antibody for binding to
the antigen. An antibody fragment may be produced by any means. For
example, an antibody fragment may be enzymatically or chemically produced
by fragmentation of an intact antibody and/or it may be recombinantly
produced from a gene encoding the partial antibody sequence.
Alternatively, or additionally, an antibody fragment may be wholly or
partially synthetically produced. An antibody fragment may optionally
comprise a single chain antibody fragment. Alternatively, or
additionally, an antibody fragment may comprise multiple chains that are
linked together, for example, by disulfide linkages. An antibody fragment
may optionally comprise a multi-molecular complex. A functional antibody
fragment typically comprises at least about 50 amino acids and more
typically comprises at least about 200 amino acids.
[0247] The term "bispecific antibody" includes an antibody capable of
selectively binding two or more epitopes. Bispecific antibodies generally
comprise two different heavy chains with each heavy chain specifically
binding a different epitope--either on two different molecules (e.g.,
antigens) or on the same molecule (e.g., on the same antigen). If a
bispecific antibody is capable of selectively binding two different
epitopes (a first epitope and a second epitope), the affinity of the
first heavy chain for the first epitope will generally be at least one to
two or three or four orders of magnitude lower than the affinity of the
first heavy chain for the second epitope, and vice versa. The epitopes
recognized by the bispecific antibody can be on the same or a different
target (e.g., on the same or a different protein). Bispecific antibodies
can be made, for example, by combining heavy chains that recognize
different epitopes of the same antigen. For example, nucleic acid
sequences encoding heavy chain variable sequences that recognize
different epitopes of the same antigen can be fused to nucleic acid
sequences encoding different heavy chain constant regions and such
sequences can be expressed in a cell that expresses an immunoglobulin
light chain.
[0248] A typical bispecific antibody has two heavy chains each having
three heavy chain CDRs, followed by a CH1 domain, a hinge, a CH2 domain,
and a CH3 domain, and an immunoglobulin light chain that either does not
confer antigen-binding specificity but that can associate with each heavy
chain, or that can associate with each heavy chain and that can bind one
or more of the epitopes bound by the heavy chain antigen-binding regions,
or that can associate with each heavy chain and enable binding or one or
both of the heavy chains to one or both epitopes. BsAbs can be divided
into two major classes, those bearing an Fc region (IgG-like) and those
lacking an Fc region, the latter normally being smaller than the IgG and
IgG-like bispecific molecules comprising an Fc. The IgG-like bsAbs can
have different formats such as, but not limited to, triomab, knobs into
holes IgG (kih IgG), crossMab, orth-Fab IgG, Dual-variable domains Ig
(DVD-Ig), two-in-one or dual action Fab (DAF), IgG-single-chain Fv
(IgG-scFv), or .kappa..lamda.-bodies. The non-IgG-like different formats
include tandem scFvs, diabody format, single-chain diabody, tandem
diabodies (TandAbs), Dual-affinity retargeting molecule (DART), DART-Fc,
nanobodies, or antibodies produced by the dock-and-lock (DNL) method
(Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their
applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Muller &
Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC
ANTIBODIES 265-310 (2014), the entire teachings of which are herein
incorporated). The methods of producing bsAbs are not limited to quadroma
technology based on the somatic fusion of two different hybridoma cell
lines, chemical conjugation, which involves chemical cross-linkers, and
genetic approaches utilizing recombinant DNA technology. Examples of
bsAbs include those disclosed in the following patent applications, which
are hereby incorporated by reference: U.S. Ser. No. 12/823,838, filed
Jun. 25, 2010; U.S. Ser. No. 13/488,628, filed Jun. 5, 2012; U.S. Ser.
No. 14/031,075, filed Sep. 19, 2013; U.S. Ser. No. 14/808,171, filed Jul.
24, 2015; U.S. Ser. No. 15/713,574, filed Sep. 22, 2017; U.S. Ser. No.
15/713,569, field Sep. 22, 2017; U.S. Ser. No. 15/386,453, filed Dec. 21,
2016; U.S. Ser. No. 15/386,443, filed Dec. 21, 2016; U.S. Ser. No.
15/223,43 filed Jul. 29, 2016; and U.S. Ser. No. 15/814,095, filed Nov.
15, 2017. Low levels of homodimer impurities can be present at several
steps during the manufacturing of bispecific antibodies. The detection of
such homodimer impurities can be challenging when performed using intact
mass analysis due to low abundances of the homodimer impurities and the
co-elution of these impurities with main species when carried out using a
regular liquid chromatographic method.
[0249] As used herein "multispecific antibody" refers to an antibody with
binding specificities for at least two different antigens. While such
molecules normally will only bind two antigens (i.e., bispecific
antibodies, bsAbs), antibodies with additional specificities such as
trispecific antibody and KIH Trispecific can also be addressed by the
system and method disclosed herein.
[0250] The term "monoclonal antibody" as used herein is not limited to
antibodies produced through hybridoma technology. A monoclonal antibody
can be derived from a single clone, including any eukaryotic,
prokaryotic, or phage clone, by any means available or known in the art.
Monoclonal antibodies useful with the present disclosure can be prepared
using a wide variety of techniques known in the art including the use of
hybridoma, recombinant, and phage display technologies, or a combination
thereof.
[0251] In some exemplary embodiments, the protein of interest can have a
pI in the range of about 4.5 to about 9.0. In one exemplary specific
embodiment, the pI can be about 4.5, about 5.0, about 5.5, about 5.6,
about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 about 6.2, about
6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9,
about 7.0, about 7.1 about 7.2, about 7.3, about 7.4, about 7.5, about
7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 about 8.2,
about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about
8.9, or about 9.0. In some exemplary embodiments, the types of protein of
interest in the compositions can be more than one.
[0252] In some exemplary embodiments, the protein of interest can be
produced from mammalian cells. The mammalian cells can be of human origin
or non-human origin can include primary epithelial cells (e.g.,
keratinocytes, cervical epithelial cells, bronchial epithelial cells,
tracheal epithelial cells, kidney epithelial cells and retinal epithelial
cells), established cell lines and their strains (e.g., 293 embryonic
kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6
retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO
cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa
S3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells, NCI-H-548
cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells,
WISH cells, BS-C-I cells, LLC-MK2 cells, Clone M-3 cells, 1-10 cells, RAG
cells, TCMK-1 cells, Y-1 cells, LLC-PKi cells, PK(15) cells, GHi cells,
GH3 cells, L2 cells, LLC-RC 256 cells, MHiCi cells, XC cells, MDOK cells,
VSW cells, and TH-I, B1 cells, BSC-1 cells, RAf cells, RK-cells, PK-15
cells or derivatives thereof), fibroblast cells from any tissue or organ
(including but not limited to heart, liver, kidney, colon, intestines,
esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular
tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid,
tonsil, bone marrow, and blood), spleen, and fibroblast and
fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells,
Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551
cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit
532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL
299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, Midi
cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero
cells, DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells,
M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells,
C3H/IOTI/2 cells, HSDMiC3 cells, KLN205 cells, McCoy cells, Mouse L
cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells, L-MTK'
(Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3
cells, Indian muntjac cells, SIRC cells, Cn cells, and Jensen cells,
Sp2/0, NS0, NS1 cells or derivatives thereof).
[0253] As used herein, the term "protein alkylating agent" refers to an
agent used for alkylating certain free amino acid residues in a protein.
Non-limiting examples of the protein alkylating agents are iodoacetamide
(IOA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM),
methyl methanethiosulfonate (MMTS), and 4-vinylpyridine or combinations
thereof.
[0254] As used herein, "protein denaturing" can refer to a process in
which the three-dimensional shape of a molecule is changed from its
native state. Protein denaturation can be carried out using a protein
denaturing agent. Non-limiting examples of a protein denaturing agent
include heat, high or low pH, reducing agents like DTT (see below) or
exposure to chaotropic agents. Several chaotropic agents can be used as
protein denaturing agents. Chaotropic solutes increase the entropy of the
system by interfering with intramolecular interactions mediated by
non-covalent forces such as hydrogen bonds, van der Waals forces, and
hydrophobic effects. Non-limiting examples for chaotropic agents include
butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium
acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate,
thiourea, N-lauroylsarcosine, urea, and salts thereof.
[0255] As used herein, the term "protein reducing agent" refers to the
agent used for reduction of disulfide bridges in a protein. Non-limiting
examples of the protein reducing agents used to reduce the protein are
dithiothreitol (DTT), -mercaptoethanol, Ellman's reagent, hydroxylamine
hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine
hydrochloride (TCEP-HCl), or combinations thereof.
[0256] As used herein, the term "variant" of a polypeptide (e.g., of a
VEGFR Ig domain) refers to a polypeptide comprising an amino acid
sequence that is at least about 70-99.9% (e.g., 70, 71, 72, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to a referenced or
native amino acid sequence of a protein of interest. A sequence
comparison can be performed by, for example, a BLAST algorithm wherein
the parameters of the algorithm are selected to give the largest match
between the respective sequences over the entire length of the respective
reference sequences (e.g., expect threshold: 10; word size: 3; max
matches in a query range: 0; BLOSUM 62 matrix; gap costs: existence 11,
extension 1; conditional compositional score matrix adjustment). Variants
of a polypeptide (e.g., of a VEGFR Ig domain) may also refer to a
polypeptide comprising a referenced amino acid sequence except for one or
more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations such as, for
example, missense mutations (e.g., conservative substitutions), non-sense
mutations, deletions, or insertions. The following references relate to
BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS:
Altschul et al. (2005) FEBS J. 272(20): 5101-5109; Altschul, S. F., et
al., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature
Genet. 3:266-272; Madden, T. L., et al., (1996) Meth. Enzymol.
266:131-141; Altschul, S. F., et al., (1997) Nucleic Acids Res.
25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton,
J. C., et al., (1993) Comput. Chem. 17:149-163; Hancock, J. M. et al.,
(1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS:
Dayhoff, M. O., et al., "A model of evolutionary change in proteins." in
Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M. O.
Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, D.C.;
Schwartz, R. M., et al., "Matrices for detecting distant relationships."
in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3." M.
O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington,
D.C.; Altschul, S. F., (1991) J. Mol. Biol. 219:555-565; States, D. J.,
et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl.
Acad. Sci. USA 89:10915-10919; Altschul, S. F., et al., (1993) J. Mol.
Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc.
Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl.
Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob.
22:2022-2039; and Altschul, S. F. "Evaluating the statistical
significance of multiple distinct local alignments." in Theoretical and
Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp.
1-14, Plenum, N.Y.; the entire teachings of which are herein
incorporated.
[0257] Some variants can be covalent modifications that polypeptides
undergo, either during (co-translational modification) or after
(post-translational modification "PTM") their ribosomal synthesis. PTMs
are generally introduced by specific enzymes or enzyme pathways. Many
occur at the site of a specific characteristic protein sequence (e.g.,
signature sequence) within the protein backbone. Several hundred PTMs
have been recorded and these modifications invariably influence some
aspect of a protein's structure or function (Walsh, G. "Proteins" (2014)
second edition, published by Wiley and Sons, Ltd., ISBN: 9780470669853,
the entire teachings of which are herein incorporated). In certain
exemplary embodiments, a protein composition can comprise more than one
type of protein variant of a protein of interest.
[0258] Protein variants in the case of aflibercept (and proteins sharing
structural characteristics of aflibercept, for example, one or more heavy
or light chain regions of aflibercept) can comprise, but are not limited
to, oxidation variants which can result from oxidation of one or more
amino acid residues occurring at, for example, histidine, cysteine,
methionine, tryptophan, phenylalanine and/or tyrosine residues;
deamidation variants which can result from deamidation at asparagine
residues and/or deoxyglucosonation arginine residue.
[0259] With respect to aflibercept (and proteins sharing structural
characteristics of aflibercept for example, one or more heavy or light
chain regions of aflibercept) oxidation variants can comprise oxidation
of histidine residue at His86, His110, His145, His209, His95, His19
and/or His203 (or equivalent residue positions on proteins sharing
certain structural characteristics of aflibercept); oxidation of
tryptophan residues at Trp58 and/or Trp138 (or equivalent residue
positions on proteins sharing certain structural characteristics of
aflibercept); oxidation of tyrosine residues at Tyr64 (or equivalent
positions on proteins sharing certain structural characteristics of
aflibercept); oxidation of phenylalanine residues at Phe44 and/or Phe166
(or equivalent residue positions on proteins sharing certain structural
characteristics of aflibercept); and/or oxidation of methionine residues
at Met10, Met20, Met163 and/or Met192 (or equivalent residue positions on
proteins sharing certain structural characteristics of aflibercept).
[0260] With respect to aflibercept (and proteins sharing structural
characteristics of aflibercept, for example, one or more heavy or light
chain regions of aflibercept) deamidation variants can comprise
deamidation of asparagine residue at Asn84 and/or Asn99 (or equivalent
residue positions on proteins sharing certain structural characteristics
of aflibercept).
[0261] With respect to aflibercept (and proteins sharing structural
characteristics of aflibercept for example, one or more heavy or light
chain regions of aflibercept) deoxyglucosonation variant can comprise
3-deoxyglucosonation of arginine residue at Arg5 (or equivalent residue
position on proteins sharing certain structural characteristics of
aflibercept).
[0262] Protein variants can include both acidic species and basic species.
Acidic species are typically the variants that elute earlier than the
main peak from CEX or later than the main peak from AEX, while basic
species are the variants that elute later than the main peak from CEX or
earlier than the main peak from AEX.
[0263] As used herein, the terms "acidic species," "AS," "acidic region,"
and "AR," refer to the variants of a protein which are characterized by
an overall acidic charge. For example, in recombinant protein
preparations such acidic species can be detected by various methods, such
as ion exchange, for example, WCX-10 HPLC (a weak cation exchange
chromatography), or IEF (isoelectric focusing). Acidic species of an
antibody may include variants, structure variants, and/or fragmentation
variants. Exemplary variants can include, but are not limited to,
deamidation variants, afucosylation variants, oxidation variants,
methylglyoxal (MGO) variants, glycation variants, and citric acid
variants. Exemplary structure variants include, but are not limited to,
glycosylation variants and acetonation variants. Exemplary fragmentation
variants include any modified protein species from the target molecule
due to dissociation of peptide chain, enzymatic and/or chemical
modifications, including, but not limited to, Fc and Fab fragments,
fragments missing a Fab, fragments missing a heavy chain variable domain,
C-terminal truncation variants, variants with excision of N-terminal Asp
in the light chain, and variants having N-terminal truncation of the
light chain. Other acidic species variants include variants comprising
unpaired disulfides, host cell proteins, and host nucleic acids,
chromatographic materials, and media components. Commonly, acidic species
elute earlier than the main peak during CEX or later than the main peak
during AEX analysis (See FIGS. 16 and 17).
[0264] In certain embodiments, a protein composition can comprise more
than one type of acidic species variant. For example, but not by way of
limitation, the total acidic species can be categorized based on
chromatographic retention time of the peaks appearing. Another example in
which the total acidic species can be categorized can be based on the
type of variant--variants, structure variants, or fragmentation variant.
[0265] The term "acidic species" or "AS" does not refer to process-related
impurities. The term "process-related impurity," as used herein, refers
to impurities that are present in a composition comprising a protein, but
are not derived from the protein itself. Process-related impurities
include, but are not limited to, host cell proteins (HCPs), host cell
nucleic acids, chromatographic materials, and media components.
[0266] In one exemplary embodiment, the amount of acidic species in the
anti-VEGF composition compared to the protein of interest can be at most
about 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%,
3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%,
1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and
ranges within one or more of the preceding. Examples of anti-VEGF
compositions are discussed in Section III below. In one aspect, the
anti-VEGF composition can comprise an anti-VEGF protein selected from the
group consisting of aflibercept, recombinant MiniTrap (examples of which
are disclosed in U.S. Pat. No. 7,279,159), a scFv and other anti-VEGF
proteins. In a preferred aspect, the recombinant protein of interest is
aflibercept.
[0267] Among the chemical degradation pathways responsible for acidic or
basic species, the two most commonly observed covalent modifications
occurring in proteins and peptides are deamination and oxidation.
Methionine, cysteine, histidine, tryptophan, and tyrosine are of the
amino acids that are most susceptible to oxidation: Met and Cys because
of their sulfur atoms and His, Trp, and Tyr because of their aromatic
rings.
[0268] As used herein, the terms "oxidative species," "OS," or "oxidation
variant" refer to the variants of a protein formed by oxidation. Such
oxidative species can also be detected by various methods, such as ion
exchange, for example, WCX-10 HPLC (a weak cation exchange
chromatography), or IEF (isoelectric focusing). Oxidation variants can
result from oxidation occurring at histidine, cysteine, methionine,
tryptophan, phenylalanine and/or tyrosine residues. With respect, in
particular, to aflibercept (and proteins sharing structural
characteristics of aflibercept e.g., one or more heavy or light chain
regions of aflibercept), oxidation variants can comprise oxidation of
histidine residue at His86, His110, His145, His209, His95, His19 and/or
His203 (or equivalent residue positions on proteins sharing certain
structural characteristics of aflibercept); oxidation of tryptophan
residues at Trp58 and/or Trp138 (or equivalent residue positions on
proteins sharing certain structural characteristics of aflibercept);
oxidation of tyrosine residues at Tyr64 (or equivalent positions on
proteins sharing certain structural characteristics of aflibercept);
oxidation of phenylalanine residues at Phe44 and/or Phe166 (or equivalent
residue positions on proteins sharing certain structural characteristics
of aflibercept); and/or oxidation of methionine residues at Met10, Met
20, Met163 and/or Met192 (or equivalent residue positions on proteins
sharing certain structural characteristics of aflibercept).
[0269] In one exemplary embodiment, the amount of oxidative species in the
anti-VEGF composition compared to the protein of interest can be at most
about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%,
3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%,
0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and ranges
within one or more of the preceding. Examples of anti-VEGF compositions
are discussed in Section III below. In one aspect, the anti-VEGF
composition can comprise an anti-VEGF protein selected from the group
consisting of aflibercept, recombinant MiniTrap (examples of which are
disclosed in U.S. Pat. No. 7,279,159), a scFv and other anti-VEGF
proteins. In a preferred aspect, the recombinant protein of interest is
aflibercept or MiniTrap.
[0270] Cysteine residues may undergo spontaneous oxidation to form either
intra- or intermolecular disulfide bonds or monomolecular byproducts such
as sulfenic acid.
[0271] Histidine residues are also highly sensitive to oxidation through
reaction with their imidazole rings, which can subsequently generate
additional hydroxyl species (Li, S, C Schoneich, and RT. Borchardt. 1995.
Chemical Instability of Protein Pharmaceuticals: Mechanisms of Oxidation
and Strategies for Stabilization. Biotechnol. Bioeng. 48:490-500, the
entire teaching of which is herein incorporated). Proposed mechanisms for
histidine oxidation are highlighted in FIG. 2 and FIG. 3. Detailed
mechanistic studies are available in Anal. Chem. 2014, 86, 4940-4948 and
J. Pharm. Biomed. Anal. 21 (2000) 1093-1097, the entire teaching of which
is herein incorporated.
[0272] Oxidation of methionine can lead to formation of methionine
sulfoxide (Li, S, C Schoneich, and RT. Borchardt. 1995. Chemical
Instability of Protein Pharmaceuticals: Mechanisms of Oxidation and
Strategies for Stabilization. Biotechnol. Bioeng. 48:490-500). The
various possible oxidation mechanisms of the methionine residues have
been discussed in the literature (Brot, N., Weissbach, H. 1982. The
biochemistry of methionine sulfoxide residues in proteins. Trends
Biochem. Sci. 7: 137-139, the entire teaching of which is herein
incorporated).
[0273] Oxidation of tryptophan can give a complex mixture of products. The
primary products can be N-formylkynurenine and kynurenine along with
mono-oxidation, di-oxidation and/or tri-oxidation products (FIG. 4).
Peptides bearing oxidized Trp modifications generally exhibit mass
increases of 4, 16, 32 and 48 Da, corresponding to the formation of
kynurenine (KYN), hydroxytryptophan (W.sub.ox1), and
N-formylkynurenine/dihydroxytryptophan (NFK/W.sub.ox2, referred to also
as "doubly oxidized Trp"), trihydroxytryptophan (W.sub.ox3, referred to
also as "triply oxidized Trp"), and their combinations, such as
hydroxykynurenine (KYN.sub.ox1, +20 Da). Oxidation to hydroxytryptophan
(W.sub.ox1) (Mass spectrometric identification of oxidative modifications
of tryptophan residues in proteins: chemical artifact or
post-translational modification? J. Am. Soc. Mass Spectrom. 2010 July;
21(7): 1114-1117, the entire teaching of which is herein incorporated).
Tryptophan oxidation, but not methionine and histidine oxidation have
been found to produce a color change in protein products
(Characterization of the Degradation Products of a Color-Changed
Monoclonal Antibody: Tryptophan-Derived Chromophores.
dx.doi.org/10.1021/ac404218t Anal. Chem. 2014, 86, 6850-6857). Similar to
tryptophan, oxidation of tyrosine primarily yields
3,4-dihydroxyphenylalanine (DOPA) and dityrosine (Li, S, C Schoneich, and
RT. Borchardt. 1995. Chemical Instability of Protein Pharmaceuticals:
Mechanisms of Oxidation and Strategies for Stabilization. Biotechnol.
Bioeng. 48:490-500).
[0274] As used herein, the terms "basic species," "basic region," and
"BR," refer to the variants of a protein, for example, an antibody or
antigen-binding portion thereof, which are characterized by an overall
basic charge, relative to the primary charge variant species present
within the protein. For example, in recombinant protein preparations,
such basic species can be detected by various methods, such as ion
exchange, for example, WCX-10 HPLC (a weak cation exchange
chromatography), or IEF (isoelectric focusing). Exemplary variants can
include, but are not limited to, lysine variants, isomerization of
aspartic acid, succinimide formation at asparagine, methionine oxidation,
amidation, incomplete disulfide bond formation, mutation from serine to
arginine, aglycosylation, fragmentation and aggregation. Commonly, basic
species elute later than the main peak during CEX or earlier than the
main peak during AEX analysis. (Chromatographic analysis of the acidic
and basic species of recombinant monoclonal antibodies. MAbs. 2012 Sep.
1; 4(5): 578-585. doi: 10.4161/mabs.21328, the entire teaching of which
is herein incorporated.)
[0275] In certain embodiments, a protein composition can comprise more
than one type of basic species variant. For example, but not by way of
limitation, the total basic species can be divided based on
chromatographic retention time of the peaks appearing. Another example in
which the total basic species can be divided can be based on the type of
variant--variants, structure variants, or fragmentation variant.
[0276] As discussed for acidic species, the term "basic species" does not
include process-related impurities and the basic species may be the
result of product preparation (referred to herein as "preparation-derived
basic species"), or the result of storage (referred to herein as
"storage-derived basic species").
[0277] In one exemplary embodiment, the amount of basic species in the
anti-VEGF composition compared to the protein of interest can be at most
about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%,
3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%,
0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and ranges
within one or more of the preceding. Examples of anti-VEGF compositions
are discussed in Section III below. In one aspect, the anti-VEGF
composition can comprise an anti-VEGF protein selected from the group
consisting of aflibercept, recombinant MiniTrap (examples of which are
disclosed in U.S. Pat. No. 7,279,159), a scFv and other anti-VEGF
proteins. In a preferred aspect, the recombinant protein of interest is
aflibercept.
[0278] As used herein, "sample matrix" or "biological sample" can be
obtained from any step of the bioprocess, such as, cell culture fluid
(CCF), harvested cell culture fluid (HCCF), any step in the downstream
processing, drug substance (DS), or a drug product (DP) comprising the
final formulated product. In some other specific exemplary embodiments,
the biological sample can be selected from any step of the downstream
process of clarification, chromatographic production, viral inactivation,
or filtration. In some specific exemplary embodiments, the drug product
can be selected from manufactured drug product in the clinic, shipping,
storage, or handling.
[0279] As used herein, the term "subject" refers to a mammal (e.g., rat,
mouse, cat, dog, cow, sheep, horse, goat, rabbit), preferably a human in
need of prevention and/or treatment of a cancer or an angiogenic eye
disorder. The subject may have cancer or angiogenic eye disorder or be
predisposed to developing cancer or angiogenic eye disorder.
[0280] In terms of protein formulation, the term "stable," as used herein
refers to the protein of interest within the formulation being able to
retain an acceptable degree of chemical structure or biological function
after storage under exemplary conditions defined herein. A formulation
may be stable even though the protein of interest contained therein does
not maintain 100% of its chemical structure or biological function after
storage for a defined amount of time. Under certain circumstances,
maintenance of about 90%, about 95%, about 96%, about 97%, about 98% or
about 99% of a protein's structure or function after storage for a
defined amount of time may be regarded as "stable."
[0281] The term "treat" or "treatment" refers to a therapeutic measure
that reverses, stabilizes or eliminates an undesired disease or disorder
(e.g., an angiogenic eye disorder or cancer), for example, by causing the
regression, stabilization or elimination of one or more symptoms or
indicia of such disease or disorder by any clinically measurable degree,
for example, with regard to an angiogenic eye disorder, by causing a
reduction in or maintenance of diabetic retinopathy severity score
(DRSS), by improving or maintaining vision (e.g., in best corrected
visual acuity, for example, as measured by an increase in ETDRS letters),
increasing or maintaining visual field and/or reducing or maintaining
central retinal thickness and, with respect to cancer, stopping or
reversing the growth, survival and/or metastasis of cancer cells in the
subject. Typically, the therapeutic measure is administration of one or
more doses of a therapeutically effective amount of VEGF MiniTrap to the
subject with the disease or disorder.
[0282] As used herein, the term "upstream process technology," in the
context of protein preparation, refers to activities involving the
production and collection of proteins from cells during or following the
cell culture of a protein of interest. As used herein, the term "cell
culture" refers to methods for generating and maintaining a population of
host cells capable of producing a recombinant protein of interest, as
well as the methods and techniques for optimizing the production and
collection of the protein of interest. For example, once an expression
vector has been incorporated into an appropriate host cell, the host cell
can be maintained under conditions suitable for expression of the
relevant nucleotide coding sequences, and the collection and production
of the desired recombinant protein.
[0283] When using the cell culture techniques of the instant invention, a
protein of interest can be produced intracellularly, in the periplasmic
space, or directly secreted into the medium. In embodiments where the
protein of interest is produced intracellularly, particulate
debris--either host cells or lysed cells (e.g., resulting from
homogenization) can be removed by a variety of means, including, but not
limited to, centrifugation or ultrafiltration. Where the protein of
interest is secreted into the medium, supernatants from such expression
systems can be first concentrated using a commercially available protein
concentration filter, for example, using an Amicon.TM. or Millipore
Pellicon.TM. ultrafiltration unit. In one aspect, the protein of interest
may be harvested by centrifugation followed by depth filtration and then
affinity capture chromatography.
[0284] As used herein, a "VEGF antagonist" is any protein or peptide that
binds to or interacts with VEGF. Typically, this binding to or
interacting with inhibits the binding of VEGF to its receptors (VEGFR1
and VEGFR2), and/or inhibits the biological signaling and activity of
VEGF. VEGF antagonists include molecules which interfere with the
interaction between VEGF and a natural VEGF receptor, for example,
molecules which bind to VEGF or a VEGF receptor and prevent or otherwise
hinder the interaction between VEGF and a VEGF receptor. Specific
exemplary VEGF antagonists include anti-VEGF antibodies (e.g.,
ranibizumab [LUCENTIS.RTM.]), anti-VEGF receptor antibodies (e.g.,
anti-VEGFR1 antibodies, anti-VEGFR2 antibodies and the like), and VEGF
receptor-based chimeric molecules or VEGF-inhibiting fusion proteins
(also referred to herein as "VEGF-Traps" or "VEGF MiniTraps"), such as
aflibercept, ziv-aflibercept and a protein having an amino acid having
SEQ ID NO.: 60. Other examples of VEGF-Traps are ALT-L9, M710, FYB203 and
CHS-2020. Additional examples of VEGF-Traps can be found in U.S. Pat.
Nos. 7,070,959; 7,306,799; 7,374,757; 7,374,758; 7,531,173; 7,608,261;
5,952,199; 6,100,071; 6,383,486; 6,897,294 & 7,771,721, which are
specifically incorporated herein by reference in their entirety.
[0285] VEGF receptor-based chimeric molecules include chimeric
polypeptides which comprise two or more immunoglobulin (Ig)-like domains
of a VEGF receptor such as VEGFR1 (also referred to as Flt1) and/or
VEGFR2 (also referred to as Flk1 or KDR), and may also comprise a
multimerizing domain (e.g., an Fc domain which facilitates the
multimerization [e.g., dimerization] of two or more chimeric
polypeptides). An exemplary VEGF receptor-based chimeric molecule is a
molecule referred to as VEGFR1R2-Fc.DELTA.C1(a) (also known as
aflibercept; marketed under the product name EYLEA.RTM.). In certain
exemplary embodiments, aflibercept comprises the amino acid sequence set
forth as
[0286] As used herein, "viral filtration" can include filtration using
suitable filters including, but not limited to, Planova 20N.TM., 50 N or
BioEx from Asahi Kasei Pharma, Viresolve.TM. filters from EMD Millipore,
ViroSart CPV from Sartorius, or Ultipor DV20 or DV50.TM. filter from Pall
Corporation. It will be apparent to one of ordinary skill in the art to
select a suitable filter to obtain desired filtration performance.
II. Color Determination
[0287] As used herein, color observed during the production of a
recombinant protein, specifically, an anti-VEGF protein, can be measured
by various methods. Non-limiting examples include using the iodine color
number, hazen color number, gardner color number, lovibond color number,
Saybolt color number, Mineral oil color number, European pharmacopoeia
color number, US pharmacopoeia color number, CIE L*, a*, b* (or CIELAB),
Klett color number, Hess-Ives color number, the yellowness index, ADMI
color number, and ASBC and EBC brewery color number. Details on such
scales can be found in Application Report No. 3.9 e by Lange, the entire
teaching of which is herein incorporated.
[0288] Visual color matching on the basis of the European Pharmacopoeia
(Ph Eur) (European Color Standards, see European Pharmacopoeia. Chapter
2.2.2. Degree of coloration of liquids. 8.sup.th ed. EP, the entire
teaching of which is herein incorporated) can include preparing a color
reference solution as described in Ph. Eur. (EP 2.2.2. Degree of
Coloration of Liquids 2)--three parent solutions for red (cobaltous (II)
chloride), yellow (ferrous (III) chloride) and blue colors (cuprous (II)
sulphate) and 1% hydrochloric acid, five color reference solutions for
yellow (Y), greenish-yellow (GY), brownish-yellow (BY), brown (B) and red
(R) hues are prepared. With these five reference solutions in turn, a
total of thirty-seven color reference solutions are prepared (Y1-Y7,
GY1-GY7, BY1-BY7, B1-B9 and R1-R7). Each reference solution is clearly
defined in the CIE-Lab color space, for example, by lightness, hue and
chroma. Of the seven yellow-brown standards (BY standards), BY1 is the
darkest standard and BY7 is the least dark. Matching a given sample to
that of a BY color standard is typically done under diffused day light.
The compositions of European yellow-brown color standards are described
in Table 1, below.
TABLE-US-00010
TABLE 1
Composition of European Brown-Yellow Standards
Reference Volumes in mL
Solution Standard Solution BY Hydrochloric acid (10 g/1HCl)
BY1 100.0 0.0
BY2 75.0 25.0
BY3 50.0 50.0
BY4 25.0 75.0
BY5 12.5 87.5
BY6 5.0 95.0
BY7 2.5 97.5
Brownish-Yellow Standard Solution (BY): 10.8 g/L
FeCl.sub.3.cndot.6H.sub.2O, 6.0 g/L CoCl.sub.2.cndot.6H.sub.2O and 2.5
g/L CuSO.sub.4.cndot.5H.sub.2O
[0289] The test for color of liquids is carried out by comparing a test
solution with a standard color solution. The composition of the standard
color solution is selected depending on the hue and intensity of the
color of the test solution. Typically, comparison is carried out in
flat-bottomed tubes of colorless, transparent, neutral glass that are
matched as closely as possible in internal diameter and in all other
respects (e.g., tubes of about 12, 15, 16 or 25 mm diameter). For
example, a comparison can be between 2 or 10 mL of the test solution and
standard color solution. The depth of liquids, for example, can be about
15, 25, 40 or 50 mm. The color assigned to the test solution should not
be more intense than that of the standard color. Color comparisons are
typically carried out in diffused light (e.g., daylight) against a white
background. Colors can be compared down the vertical axis or horizontal
axis of the tubes.
[0290] In contrast to the EP color measurement, the USP Monograph 1061
Color--Instrumental Measurement references the use of CIE L*, a*, b* (or
CIELAB) color measurement to quantify colors precisely and objectively. A
total of twenty color reference solutions (identified sequentially by the
letters A to T) are defined in U.S. Pharmacopoeia. The color of the
measured sample is automatically correlated to the color reference
solutions. This means that the color reference solution that is closest
to the sample (i.e., the reference solution with the smallest color
difference .DELTA.E* to the color of the sample) is displayed. The
.DELTA.L*, .DELTA.a* and .DELTA.b* values give the quantitative
differences between the L*, a* and b* values of the sample and those of
the displayed USP solutions. The CIE L*a*b* coordinate system, L*
represents the degree of lightness of a color on a scale of 0-100, with 0
being the darkest and 100 the lightest, a* represents the redness or
greenness of a color (positive values of a* represent red, whereas
negative values of a* represent green), and b* represents the yellowness
or blueness of a sample, with positive values of b* representing yellow
and negative values of b* representing blue. Color difference from a
standard, or from an initial sample in an evaluation, can be represented
by a change in the individual color components .DELTA.L*, .DELTA.a*, and
.DELTA.b*. The composite change, or difference in color, can be
calculated as a simple Euclidian distance in space using the formula:
dE*= {square root over
((.DELTA.L*).sup.2+(.DELTA.a*).sup.2+(.DELTA.b*).sup.2)}. CIE L*, a*, b*
color coordinates can be generated, for example, using the Hunter Labs
UltrascanPro (Hunter Associates Laboratory, Reston, Va.) or on the BYK
Gardner LCS IV (BYK-Gardner, Columbia, Md.). For the Hunter Labs
UltraScan Pro, the Didymium Filter Test can be executed for wavelength
calibration. The instrument can be standardized in TTRAN with the
0.780-inch port insert and DIW before use; thus, establishing the top
(L=100) and bottom (L=0) of the photometric scale using a light trap and
black card. See Pack et al., Modernization of Physical Appearance and
Solution Color Tests Using Quantitative Tristimulus Colorimetry:
Advantages, Harmonization, and Validation Strategies, J. Pharmaceutical
Sci. 104: 3299-3313 (2015), the entire teaching of which is herein
incorporated. The color of the BY standards can also be expressed under
the CIE L*, a*, b* color space ("CIELAB" or "CIELab" color space). See
Table 2.
TABLE-US-00011
TABLE 2
Characterization of European Brown-Yellow Color
Standards in the CIE L*, a*, b* Color Space
Std. L*{circumflex over ( )} a*{circumflex over ( )} b*{circumflex over (
)} L*.sup.~ a*.sup.~ b*.sup.~
BY1 93.95 -2.76 28.55 92.84 -3.16 31.15
BY2 94.76 -2.96 22.69 94.25 -3.77 26.28
BY3 96.47 -2.84 16.41 95.92 -3.44 18.52
BY4 97.17 -1.94 9.07 97.67 -2.63 10.70
BY5 98.91 -1.19 4.73 98.75 -1.61 5.77
BY6 99.47 -0.59 2.09 99.47 -0.71 2.38
BY7 99.37 -0.31 1.13 99.71 -0.37 1.17
{circumflex over ( )}Reported by Pack et al.
.sup.~Measured experimentally herein-the L* and b* values, for each BY
color standard
[0291] To enable a high throughput screening for the color assay, the
spectrophometric assay method (CIELAB) is more suitable and quantitative
measure than BY color standards. The surrogate assay was further
optimized as described in the Example section.
[0292] For any of the samples evaluated for color, the protein
concentration of the test samples must be standardized for protein
concentration in the samples, for example, 5 g/L, 10 g/L and the like for
comparison.
III. Anti-VEGF Compositions
[0293] There are at least five members of the VEGF family of proteins that
regulate the VEGF signaling pathway: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and
placental growth factor (P1GF). Anti-VEGF compositions can comprise a
VEGF antagonist, which specifically interacts with one or more members of
the VEGF family of proteins and inhibits one or more of its biological
activities, for example, its mitogenic, angiogenic and/or vascular
permeability activity.
[0294] In one embodiment, a method of producing an anti-VEGF protein
comprises: (a) providing a host cell genetically engineered to express
the anti-VEGF protein; (b) culturing the host cell in a CDM under
conditions suitable in which the cell expresses the anti-VEGF protein;
and (c) harvesting a preparation of the anti-VEGF protein produced by the
cell. In one aspect, the anti-VEGF protein is selected from the group
consisting of aflibercept, recombinant MiniTrap (examples of which are
disclosed in U.S. Pat. No. 7,279,159), a scFv and other anti-VEGF
proteins. In a preferred aspect, the recombinant protein of interest is
aflibercept.
[0295] The inventors discovered that manufacturing anti-VEGF proteins
(e.g., aflibercept) in certain CDMs produced a biological sample
exhibiting a distinctive color. The distinct color properties were
observed in different manufacturing steps and even in the final
formulation comprising the anti-VEGF protein. As observed in Example 9,
for the production of VEGF MiniTrap, culturing cells in a CDM produced
anti-VEGF proteins (e.g., aflibercept) with an intense yellow-brown
color. The affinity capture step following harvesting also produced an
eluate exhibiting a certain color--a yellow-brown color. Further
production steps using AEX also exhibited a yellow-brown color however
with reduced intensity.
[0296] As described in more detail below, color may be assessed using (i)
the European Color Standard BY in which a qualitative visual inspection
is made or (ii) a colorimetric assay, CIELAB, which is more quantitative
than the BY system. However, in either case, color assessment between
multiple samples were normalized against protein concentration in order
to assure a meaningful assessment/comparison. For example, referring to
Example 9, in particular Table 9-2, the Protein A eluate has a b* value
of around 2.52 which corresponds to approximately a BY value of BY5 (when
measured at a concentration of 5 g/L protein in the Protein A eluate). If
the color of the Protein A eluate is to be compared to another sample,
then the comparison should be made using the same protein concentration.
Thus, comparing the Protein A eluate to the AEX pool which has a b* value
of around 0.74 (when measured at a concentration of 5 g/L protein in the
protein A eluate), the method of production shows a substantial reduction
in the yellow-brown color of the sample from the Protein A eluate to the
AEX pool following AEX chromatography.
[0297] Compositions of the present invention can be characterized by a
yellow-brown color as discussed herein, for example, no darker/intense
than the European Brown-Yellow Color Standard BY2-BY3, BY3-BY4, BY4-BY5
or BY5-BY6 and/or having a b* value 17-23, 10-17, 5-10, 3-5, or 1-3,
wherein the composition comprises about 5 g/L of the anti-VEGF protein or
about 10 g/L of the anti-VEGF protein and wherein the composition is
obtained as a sample from a clarified harvest or a Protein A eluate of
the clarified harvest.
[0298] In one embodiment, the compositions of the invention produced using
CDM produces a biological sample having a distinct yellow-brown color,
wherein the sample may be characterized by a recognized standard color
characterization:
(i) no more yellow-brown than European Color Standard BY2; (ii) no more
yellow-brown than European Color Standard BY3; (iii) no more yellow-brown
than European Color Standard BY4; (iv) no more yellow-brown than European
Color Standard BY5; (v) between European Color Standard BY2 and BY3; (vi)
between European Color Standard BY3 and BY4; (vii) between European Color
Standard BY4 and BY5, wherein the composition comprises about 5 g/L or
about 10 g/L of the anti-VEGF protein and wherein the composition is
obtained as a sample from a Protein A eluate of a clarified harvest.
[0299] In another embodiment, the compositions of the invention produced
using a CDM produces a biological sample having a distinct yellow-brown
color, wherein the composition is characterized by a recognized standard
color characterization in the CIELAB scale:
(i) no more yellow-brown than b* value of about 22-23; (ii) no more
yellow-brown than b* value of about 16-17; (iii) no more yellow-brown
than b* value of 9-10; (iv) no more yellow-brown than b* value of 4-5;
(v) no more yellow-brown than b* value of 2-3; (vi) between b* value of
17-23; (vii) between b* value of 10-17; (viii) between b* value of 5-10;
(ix) between b* value of 3-5; or (x) between b* value of 1-3, wherein the
composition comprises about 5 g/L or about 10 g/L of the anti-VEGF
protein and wherein the composition is obtained as a sample from a
Protein A eluate of a clarified harvest.
[0300] In one embodiment, the compositions of the invention produced using
CDM can comprise other species or variants of the anti-VEGF protein.
These variants include anti-VEGF protein isoforms that comprise one or
more oxidized amino acid residues collectively referred to as
oxo-variants. The enzymatic digestion of such compositions comprising the
anti-VEGF protein and its oxo-variants can comprise one or more of:
EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18) which comprises about 0.004-0.013%
2-oxo-histidines, QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) which
comprises about 0.006-0.028% 2-oxo-histidines, TELNVGIDFNWEYPSSKH*QHK
(SEQ ID NO.: 20) which comprises about 0.049-0.085% 2-oxo-histidines,
DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17) which comprises about 0.057-0.092%
2-oxo-histidines, TNYLTH*R (SEQ ID NO.: 21) which comprises about
0.008-0.022% 2-oxo-histidines, and/or IIWDSR (SEQ ID NO.: 56) which
comprises about 0.185-0.298% dioxidized tryptophan; or
EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18) which comprises about 0.008%
2-oxo-histidines, QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) which
comprises about 0.02% 2-oxo-histidines, TELNVGIDFNWEYPSSKH*QHK (SEQ ID
NO.: 20) which comprises about 0.06% 2-oxo-histidines,
DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17) which comprises about 0.07%
2-oxo-histidines, TNYLTH*R (SEQ ID NO.: 21) which comprises about 0.01%
2-oxo-histidines, and/or IIWDSR (SEQ ID NO.: 56) which comprises about
0.23% di-oxo-tryptophans, wherein H* is a histidine that may be oxidized
to 2-oxo-histidine and wherein C* is a cysteine which may be
carboxymethylated. In a particular embodiment, the anti-VEGF protein is
aflibercept. In another embodiment, the anti-VEGF protein is a VEGF
MiniTrap.
[0301] In one exemplary embodiment of the invention, the compositions of
the invention can comprise an anti-VEGF protein, wherein no more than
about 1%, no more than about 0.1% or about 0.1-1%, 0.2-1%, 0.3-1%,
0.4-1%, 0.5-1%, 0.6-1%, 0.7-1%, 0.8-1% or 0.9-1% of histidine residues of
the anti-VEGF protein are 2-oxo-histidine. In such compositions, there
can be a heterogeneous population of the anti-VEGF protein variants each
having a varying amount of 2-oxo-histidine residues and un-oxidized
histidine residues. Thus, the percentage of 2-oxo-histidine anti-VEGF
protein in a composition refers to the site-specific 2-oxo-histidines
among the anti-VEGF molecules divided by total site-specific histidines
in the molecules of the anti-VEGF protein (oxidized plus un-oxidized)
times 100. One method to quantitate the level of 2-oxo-histidines in a
composition is to digest the polypeptide with a protease (e.g., Lys-C
and/or trypsin) and analyze the quantity of 2-oxo-histidines in the
resulting peptides by, for example, mass spectrometry (ms).
[0302] Before digestion of the anti-VEGF protein, cysteines sulfhydryl
groups are blocked by reaction with iodoacetamide (IAM) resulting in a
residue represented by the following chemical structure:
##STR00001##
Such modification protects free thiols from reforming disulfide bridges
and prevents disulfide bond scrambling. The present invention includes
compositions (e.g., aqueous compositions) comprising anti-VEGF protein
and its variants which, when modified with IAM and digested with protease
(e.g., Lys-C and trypsin) and analyzed by mass spectrometry comprise the
following peptides: EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18) which comprises
about 0.004-0.013% 2-oxo-histidines, QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID
NO.: 19) which comprises about 0.006-0.028% 2-oxo-histidines,
TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20) which comprises about
0.049-0.085% 2-oxo-histidines, DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17) which
comprises about 0.057-0.092% 2-oxo-histidines, TNYLTH*R (SEQ ID NO.: 21)
which comprises about 0.008-0.022% 2-oxo-histidines, and/or IIWDSR (SEQ
ID NO.: 56) which comprises about 0.185-0.298% dioxidized tryptophan; or
EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18) which comprises about 0.008%
2-oxo-histidines, QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) which
comprises about 0.02% 2-oxo-histidines, TELNVGIDFNWEYPSSKH*QHK (SEQ ID
NO.: 20) which comprises about 0.06% 2-oxo-histidines,
DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17) which comprises about 0.07%
2-oxo-histidines, TNYLTH*R (SEQ ID NO.: 21) which comprises about 0.01%
2-oxo-histidines, and/or IIWDSR (SEQ ID NO.: 56) which comprises about
0.23% di-oxo-tryptophans, wherein H* is 2-oxo-histidine and wherein C* is
carboxymethylated cysteine. In one embodiment of the invention, the
peptides are deglycosylated with PNGase F.
[0303] The present invention includes compositions comprising anti-VEGF
protein, wherein about 0.1%-10% of all histidines of the anti-VEGF
protein are modified to 2-oxo-histidine. Further, the color of the
composition is no darker/intense than, for example, the European
Brown-Yellow Color Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6,
alternatively, having a b* value, as characterized using CIE L*, a*, b*
of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition
comprises about 5 g/L or about 10 g/L of the anti-VEGF protein. The
composition is obtained either as a sample from a clarified harvest or a
Protein A eluate of the clarified harvest. Such compositions can be
obtained from the clarified harvest when the harvest material is
subjected to a capture chromatography procedure. In one aspect, the
capture step is an affinity chromatography procedure using, for example,
a Protein A affinity column. When an affinity sample is analyzed using
liquid chromatography--mass spectrophotometry (LC-MS), one or more
variants may be detected.
[0304] The present invention includes compositions comprising anti-VEGF
protein, wherein about 0.1%-10% of all tryptophans of the anti-VEGF
protein are modified to kynurenine. Further, the color of the composition
is no darker/intense than the European Brown-Yellow Color Standard
BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value, as
characterized by CIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or 1-3,
wherein the composition comprises about 5 g/L of the anti-VEGF protein or
about 10 g/L of the anti-VEGF protein. The composition is obtained as a
sample from a clarified harvest or a Protein A eluate of the clarified
harvest. Such compositions can be obtained from the clarified harvest
when subjected to a capture chromatography procedure. The capture step is
an affinity chromatography procedure using, for example, a Protein A
affinity column. When an affinity sample is analyzed using liquid
chromatography--mass spectrophotometry (LC-MS), one or more these
variants may be detected.
[0305] The present invention includes compositions comprising anti-VEGF
protein, wherein about 0.1%-10% of all tryptophans of the anti-VEGF
protein are modified to mono-hydroxyl tryptophan. Further, the color of
the composition is no darker/intense than the European Brown-Yellow Color
Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value
characterized by CIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or 1-3,
wherein the composition comprises about 5 g/L of the anti-VEGF protein or
about 10 g/L of the anti-VEGF protein. The composition is obtained as a
sample from a clarified harvest or a Protein A eluate of the clarified
harvest. Such compositions can be obtained from the clarified harvest
when subjected to a capture chromatography procedure. The capture step is
an affinity chromatography procedure using, for example, a Protein A
affinity column. When a sample extracted from the affinity step is
analyzed using liquid chromatography--mass spectrophotometry (LC-MS), one
or more these variants may be detected.
[0306] The present invention includes compositions comprising anti-VEGF
protein, wherein about 0.1%-10% of all tryptophans of the anti-VEGF
protein are modified to di-hydroxyl tryptophan. Further, the color is no
darker/intense than the European Brown-Yellow Color Standard BY2-BY3,
BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value characterized using
CIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the
composition comprises about 5 g/L of the anti-VEGF protein or about 10
g/L of the anti-VEGF protein. The composition is obtained as a sample
from a clarified harvest or a Protein A eluate of the clarified harvest.
Such compositions can be obtained from the clarified harvest made using
CDM comprising the anti-VEGF protein as well as its oxo-variants
subjected to a capture chromatography procedure. The capture step is an
affinity chromatography procedure using, for example, a Protein A
affinity column. When a sample extracted from the affinity step is
analyzed using liquid chromatography--mass spectrophotometry (LC-MS), one
or more these variants may be detected.
[0307] The present invention includes compositions comprising anti-VEGF
protein, wherein about 0.1%-10% of all tryptophans of the anti-VEGF
protein are modified to tri-hydroxyl tryptophan. Further, the color of
the composition is no darker/intense than the European Brown-Yellow Color
Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value
characterized by CIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or 1-3,
wherein the composition comprises about 5 g/L of the anti-VEGF protein or
about 10 g/L of the anti-VEGF protein. The composition is obtained as a
sample from a clarified harvest or a Protein A eluate of the clarified
harvest. Such compositions can be obtained using capture chromatography.
The capture step is an affinity chromatography procedure using, for
example, a Protein A affinity column. When a sample extracted from the
affinity is analyzed using liquid chromatography--mass spectrophotometry
(LC-MS), one or more these variants may be detected.
[0308] In one embodiment, the compositions of the invention can comprise
an anti-VEGF protein, wherein the anti-VEGF protein can comprise
modifications of one or more residues as follows: one or more asparagines
are deamidated; one or more aspartic acids are converted to iso-aspartate
and/or Asn; one or more methionines are oxidized; one or more tryptophans
are converted to N-formylkynurenin; one or more tryptophans are
mono-hydroxyl tryptophan; one or more tryptophans are di-hydroxyl
tryptophan; one or more tryptophans are tri-hydroxyl tryptophan; one or
more arginines are converted to Arg 3-deoxyglucosone; the C-terminal
glycine is not present; and/or there are one or more non-glycosylated
glycosites.
[0309] Such compositions can be obtained from a clarified harvest made
using CDM comprising the anti-VEGF protein as well as its variants
subjected to, for example, a capture chromatography procedure. The
capture step is an affinity chromatography procedure using, for example,
a Protein A column. When a sample extracted from the affinity step is
analyzed using, for example, liquid chromatography--mass
spectrophotometry (LC-MS), one or more these variants may be detected.
[0310] In one exemplary embodiment, the compositions of the invention can
comprise an anti-VEGF protein sharing structural characteristics of
aflibercept which can be oxidized at one or more of the following: His86,
His110, His145, His209, His95, His19 and/or His203 (or equivalent residue
positions on proteins sharing certain structural characteristics of
aflibercept); Trp58 and/or Trp138 (or equivalent residue positions on
proteins sharing certain structural characteristics of aflibercept);
Tyr64 (or equivalent positions on proteins sharing certain structural
characteristics of aflibercept); Phe44 and/or Phe166 (or equivalent
residue positions on proteins sharing certain structural characteristics
of aflibercept); and/or Met10, Met 20, Met163 and/or Met192 (or
equivalent residue positions on proteins sharing certain structural
characteristics of aflibercept. Such compositions can be obtained from a
clarified harvest made using CDM comprising aflibercept as well as its
oxo-variants subjected to a capture chromatography procedure. The capture
step can be an affinity chromatography procedure using, for example, a
Protein A column. When a sample extracted from the affinity step is
analyzed using, for example, liquid chromatography--mass
spectrophotometry (LC-MS), one or more these variants may be detected.
[0311] In one embodiment, the compositions of the invention can comprise a
VEGF MiniTrap having amino acid sequence of SEQ ID NO.: 46, which can be
oxidized at His86, His110, His145, His209, His95, His19 and/or His203;
Trp58 and/or Trp138; Tyr64; Phe44 and/or Phe166; and/or Met10, Met 20,
Met163 and/or Met192. Such compositions can be obtained from the
clarified harvest made using CDM comprising the VEGF MiniTrap as well as
its oxo-variants subjected to a capture chromatography procedure. The
capture step is an affinity chromatography procedure using, for example,
a Protein A column--when analyzed using liquid chromatography--mass
spectrophotometry (LC-MS), one or more these variants may be detected.
[0312] In some exemplary embodiments, compositions of the present
invention can comprise an anti-VEGF protein and its variants (including
oxo-variants), wherein the amount of the protein variants in the
composition can be at most about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,
12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%,
1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%,
0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and ranges within one or more
of the preceding. Such compositions can be obtained from the clarified
harvest made using CDM comprising the anti-VEGF protein as well as its
variants subjected to a capture chromatography procedure. The capture
step is an affinity chromatography procedure using, for example, a
Protein A column--when analyzed using liquid chromatography--mass
spectrophotometry (LC-MS), one or more these variants may be detected. In
one aspect, the color of such a composition is no darker/intense than,
for example, the European Brown-Yellow Color Standard BY2-BY3, BY3-BY4,
BY4-BY5 or BY5-BY6 and/or having a b* value characterized by CIE L*, a*,
b* of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition
comprises about 5 g/L or about 10 g/L of the anti-VEGF protein.
[0313] In other exemplary embodiments, compositions of the present
invention can comprise an anti-VEGF protein and its variants, wherein the
amount of the protein variants in the composition can be about 0% to
about 20%, for example, about 0% to about 20%, about 0.05% to about 20%,
about 0.1% to about 20%, about 0.2% to about 20%, about 0.3% to about
20%, about 0.4% to about 20%, about 0.5% to about 20%, about 0.6% to
about 20%, about 0.7% to about 20%, about 0.8% to about 20%, about 0.9%
to about 20%, about 1% to about 20%, about 1.5% to about 20%, about 2% to
about 20%, about 3% to about 20%, about 4% to about 20%, about 5% to
about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to
about 20%, about 9% to about 20%, about 10% to about 20%, about 0% to
about 10%, about 0.05% to about 10%, about 0.1% to about 10%, about 0.2%
to about 10%, about 0.3% to about 10%, about 0.4% to about 10%, about
0.5% to about 10%, about 0.6% to about 10%, about 0.7% to about 10%,
about 0.8% to about 10%, about 0.9% to about 10%, about 1% to about 10%,
about 1.5% to about 10%, about 2% to about 10%, about 3% to about 10%,
about 4% to about 10%, about 5% to about 10%, about 6% to about 10%,
about 7% to about 10%, about 8% to about 10%, about 9% to about 10%,
about 0% to about 7.5%, about 0.05% to about 7.5%, about 0.1% to about
7.5%, about 0.2% to about 7.5%, about 0.3% to about 7.5%, about 0.4% to
about 7.5%, about 0.5% to about 7.5%, about 0.6% to about 7.5%, about
0.7% to about 7.5%, about 0.8% to about 7.5%, about 0.9% to about 7.5%,
about 1% to about 7.5%, about 1.5% to about 7.5%, about 2% to about 7.5%,
about 3% to about 7.5%, about 4% to about 7.5%, about 5% to about 7.5%,
about 6% to about 7.5%, about 7% to about 7.5%, about 0% to about 5%,
about 0.05% to about 5%, about 0.1% to about 5%, about 0.2% to about 5%,
about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%,
about 0.6% to about 5%, about 0.7% to about 5%, about 0.8% to about 5%,
about 0.9% to about 5%, about 1% to about 5%, about 1.5% to about 5%,
about 2% to about 5%, about 3% to about 5%, about 4% to about 5% and
ranges within one or more of the preceding. Such compositions can be
obtained performing capture chromatography on a harvest sample. The
capture step is an affinity chromatography procedure using, for example,
a Protein A column. When a sample is analyzed using liquid
chromatography--mass spectrophotometry (LC-MS), one or more these
variants may be detected. In one aspect, the color of such a composition
is no darker/intense than, for example, the European Brown-Yellow Color
Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value
characterized by CIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or 1-3,
wherein the composition comprises about 5 g/L or about 10 g/L of the
anti-VEGF protein.
[0314] In one embodiment, compositions of the present invention can
comprise an anti-VEGF protein including its acidic species, wherein the
amount of the acidic species in the composition can be about 20%, 19%,
18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%,
4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%,
1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%
and ranges within one or more of the preceding. As discussed supra, such
acidic species can be detected by various methods such as ion exchange,
for example, WCX (WCX-10 HPLC, a weak cation exchange chromatography), or
IEF (isoelectric focusing). Commonly, acidic species elute earlier than
the main peak during CEX or later than the main peak during AEX analysis
(see FIG. 16 and FIG. 17). Compositions comprising acidic species can be
obtained from biological material such as harvest or affinity produced
material using ion exchange chromatography.
[0315] In one aspect, the color of such a composition is no darker/intense
than, for example, the European Brown-Yellow Color Standard BY2-BY3,
BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value characterized by CIE
L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the
composition comprises about 5 g/L or about 10 g/L. As an example,
referring to FIG. 16 and FIG. 17, fractions F1 and F2 represent acidic
fractions which comprises the majority of the acidic species. Peaks 1 and
2 of MT1 in FIG. 17 comprise the acidic species and fractions F1 and F2
comprises the majority of the acidic fractions. The fractions comprising
such acidic species (F1 and F2) also showed a yellow-brown color compared
to other fractions (FIG. 18B and FIG. 18C).
[0316] In another embodiment, compositions of the instant invention
comprise an anti-VEGF protein including its acidic species, wherein the
amount of acidic species in the composition can be about 0% to about 20%,
for example, about 0% to about 20%, about 0.05% to about 20%, about 0.1%
to about 20%, about 0.2% to about 20%, about 0.3% to about 20%, about
0.4% to about 20%, about 0.5% to about 20%, about 0.6% to about 20%,
about 0.7% to about 20%, about 0.8% to about 20%, about 0.9% to about
20%, about 1% to about 20%, about 1.5% to about 20%, about 2% to about
20%, about 3% to about 20%, about 4% to about 20%, about 5% to about 20%,
about 6% to about 20%, about 7% to about 20%, about 8% to about 20%,
about 9% to about 20%, about 10% to about 20%, about 0% to about 10%,
about 0.05% to about 10%, about 0.1% to about 10%, about 0.2% to about
10%, about 0.3% to about 10%, about 0.4% to about 10%, about 0.5% to
about 10%, about 0.6% to about 10%, about 0.7% to about 10%, about 0.8%
to about 10%, about 0.9% to about 10%, about 1% to about 10%, about 1.5%
to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to
about 10%, about 5% to about 10%, about 6% to about 10%, about 7% to
about 10%, about 8% to about 10%, about 9% to about 10%, about 0% to
about 7.5%, about 0.05% to about 7.5%, about 0.1% to about 7.5%, about
0.2% to about 7.5%, about 0.3% to about 7.5%, about 0.4% to about 7.5%,
about 0.5% to about 7.5%, about 0.6% to about 7.5%, about 0.7% to about
7.5%, about 0.8% to about 7.5%, about 0.9% to about 7.5%, about 1% to
about 7.5%, about 1.5% to about 7.5%, about 2% to about 7.5%, about 3% to
about 7.5%, about 4% to about 7.5%, about 5% to about 7.5%, about 6% to
about 7.5%, about 7% to about 7.5%, about 0% to about 5%, about 0.05% to
about 5%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to
about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 0.6% to
about 5%, about 0.7% to about 5%, about 0.8% to about 5%, about 0.9% to
about 5%, about 1% to about 5%, about 1.5% to about 5%, about 2% to about
5%, about 3% to about 5%, about 4% to about 5% and ranges within one or
more of the preceding. As discussed above, such acidic species can be
detected by various methods, such as ion exchange, for example, WCX
(WCX-10 HPLC, a weak cation exchange chromatography), or IEF (isoelectric
focusing). Typically, acidic species elute earlier than the main peak
during CEX or later than the main peak during AEX analysis (See FIG. 16
and FIG. 17).
[0317] Using a cation exchange column, all peaks eluting prior to the main
peak of interest were summed as the acidic region, and all peaks eluting
after the protein of interest were summed as the basic region. In
exemplary embodiments, the acidic species can be eluted as two or more
acidic regions and can be numbered AR1, AR2, AR3 and so on based on a
certain retention time of the peaks and on the ion exchange column used.
[0318] In one embodiment, compositions can comprise an anti-VEGF protein
including acidic species, wherein AR1 is 20%, 15%, 14%, 13%, 12%, 11%,
10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%,
1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%,
0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of the
preceding. In one aspect, compositions can comprise an anti-VEGF protein
including its acidic species, wherein AR1 is about 0.0% to about 10%,
about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%,
about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, or
about 8% to about 10%, or about 10% to about 15%, and ranges within one
or more of the preceding. As discussed above, such acidic regions can be
detected by various methods, such as ion exchange, for example, WCX
(WCX-10 HPLC, a weak cation exchange chromatography), or IEF (isoelectric
focusing). Commonly, acidic species elute earlier than the main peak
during CEX or later than the main peak during AEX analysis (See FIG. 16
and FIG. 17).
[0319] In another embodiment, compositions can comprise an anti-VEGF
protein including acidic species, wherein AR2 is 20%, 15%, 14%, 13%, 12%,
11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%,
1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%,
0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of
the preceding. In one aspect, compositions can comprise an anti-VEGF
protein including acidic species, wherein AR2 is about 0.0% to about 10%,
about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%,
about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, or
about 8% to about 10%, or about 10% to about 15%, and ranges within one
or more of the preceding.
[0320] In one embodiment, compositions can comprise an anti-VEGF protein
including basic species, wherein the amount of the basic species in the
composition can be at most about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,
12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%,
1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%,
0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and ranges within one or more
of the preceding. In one aspect, compositions can comprise an anti-VEGF
protein and its basic species, wherein the amount of the basic species in
the composition compared to the anti-VEGF protein can be 0% to about 20%
e.g., about 0% to about 20%, about 0.05% to about 20%, about 0.1% to
about 20%, about 0.2% to about 20%, about 0.3% to about 20%, about 0.4%
to about 20%, about 0.5% to about 20%, about 0.6% to about 20%, about
0.7% to about 20%, about 0.8% to about 20%, about 0.9% to about 20%,
about 1% to about 20%, about 1.5% to about 20%, about 2% to about 20%,
about 3% to about 20%, about 4% to about 20%, about 5% to about 20%,
about 6% to about 20%, about 7% to about 20%, about 8% to about 20%,
about 9% to about 20%, about 10% to about 20%, about 0% to about 10%,
about 0.05% to about 10%, about 0.1% to about 10%, about 0.2% to about
10%, about 0.3% to about 10%, about 0.4% to about 10%, about 0.5% to
about 10%, about 0.6% to about 10%, about 0.7% to about 10%, about 0.8%
to about 10%, about 0.9% to about 10%, about 1% to about 10%, about 1.5%
to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to
about 10%, about 5% to about 10%, about 6% to about 10%, about 7% to
about 10%, about 8% to about 10%, about 9% to about 10%, about 0% to
about 7.5%, about 0.05% to about 7.5%, about 0.1% to about 7.5%, about
0.2% to about 7.5%, about 0.3% to about 7.5%, about 0.4% to about 7.5%,
about 0.5% to about 7.5%, about 0.6% to about 7.5%, about 0.7% to about
7.5%, about 0.8% to about 7.5%, about 0.9% to about 7.5%, about 1% to
about 7.5%, about 1.5% to about 7.5%, about 2% to about 7.5%, about 3% to
about 7.5%, about 4% to about 7.5%, about 5% to about 7.5%, about 6% to
about 7.5%, about 7% to about 7.5%, about 0% to about 5%, about 0.05% to
about 5%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to
about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 0.6% to
about 5%, about 0.7% to about 5%, about 0.8% to about 5%, about 0.9% to
about 5%, about 1% to about 5%, about 1.5% to about 5%, about 2% to about
5%, about 3% to about 5%, about 4% to about 5% and ranges within one or
more of the preceding.
[0321] The basic species can be eluted as two or more basic regions and
can be numbered BR1, BR2, BR3 and so on based on a certain retention time
of the peaks and ion exchange used.
[0322] In one embodiment, compositions can comprise an anti-VEGF protein
including its basic species, wherein BR1 is 15%, 14%, 13%, 12%, 11%, 10%,
9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%,
1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%,
0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of the
preceding. In one aspect, compositions can comprise an anti-VEGF protein
and its basic species, wherein BR1 is about 0.0% to about 10%, about 0.0%
to about 5%, about 0.0% to about 4%, about 0.0% to about 3%, about 0.0%
to about 2%, about 3% to about 5%, about 5% to about 8%, or about 8% to
about 10%, or about 10% to about 15%, and ranges within one or more of
the preceding.
[0323] In another embodiment, the composition can comprise an anti-VEGF
protein and its basic species, wherein BR2 is 15%, 14%, 13%, 12%, 11%,
10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%,
1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%,
0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of the
preceding. In one aspect, compositions can comprise an anti-VEGF protein
and its basic species of the anti-VEGF protein, wherein BR2 is about 0.0%
to about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0%
to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to
about 8%, or about 8% to about 10%, or about 10% to about 15%, and ranges
within one or more of the preceding.
[0324] In another embodiment, the composition can comprise an anti-VEGF
protein and its basic species, wherein BR3 is 15%, 14%, 13%, 12%, 11%,
10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%,
1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%,
0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of the
preceding. In one aspect, compositions can comprise an anti-VEGF protein
and its basic species of the anti-VEGF protein, wherein BR3 is about 0.0%
to about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0%
to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to
about 8%, or about 8% to about 10%, or about 10% to about 15%, and ranges
within one or more of the preceding.
[0325] Photo-Induced Oxidation of Aflibercept
[0326] In addition to discovering the different color characteristics or
variants of the anti-VEGF protein compositions produced using CDM, the
inventors also discovered that such compositions can be artificially
produced in the laboratory by exposure to light.
[0327] Modified, including oxidized, variants of an anti-VEGF composition
can be produced by exposing an anti-VEGF protein to cool-white light
white or ultraviolet light. In one aspect, the anti-VEGF composition can
comprise about 1.5 to about 50-fold increase in one or more modified
oligopeptides, compared to the sample, wherein the oligopeptides are
selected from the group consisting of:
DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), EIGLLTC*EATVNGH*LYK (SEQ ID NO.:
18), QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), TELNVGIDFNWEYPSSKH*QHK
(SEQ ID NO.: 20), TNYLTH*R (SEQ ID NO.: 21), SDTGRPFVEMYSEIPEIIH*MTEGR
(SEQ ID NO.: 22), VH*EKDK (SEQ ID NO.: 23), SDTGRPFVEM*YSEIPEIIHMTEGR
(SEQ ID NO.: 64), SDTGRPFVEMYSEIPEIIHM*TEGR (SEQ ID NO.: 65), TQSGSEM*K
(SEQ ID NO.: 66), SDQGLYTC*AASSGLM*TK (SEQ ID NO.: 67),
IIW*DSR/RIIW*DSR/IIW*DSRK (SEQ ID NO.: 28), TELNVGIDFNW*EYPSSK (SEQ ID
NO.: 29), GFIISNATY*K (SEQ ID NO.: 69), KF*PLDTLIPDGK (SEQ ID NO.: 70)
F*LSTLTIDGVTR (SEQ ID NO.: 32), wherein H* is a histidine is oxidized to
2-oxo-histidine, wherein C* is a cysteine is carboxymethylated, wherein
M* is a oxidized methionine, wherein W* is a oxidized tryptophan, wherein
Y* is a oxidized tyrosine, and wherein F* is a oxidized phenylalanine. In
a further aspect, the anti-VEGF composition can comprise about 1.5 to
about 10-fold increase in one or more modified oligopeptides by exposing
an anti-VEGF composition to cool-white light for a period of time, for
example, about 30 hours. In another aspect, the anti-VEGF composition can
comprise about 1.5 to about 10-fold increase in one or more modified
oligopeptides by exposing a sample to cool-white light for about 75
hours. In yet another aspect, the anti-VEGF composition can comprise
about 1.5 to about 20-fold increase in one or more oligopeptides
described before by exposing the sample to cool-white light for about 100
hours. In yet another aspect, the anti-VEGF composition can comprise
about 1.5 to about 20-fold increase in one or more oligopeptides
described before by exposing the sample to cool-white light for about 150
hours. In still another aspect, the anti-VEGF composition can comprise
about 1.5 to about 50-fold increase in one or more oligopeptides by
exposing the sample to cool-white light for about 300 hours--see Example
4 below.
[0328] The anti-VEGF composition can comprise about 1.5 to about 3-fold
increase in one or more oligopeptides, as described above, by exposing a
sample of an anti-VEGF composition to ultraviolet light for about 4
hours. In another aspect, the anti-VEGF composition can comprise about
1.5 to about 10-fold increase in one or more oligopeptides by exposing
the sample to ultraviolet light for about 10 hours. In yet another
aspect, the anti-VEGF composition can comprise about 1.5 to about 10-fold
increase in one or more oligopeptides described by exposing the sample to
ultraviolet light for about 16 hours. In yet another aspect, the
anti-VEGF composition can comprise about 1.5 to about 25-fold increase in
one or more oligopeptides by exposing the sample to ultraviolet light for
about 20 hours. In yet another aspect, the anti-VEGF composition can
comprise about 1.5 to about 25-fold increase in one or more oligopeptides
by exposing the sample matrix to ultraviolet light for about 40 hours.
See Example 4.
[0329] Glycodiversity--Anti-VEGF Protein Produced Using CDM
[0330] The compositions of this invention comprise an anti-VEGF protein,
wherein the anti-VEGF protein produced in CDM has a variety of
glycodiversity. The different glycosylation profiles of the anti-VEGF
protein are within the scope of this invention.
[0331] In some exemplary embodiments of the invention, the composition can
comprise an anti-VEGF protein glycosylated at one or more asparagines as
follows: G0-GlcNAc glycosylation; G1-GlcNAc glycosylation; G1S-GlcNAc
glycosylation; GO glycosylation; G1 glycosylation; G1S glycosylation; G2
glycosylation; G2S glycosylation; G2S2 glycosylation; G0F glycosylation;
G2F2S glycosylation; G2F2S2 glycosylation; G1F glycosylation; G1FS
glycosylation; G2F glycosylation; G2FS glycosylation; G2FS2
glycosylation; G3FS glycosylation; G3FS3 glycosylation; G0-2GlcNAc
glycosylation; Man4 glycosylation; Man4_A1G1 glycosylation; Man4_A1G1S1
glycosylation; Man5 glycosylation; Man5_A1G1 glycosylation; Man5_A1G1S1
glycosylation; Man6 glycosylation; Man6_G0+Phosphate glycosylation;
Man6+Phosphate glycosylation; and/or Man7 glycosylation. In one aspect,
the protein of interest can be aflibercept, anti-VEGF antibody or VEGF
MiniTrap.
[0332] In one embodiment, the composition can have a glycosylation profile
as follows: about 40% to about 50% total fucosylated glycans, about 30%
to about 50% total sialylated glycans, about 6% to about 15% mannose-5,
and about 60% to about 79% galactosylated glycans. (Example 6).
[0333] In one embodiment, the composition can comprise an anti-VEGF
protein, wherein the protein of interest has Man5 glycosylation at about
32.4% of asparagine 123 residues and/or about 27.1% of asparagine 196
residues. In one aspect, the protein of interest can be aflibercept,
anti-VEGF antibody or VEGF MiniTrap.
[0334] In another embodiment, the composition can have about 40%, about
41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%,
about 48%, about 49% or about 50% total fucosylated glycans.
[0335] In yet another embodiment, the composition can have about 30%,
about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about
37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%,
about 44%, about 45%, about 46%, about 47%, about 48%, about 49% or about
50% total sialylated glycans.
[0336] In one embodiment, the composition can have about 6%, about 7%,
about 8%, about 8%, about 10%, about 11%, about 12%, about 13%, about
14%, or about 15% mannose-5.
[0337] In another embodiment, the composition can have about 60%, about
61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%,
about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about
74%, about 75%, about 76%, about 77%, about 78%, or about 79% total
galactosylated glycans.
[0338] In one embodiment, the anti-VEGF protein can have an decreased
level of fucosylated glycans by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%,
3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges within
one or more of the preceding values, for example, 1-10%, 1-15%, 1-20%,
1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%,
1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%,
2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%,
2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%,
3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%,
4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%,
4-46%, 4-47%, 4-48%, 4-49%, 4-50% or 1-99% compared to level of
fucosylated glycans in an anti-VEGF protein produced using a soy
hydrolysate.
[0339] In one embodiment, the anti-VEGF protein can have a decreased level
of sialylated glycans by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%,
3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges within one or
more of the preceding values, for example, 1-10%, 1-15%, 1-20%, 1-25%,
1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%,
1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%,
2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%,
3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%,
3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%,
4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%,
4-47%, 4-48%, 4-49%, 4-50% or 1-99% compared to level of sialylated
glycans in an anti-VEGF protein produced using a soy hydrolysate.
[0340] In another embodiment, the anti-VEGF protein can have a decreased
level of galactosylated glycans by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%,
3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges
within one or more of the preceding values, e.g., 1-10%, 1-15%, 1-20%,
1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%,
1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%,
2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%,
2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%,
3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%,
4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%,
4-46%, 4-47%, 4-48%, 4-49%, 4-50% or 1-99% compared to level of
galactosylated glycans in an anti-VEGF protein produced using a soy
hydrolysate.
[0341] In one embodiment, the anti-VEGF protein can have an increased
level of mannosylated glycans by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%,
3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges
within one or more of the preceding values, e.g., 1-10%, 1-15%, 1-20%,
1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%,
1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%,
2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%,
2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%,
3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%,
4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%,
4-46%, 4-47%, 4-48%, 4-49%, 4-50% or 1-99% compared to level of
mannosylated glycans in an anti-VEGF protein produced using a soy
hydrolysate.
[0342] The compositions described in this section can be produced by
several upstream and downstream parameters as described below in sections
IV and V, respectively.
IV. Preparation of Compositions Using Upstream Process Technologies
[0343] For biologics, the implementation of a robust and flexible upstream
process is desirable. An efficient upstream process can lead to desirable
production and scale-up of a protein of interest. The inventors
discovered that the compositions of the invention comprising an anti-VEGF
protein can be produced by modulating conditions during upstream protein
production, such as changes in media components of a CDM. Each step in an
upstream process may affect quality, purity and quantity of the
manufactured protein.
[0344] The present disclosure provides evidence for the existence of
certain variants of aflibercept and/or MiniTrap produced using CDM. These
variants include isoforms that comprise one or more oxidized amino acid
residues. Examples of oxidized residues include, but are not limited to,
one or more histidine, tryptophan, methionine, phenylalanine or tyrosine
residues. The compositions produced by using the modified CDM can produce
a preparation of anti-VEGF protein with a desired target value of protein
variants of aflibercept and/or MiniTrap. As alluded to above, there can
also be a yellow-brownish color associated with fractions produced using
a CDM. (As mentioned above, not all CDMs tested by the inventors
manifested a distinct discoloration.)
[0345] This invention includes culturing a host cell in a modified CDM
under suitable conditions in which the cell expresses a recombinant
protein of interest followed by harvesting a preparation of the
recombinant protein of interest produced by the cell. Such a modified CDM
can be used to produce the compositions as described above in Section
III. (Note, the CDM is a medium that when aflibercept is expressed there
is a yellow-brown color.)
[0346] In one embodiment, the method comprises culturing a host cell in a
CDM under suitable conditions, wherein the host cell expresses a
recombinant protein of interest, such as aflibercept. The method further
comprises harvesting a preparation of the recombinant protein of interest
produced by the cell, wherein the suitable conditions include a CDM with
a: cumulative concentration of iron in said CDM that is less than about
55 cumulative concentration of copper in said CDM that is less than or
equal to about 0.8 cumulative concentration of nickel in said CDM that is
less than or equal to about 0.40 cumulative concentration of zinc in said
CDM that is less than or equal to about 56 cumulative concentration of
cysteine in said CDM that is less than about 10 mM; and/or an
anti-oxidant in said CDM in a concentration of about 0.001 mM to about 10
mM for a single antioxidant and no more than about 30 mM cumulative
concentration if multiple antioxidants are added in said CDM.
[0347] In one aspect of the present embodiment, the preparation obtained
from using suitable conditions results in a reduction in protein variants
of aflibercept and VEGF MiniTrap to a desired amount of protein variants
of aflibercept and VEGF MiniTrap (referred to as a "target value" of
protein variants of aflibercept and VEGF MiniTrap). In further aspect of
this embodiment, the preparation obtained from using suitable conditions
results in a reduction in color of the preparations to a desired BY value
(referred to as a "target BY value") when the preparation of protein,
including variants of aflibercept and VEGF MiniTrap are normalized to a
concentration of 5 g/L, 10 g/L or even higher.
[0348] In a further aspect of the present embodiment, the target BY value
and/or target value of variants can be obtained in a preparation where
the titer increases or does not significantly decrease (see Example 5).
[0349] In some embodiments, the compositions produced by using the
modified CDM can produce a preparation of anti-VEGF protein with a
desired target BY value, wherein the color of the preparation is
characterized as follows:
(i) no more yellow-brown than European Color Standard BY2; (ii) no more
yellow-brown than European Color Standard BY3; (iii) no more yellow-brown
than European Color Standard BY4; (iv) no more yellow-brown than European
Color Standard BY5; (v) between European Color Standard BY2 and BY3; (vi)
between European Color Standard BY3 and BY4; (vii) between European Color
Standard BY4 and BY5, wherein the composition comprises about 5 g/L or
about 10 g/L of the anti-VEGF protein and wherein a sample of the
composition can be obtained as a sample from a Protein A eluate of a
clarified harvest. As seen in Example 9, Table 9-3 below, the Protein A
eluate comprising 5 g/L aflibercept exhibited a yellow-brown color
measured as having a b* value of 1.77. Such a sample when produced
downstream following AEX had a b* value of 0.50 demonstrating the utility
of AEX to lower the yellow-brown coloration of a sample (Table 9-3).
[0350] The compositions produced by using the modified CDM can produce a
preparation of anti-VEGF protein, wherein the color of the preparation is
characterized by a recognized standard color characterization in the
CIELAB scale:
(i) no more yellow-brown than b* value of about 22-23; (ii) no more
yellow-brown than b* value of about 16-17; (iii) no more yellow-brown
than b* value of 9-10; (iv) no more yellow-brown than b* value of 4-5;
(v) no more yellow-brown than b* value of 2-3; (vi) between b* value of
17-23; (vii) between b* value of 10-17; (viii) between b* value of 5-10;
(ix) between b* value of 3-5; or (x) between b* value of 1-3, wherein the
composition comprises about 5 g/L or about 10 g/L of the anti-VEGF
protein and wherein the composition is obtained as a sample from a
Protein A eluate of a clarified harvest. See Example 9, Table 9-3.
[0351] For components added to the cell culture to form the modified CDM,
the term "cumulative amount" refers to the total amount of a particular
component added to a bioreactor over the course of the cell culture to
form the CDM, including amounts added at the beginning of the culture
(CDM at day 0) and subsequently added amounts of the component. Amounts
of a component added to a seed-train culture or inoculum prior to the
bioreactor production (i.e., prior to the CDM at day 0) are also included
when calculating the cumulative amount of the component. A cumulative
amount is unaffected by the loss of a component over time during the
culture (for example, through metabolism or chemical degradation). Thus,
two cultures with the same cumulative amounts of a component may
nonetheless have different absolute levels, for example, if the component
is added to the two cultures at different times (e.g., if in one culture
all of the component is added at the outset, and in another culture the
component is added over time). A cumulative amount is also unaffected by
in situ synthesis of a component over time during the culture (for
example, via metabolism or chemical conversion). Thus, two cultures with
the same cumulative amounts of a given component may nonetheless have
different absolute levels, for example, if the component is synthesized
in situ in one of the two cultures by way of a bioconversion process. A
cumulative amount may be expressed in units such as, for example, grams
or moles of the component. The term "cumulative concentration" refers to
the cumulative amount of a component divided by the volume of liquid in
the bioreactor at the beginning of the production batch, including the
contribution to the starting volume from any inoculum used in the
culture. For example, if a bioreactor contains 2 liters of cell culture
medium at the beginning of the production batch, and one gram of
component X is added at days 0, 1, 2, and 3, then the cumulative
concentration after day 3 is 2 g/L (i.e., 4 grams divided by 2 liters).
If, on day 4, an additional one liter of liquid not containing component
X were added to the bioreactor, the cumulative concentration would remain
2 g/L. If, on day 5, some quantity of liquid were lost from the
bioreactor (for example, through evaporation), the cumulative
concentration would remain 2 g/L. A cumulative concentration may be
expressed in units such as, for example, grams per liter or moles per
liter.
A. Amino Acids:
[0352] In some embodiments, a modified CDM can be obtained by decreasing
or increasing cumulative concentrations of amino acids in a CDM.
Non-limiting examples of such amino acids include alanine, arginine,
asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
proline, serine, threonine, tryptophan, tyrosine, and valine (or salts
thereof). The increase or decrease in the cumulative amount of these
amino acids in the modified CDM can be of about 1%, 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 100% as compared to the starting CDM, and ranges within one or more
of the preceding. Alternatively, the increase or decrease in the
cumulative amount of the one or more amino acids in the modified CDM can
be about 5 to about 20%, about 10 to about 30%, about 30% to about 40%,
about 30% to about 50%, about 40% to about 60%, about 60% to about 70%,
about 70% to about 80%, about 80% to about 90%, or about 90% to about
100% as compared to the unmodified CDM, and ranges within one or more of
the preceding (see FIGS. 25-27 and Example 5).
[0353] In some embodiments, the modified CDM can be obtained by decreasing
the cumulative concentration of cysteine in a CDM. The decrease in the
amount of the cysteine in the CDM to form the modified CDM can be about
1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 100% as compared to the unmodified CDM, and
ranges within one or more of the preceding. Alternatively, the decrease
in the cumulative amount of the cysteine in the modified CDM can be about
5 to about 20%, about 10-about 30%, about 30% to about 40%, about 30% to
about 50%, about 40% to about 60%, about 60% to about 70%, about 70% to
about 80%, about 80% to about 90%, or about 90% to about 100% as compared
to the CDM, and ranges within one or more of the preceding. In one
aspect, the amount of cumulative cysteine in modified CDM is less than
about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM,
about 7 mM, about 8 mM, about 9 mM or about 10 mM (see FIGS. 25-27 and
Example 5).
[0354] In some embodiments, the modified CDM can be obtained by replacing
at least a certain percentage of cumulative cysteine in a CDM with
cystine. The replacement can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% as
compared to the unmodified CDM, and ranges within one or more of the
preceding. Alternatively, the replacement can be about 5 to about 20%,
about 10% to about 30%, about 30% to about 40%, about 30% to about 50%,
about 40% to about 60%, about 60% to about 70%, about 70% to about 80%,
about 80% to about 90%, or about 90% to about 100% as compared to the
unmodified CDM, and ranges within one or more of the preceding (see FIGS.
25-27 and Example 5).
[0355] In some embodiments, the modified CDM can be obtained by replacing
at least a certain percentage of cumulative cysteine in a CDM with
cysteine sulfate. The replacement can be about 1%, 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 100% as compared to the unmodified CDM, and ranges within one or
more of the preceding. Alternatively, the replacement can be about 5 to
about 20%, about 10-about 30%, about 30% to about 40%, about 30% to about
50%, about 40% to about 60%, about 60% to about 70%, about 70% to about
80%, about 80% to about 90%, or about 90% to about 100% as compared to
the unmodified CDM, and ranges within one or more of the preceding.
B. Metals:
[0356] In some embodiments, the modified CDM can be obtained by decreasing
or increasing cumulative concentration of metals in a CDM. Non-limiting
examples of metals include iron, copper, manganese, molybdenum, zinc,
nickel, calcium, potassium and sodium. The increase or decrease in the
amount of the one or more metals in the modified CDM can be of about 1%,
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 100% as compared to the unmodified CDM, and ranges
within one or more of the preceding. Alternatively, the increase or
decrease in the cumulative amount of the one or more metals in the
modified CDM can be about 5 to about 20%, about 10 to about 30%, about
30% to about 40%, about 30% to about 50%, about 40% to about 60%, about
60% to about 70%, about 70% to about 80%, about 80% to about 90%, or
about 90% to about 100% as compared to the unmodified CDM, and ranges
within one or more of the preceding (see FIGS. 25-27 and Example 5).
C. Anti-Oxidants:
[0357] In some embodiments, the modified CDM comprises one or more
anti-oxidants. Non-limiting examples of anti-oxidants can include
taurine, hypotaurine, glycine, thioctic acid, glutathione, choline
chloride, hydrocortisone, Vitamin C, Vitamin E and combinations thereof
(see FIG. 28A-E and Example 5).
[0358] In some embodiments, the modified CDM comprises about 0.01 mM to
about 20 mM of taurine, i.e., 0.01 mM to about 1 mM, about 0.01 mM to
about 5 mM, about 0.01 mM to about 10 mM, 0.1 mM to about 1 mM, about 0.1
mM to about 5 mM, about 0.1 mM to about 10 mM, about 1 mM to about 5 mM,
about 1 mM to about 10 mM, and ranges within one or more of the
preceding.
[0359] In some embodiments, the modified CDM comprises about 0.01 mM to
about 20 mM of hypotaurine, i.e., 0.01 mM to about 1 mM, about 0.01 mM to
about 5 mM, about 0.01 mM to about 10 mM, 0.1 mM to about 1 mM, about 0.1
mM to about 5 mM, about 0.1 mM to about 10 mM, about 1 mM to about 5 mM,
about 1 mM to about 10 mM, and ranges within one or more of the
preceding.
[0360] In some embodiments, the modified CDM comprises about 0.01 mM to
about 20 mM of glycine, i.e., 0.01 mM to about 1 mM, about 0.01 mM to
about 5 mM, about 0.01 mM to about 10 mM, 0.1 mM to about 1 mM, about 0.1
mM to about 5 mM, about 0.1 mM to about 10 mM, about 1 mM to about 5 mM,
about 1 mM to about 10 mM, and ranges within one or more of the
preceding.
[0361] In some embodiments, the modified CDM comprises about 0.01 .mu.M to
about 5 .mu.M of thioctic acid, i.e., about 0.01 .mu.M to about 0.1
.mu.M, about 0.1 .mu.M to about 1 .mu.M, about 1 .mu.M to about 2.5
.mu.M, about 1 .mu.M to about 3 .mu.M, about 1 .mu.M to about 5 .mu.M,
and ranges within one or more of the preceding.
[0362] In some embodiments, the modified CDM comprises about 0.01 M to
about 5 mM of glutathione, i.e., 0.01 mM to about 1 mM, 0.1 mM to about 1
mM, about 0.1 mM to about 5 mM, about 1 mM to about 5 mM, and ranges
within one or more of the preceding.
[0363] In some embodiments, the modified CDM comprises about 0.01 .mu.M to
about 5 .mu.M of hydrocortisone, i.e., about 0.01 .mu.M to about 0.1
.mu.M, about 0.1 .mu.M to about 1 .mu.M, about 1 .mu.M to about 2.5
.mu.M, about 1 .mu.M to about 3 .mu.M, about 1 .mu.M to about 5 .mu.M,
and ranges within one or more of the preceding.
[0364] In some embodiments, the modified CDM comprises about 1 .mu.M to
about 50 .mu.M of vitamin C, i.e., about 1 .mu.M to about 5 .mu.M, about
5 .mu.M to about 20 .mu.M, about 10 .mu.M to about 30 .mu.M, about 5
.mu.M to about 30 .mu.M, about 20 .mu.M to about 50 .mu.M, about 25 .mu.M
to about 50 .mu.M, and ranges within one or more of the preceding.
D. Changes to the Media to Modulate Glycosylation:
[0365] This disclosure also includes methods of modulating glycosylation
of an anti-VEGF protein by varying cumulative concentrations of certain
components in a CDM. Based on the cumulative amounts of components added
to the CDM, the total % fucosylation, total % galactosylation, total %
sialylation and mannose-5 can be varied.
[0366] In exemplary embodiments, the method of modulating glycosylation of
an anti-VEGF protein can comprise supplementing the CDM with uridine. The
anti-VEGF protein can have about 40% to about 50% total fucosylated
glycans, about 30% to about 55% total sialylated glycans, about 2% to
about 15% mannose-5, and about 60% to about 79% galactosylated glycans.
(See Example 6 below).
[0367] In some embodiments, the method of modulating glycosylation of an
anti-VEGF protein can comprise supplementing a CDM with manganese. In one
aspect, the CDM is devoid of manganese before supplementation. The
anti-VEGF protein can have about 40% to about 50% total fucosylated
glycans, about 30% to about 55% total sialylated glycans, about 2% to
about 15% mannose-5, and about 60% to about 79% galactosylated glycans.
(See Example 6 below).
[0368] In some embodiments, the method of modulating glycosylation of an
anti-VEGF protein can comprise supplementing a CDM with galactose. In one
aspect, the CDM is devoid of galactose before supplementation. The
anti-VEGF protein can have about 40% to about 50% total fucosylated
glycans, about 30% to about 55% total sialylated glycans, about 2% to
about 15% mannose-5, and about 60% to about 79% galactosylated glycans.
(Example 6).
[0369] In some embodiments, the method of modulating glycosylation of an
anti-VEGF protein can comprise supplementing a CDM with dexamethasone. In
one aspect, the CDM is devoid of dexamethasone before supplementation.
The anti-VEGF protein can have about 40% to about 50% total fucosylated
glycans, about 30% to about 55% total sialylated glycans, about 2% to
about 15% mannose-5, and about 60% to about 79% galactosylated glycans.
(See Example 6 below).
[0370] In some embodiments, the method of modulating glycosylation of an
anti-VEGF protein can comprise supplementing a CDM with one or more of
uridine, manganese, galactose and dexamethasone. In one aspect, the CDM
is devoid of one or more of uridine, manganese, galactose and
dexamethasone before supplementation. The anti-VEGF protein can have
about 40% to about 50% total fucosylated glycans, about 30% to about 55%
total sialylated glycans, about 2% to about 15% mannose-5, and about 60%
to about 79% galactosylated glycans. (Example 6).
V. Preparation of Compositions Using Downstream Process Technologies
[0371] The compositions comprising an anti-VEGF protein of the invention
can be produced by modulating conditions during downstream protein
production. The inventors discovered that optimizing the downstream
procedures can lead to minimization of certain variants of the anti-VEGF
protein as well as discoloration. Optimization of the downstream process
may produce a composition with reduced oxo-variants as well as optimized
color characteristics.
[0372] The downstream process technologies may be used alone or in
combination with the upstream process technologies described in Section
IV, supra.
A. Anion-Exchange Chromatography:
[0373] In some embodiments, a composition of the invention can involve a
process comprising: expressing an anti-VEGF protein in a host cell in a
CDM, wherein the anti-VEGF protein is secreted from the host cell into
the medium and a clarified harvest is obtained. The harvest is subjected
to the following steps: (a) loading a biological sample obtained from the
harvest onto an anion-exchange chromatography (AEX) column; (b) washing
the AEX column with a suitable wash buffer, (c) collecting the
flowthrough fraction(s), optionally, (d) washing the column with a
suitable strip buffer and (e) collecting stripped fractions.
[0374] The flowthrough fractions can comprise oxo-variants of the
anti-VEGF protein which are about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the
anti-VEGF protein sample when compared to the oxo-variants in the
stripped fraction of the anion-exchange chromatography column. For
example, referring to Table 9-5 and Table 9-6, the flowthrough fractions
comprise oxidized variants of anti-VEGF protein where several histidine
and tryptophan residues are about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% (and ranges
within one or more of the preceding) oxidized when compared against the
oxidized variants in the stripped fractions.
[0375] The pH of both the equilibration and wash buffers for the AEX
column can be from about 8.20 to about 8.60. In another aspect, the
conductivity of both the equilibration and wash buffers for the AEX
column can be from about 1.50 to about 3.0 mS/cm. In one aspect, the
equilibration and wash buffers can be about 50 mM Tris hydrochloride. In
one aspect, the strip buffer comprises 2M sodium chloride or 1N sodium
hydroxide or both (see Table 2-2). Example 2 further illustrates
optimizing the concentration and conductivity of the equilibration and
wash buffers.
[0376] A protein variants can include modifications of one or more
residues as follows: one or more asparagines are deamidated; one or more
aspartic acids are converted to iso-aspartate and/or Asn; one or more
methionines are oxidized; one or more tryptophans are converted to
N-formylkynurenin; one or more tryptophans are mono-hydroxyl tryptophan;
one or more tryptophans are di-hydroxyl tryptophan; one or more
tryptophans are tri-hydroxyl tryptophan; one or more arginines are
converted to Arg 3-deoxyglucosone; the C-terminal glycine is not present;
and/or there are one or more non-glycosylated glycosites.
[0377] The protein of interest can be aflibercept, anti-VEGF antibody or a
VEGF MiniTrap. The protein variants can be formed by one or more of (i)
oxidation of histidines from the histidine residues selected from His86,
His110, His145, His209, His95, His19 and/or His203 (or equivalent residue
positions on proteins sharing certain structural characteristics of
aflibercept); (ii) oxidation of tryptophan residues selected from
tryptophan residues at Trp58 and/or Trp138 (or equivalent residue
positions on proteins sharing certain structural characteristics of
aflibercept); (iii) oxidation tyrosine residue at Tyr64 (or equivalent
positions on proteins sharing certain structural characteristics of
aflibercept); (iv) oxidation of phenylalanine residues selected from
Phe44 and/or Phe166 (or equivalent residue positions on proteins sharing
certain structural characteristics of aflibercept); and/or (v) oxidation
of methionine residues selected from Met10, Met 20, Met163 and/or Met192
(or equivalent residue positions on proteins sharing certain structural
characteristics of aflibercept).
[0378] The flowthrough fractions can comprise one or more of the
following:
(a) a percentage of histidine residues which have been oxidized to
2-oxo-histidine wherein their color characterization is as follows: (i)
no more yellow-brown than European Color Standard BY2; (ii) no more
yellow-brown than European Color Standard BY3; (iii) no more yellow-brown
than European Color Standard BY4; (iv) no more yellow-brown than European
Color Standard BY5; (v) between European Color Standard BY2 and BY3; (vi)
between European Color Standard BY3 and BY4; (vii) between European Color
Standard BY4 and BY5, wherein the composition comprises about 5 g/L or
about 10 g/L of the anti-VEGF protein, and wherein the composition is
obtained as a sample from the flowthrough fractions. (b) a percentage of
histidine residues which have been oxidized to 2-oxo-histidine. Further,
their color is characterized by having a yellow-brown color which
approximates that of BY2, BY3, BY4, BY5, BY6, BY7; or is no
darker/intense than BY2, no darker than BY3, no darker than BY4, no
darker than BY5, no darker than BY6, no darker than BY7; or is between
that of BY2 and BY3, between that of BY2 and BY4, between that of BY3 and
BY4 or between that of BY3 and BY5. (c) a percentage of histidine
residues which have been oxidized to 2-oxo-histidine wherein their color
is characterized by a color in the CIE L*, a*, b* color space as follows:
(i) no more yellow-brown than b* value of about 22-23; (ii) no more
yellow-brown than b* value of about 16-17; (iii) no more yellow-brown
than b* value of 9-10; (iv) no more yellow-brown than b* value of 4-5;
(v) no more yellow-brown than b* value of 2-3; (vi) between b* value of
17-23; (vii) between b* value of 10-17; (viii) between b* value of 5-10;
(ix) between b* value of 3-5; or (x) between b* value of 1-3, wherein the
composition comprises about 5 g/L or about 10 g/L of the anti-VEGF
protein and wherein the composition is obtained as a sample from the
flowthrough fractions. (d) no more than about 1%, no more than about 0.1%
or about 0.1-1%, 0.2-1%, 0.3-1%, 0.4-1%, 0.5-1%, 0.6-1%, 0.7-1%, 0.8-1%
or 0.9-1% of histidine residues in the composition are oxidized to
2-oxo-histidine. The percentage calculation is described in Section II.
B. Affinity Chromatography:
[0379] In some embodiments, compositions of the invention can be produced
using a process comprising: expressing an anti-VEGF protein in a host
cell wherein anti-VEGF protein is secreted from the host cell into the
medium and a clarified harvest is obtained. The harvest is subjected to
the following steps, comprising (a) loading a biological sample obtained
from the clarified harvest onto an affinity chromatography column,
wherein the affinity chromatography comprises a protein capable of
selectively or specifically binding to the anti-VEGF protein; (b) washing
the affinity chromatography column with a suitable elution buffer, and
(c) collecting the eluted fraction(s). For example, as exemplified in
Table 7-1 and Table 7-7 through 7-10, using VEGF.sub.165 as the protein
capable of selectively or specifically binding to the anti-VEGF protein
and collecting the eluted fractions as per the method above, led to a
successful production of MT5 (an anti-VEGF protein), aflibercept and an
anti-VEGF scFv fragment. Table 7-1 also discloses successful production
of MT5 using (i) mAb1 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID
NO.: 73 is a heavy chain and SEQ ID NO.: 74 is a light chain); (ii) mAb2
(a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 75 is a heavy chain
and SEQ ID NO.: 76 is a light chain); (iii) mAb3 (a mouse anti-VEGF-R1
mAb mouse IgG1 where SEQ ID NO.: 77 is a heavy chain and SEQ ID NO.: 78
is a light chain) and (iv) mAb4 (a mouse anti-VEGFR1 mAb mouse IgG1 where
SEQ ID NO.: 79 is a heavy chain and SEQ ID NO.: 80 is a light chain) as
different proteins capable of selectively or specifically binding to MT5.
[0380] With respect to step (a) above, the biological sample to be loaded
onto the affinity column can come from a sample in which the clarified
harvest can be subjected to production prior to affinity including, but
not limited to, ion exchange chromatography (either anion or cation).
Other chromatographic procedures well known to the skilled artisan can
also be employed prior to use of the affinity step. The important point
is that a biological sample comprising an anti-VEGF protein can be
subjected to affinity chromatography.
[0381] In some embodiments, compositions of the invention can be produced
using a process comprising: expressing a VEGF MiniTrap protein in a host
cell wherein the VEGF MiniTrap is secreted from the host cell into the
medium and wherein the medium can be further processed forming a
clarified harvest. This harvest can be further processed by known
chromatographic procedures yielding a biological sample comprising a VEGF
MiniTrap. This biological sample can be further processed by employing
the following steps, comprising (a) loading the biological sample onto an
affinity chromatography column, wherein the affinity chromatography
comprises a protein capable of selectively or specifically binding to or
interacting with the VEGF MiniTrap protein; (b) washing the affinity
chromatography column with a suitable elution buffer and (c) collecting
the eluted fraction(s). Referring again to Table 7-1, disclosed in this
Table is a successful production of MT5 (VEGF MiniTrap) using (i)
VEGF.sub.165; (ii) mAb1 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID
NO.: 73 is a heavy chain and SEQ ID NO.: 74 is a light chain); (iii) mAb2
(a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 75 is a heavy chain
and SEQ ID NO.: 76 is a light chain); (iv) mAb3 (a mouse anti-VEGF-R1 mAb
mouse IgG1 where SEQ ID NO.: 77 is a heavy chain and SEQ ID NO.: 78 is a
light chain) and (v) mAb4 (a mouse anti-VEGFR1 mAb mouse IgG1 where SEQ
ID NO.: 79 is a heavy chain and SEQ ID NO.: 80 is a light chain) as
different proteins capable of selectively or specifically binding to of
interacting with MT5.
[0382] In one embodiment, affinity chromatography can also be used to
isolate other MiniTrap proteins. Following cleavage of an aflibercept, a
sample comprising the cleaved aflibercept can be subjected to affinity
chromatography using a binder specific for the cleaved aflibercept. In
one aspect, the binder can be an antibody or portion thereof.
[0383] Cleaving of the aflibercept can be facilitated using proteolytic
digestion of aflibercept with, for example, IdeS protease (FabRICATOR) or
a variant thereof to generate the VEGF MiniTrap. Cleaving of the
aflibercept with IdeS protease or a variant thereof can produce a mixture
of products including a Fc fragment and the VEGF MiniTrap. The VEGF
MiniTrap can be further processed by using one or more of the production
strategies described herein.
[0384] In some exemplary embodiments, a protein capable of selectively or
specifically binding ("binder") to or interacting with an anti-VEGF
protein, such as aflibercept or MiniTrap, can originate from a human or a
mouse.
[0385] The affinity production process can further comprise equilibrating
an affinity column using an equilibration buffer before loading the
biological sample. Exemplary equilibration buffers can be 20 mM sodium
phosphate, pH 6-8 (esp. 7.2), 10 mM sodium phosphate, 500 mM NaCl, pH 6-8
(esp. 7.2), 50 mM Tris pH 7-8, DPBS pH 7.4.
[0386] The biological sample can be loaded using a suitable buffer, such
as, DPBS.
[0387] This affinity production process can further comprise washing an
affinity column with one or more wash buffers. The column can be washed
one or multiple times. Further, the washes can also be collected as wash
fractions. The pH of both the wash buffer can be from about 7.0 to about
8.60. In one aspect, the wash buffer can be DPBS. In another aspect, the
wash buffer can be 20 mM sodium phosphate, pH 6-8 (esp. 7.2), 10 mM
sodium phosphate, 500 mM NaCl, pH 6-8 (esp. 7.2), 50 mM Tris pH 7-8, or
DPBS pH 7.4.
[0388] This affinity process can further comprise washing an affinity
column with one or more suitable elution buffers and collecting the
eluted fractions. The column can be washed one or multiple times.
Non-limiting examples of such a suitable elution buffer includes:
ammonium acetate (pH of about 2.0 to about 3.0), acetic acid (pH of about
2.0 to about 3.2), glycine-HCl (pH of about 2.0 to about 3.0), sodium
citrate (pH of about 2.0 to about 3.0), citric acid (pH of about 2.0 to
about 3.0), potassium isothiocyanate (pH of about 2.0 to about 3.0), or
combinations thereof.
[0389] In some aspects, the eluted fractions can be neutralized using a
neutralizing buffer. An example of such a neutralizing buffer is Tris to
Tris-HCl (pH of about 7.0 to about 9.0).
C. IdeS Mutants:
[0390] The IdeS protease used for the cleavage of an Fc fusion protein
such as aflibercept will rapidly lose enzymatic activity under basic pH
conditions, which can limit its use during the manufacture of VEGF
MiniTrap. Thus, variants have been developed to be more stable at basic
pH, for example, in the presence of a strong base such as NaOH. Such
basic conditions can be 0.05N NaOH for 1 hr or 0.1N NaOH for 0.5 hr.
[0391] In some embodiments, IdeS mutants can have an amino acid sequence
comprising at least about 70% sequence identity over its full length to
the amino acid sequences set forth in the group consisting of SEQ ID NO.:
2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID
NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ
ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID
NO.: 16. In some aspects, the amino acid sequence has about 75%, 80%,
85%, 90%, 95% or about 100% sequence identity over its full length to the
amino acid sequences mentioned directly above.
[0392] In some embodiments, IdeS mutants can have an isolated nucleic acid
molecule encoding a polypeptide with an amino acid sequence comprising at
least 70% sequence identity over its full length to the amino acid
sequences as set forth in the group consisting of SEQ ID NO.: 2, SEQ ID
NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ
ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12,
SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16. In
some aspects, the amino acid sequence has about 75%, 80%, 85%, 90%, 95%
or about 100% sequence identity over its full length to the amino acid
sequences mentioned directly above.
[0393] In some embodiments, the polypeptide has an amino acid sequence
comprising at least 70% sequence identity over its full length to the
amino acid sequences as set forth in the group consisting of SEQ ID NO.:
2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID
NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ
ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID
NO.: 16 and be expressed by a host cell with a suitable vector comprising
nucleic acid coding for the identified peptides. In one aspect, the
nucleic acid molecule is operatively linked to an expression control
sequence capable of directing its expression in a host cell. In one
aspect, the vector can be a plasmid. In some aspects, the amino acid
sequence has about 75%, 80%, 85%, 90%, 95% or about 100% sequence
identity over its full length to the amino acid sequences mentioned
directly above. In some aspects, an isolated nucleic acid molecule can be
used to encode the polypeptide.
[0394] In some embodiments, IdeS mutants can have an amino acid sequence
comprising a parental amino acid sequence defined by SEQ ID NO.: 1 (IdeS)
with asparagine residue at position 87, 130, 182 and/or 274 mutated to an
amino acid other than asparagine. In one aspect, the mutation can confer
an increased chemical stability at alkaline pH-values compared to the
parental amino acid sequence. In another aspect, the mutation can confer
an increase in chemical stability by 50% at alkaline pH-values compared
to the parental amino acid sequence. In one aspect, the amino acid can be
selected from aspartic acid, leucine, and arginine. In a particular
aspect, the asparagine residue at position 87 is mutated to aspartic acid
residue. In another particular aspect, the asparagine residue at position
130 is mutated to arginine residue. In a yet another particular aspect,
the asparagine residue at position 182 is mutated to a leucine residue.
In a yet another particular aspect, the asparagine residue at position
274 is mutated to aspartic acid residue. In a yet another particular
aspect, the asparagine residue at position 87 and 130 are mutated. In a
yet another particular aspect, the asparagine residue at position 87 and
182 are mutated. In a yet another particular aspect, the asparagine
residue at position 87 and 274 are mutated. In a yet another particular
aspect, the asparagine residue at position 130 and 182 are mutated. In a
yet another particular aspect, the asparagine residue at position 130 and
274 are mutated. In a yet another particular aspect, the asparagine
residue at position 182 and 274 are mutated. In a yet another particular
aspect, the asparagine residue at position 87, 130 and 182 are mutated.
In a yet another particular aspect, the asparagine residue at position
87, 182 and 274 are mutated. In a yet another particular aspect, the
asparagine residue at position 130, 182 and 274 are mutated. In a yet
another particular aspect, the asparagine residue at position 87, 130,
182 and 274 are mutated. In some aspects, the amino acid sequence has
about 75%, 80%, 85%, 90%, 95% or about 100% sequence identity over its
full length to the amino acid sequences described above. In some aspects,
an isolated nucleic acid molecule can be used to encode the polypeptide.
[0395] Those of ordinary skill in the art familiar with standard molecular
biology techniques can without undue burden prepare and use IdeS mutants
of the present invention. Standard techniques can be used for recombinant
DNA, oligonucleotide synthesis, tissue culture, and transformation (e.g.,
electroporation, lipofection). See, for example, Sambrook et al.,
Molecular Cloning: A Laboratory Manual, supra, which is incorporated
herein by reference for any purpose. Enzymatic reactions and production
techniques can be performed according to manufacturer's specification or
as described herein.
VI. Protein Production Generally
[0396] A variety of different production techniques, including, but not
limited to, affinity, ion exchange, mixed mode, size exclusion, and
hydrophobic interaction chromatography, singularly or in combination, are
envisaged to be within the scope of the present invention. These
chromatographic steps separate mixtures of proteins of a biological
sample on the basis of their charge, degree of hydrophobicity, or size,
or a combination thereof, depending on the particular form of separation.
Several different chromatography resins are available for each of the
techniques alluded to supra, allowing accurate tailoring of the
production scheme to a particular protein involved. Each separation
method results in the protein traversing at different rates through a
column to achieve a physical separation that increases as they pass
further through the column or adhere selectively to a separation medium.
The proteins are then either (i) differentially eluted using an
appropriate elution buffers and/or (ii) collected from flowthrough
fractions obtained from the column used, optionally, from washing the
column with an appropriate equilibration buffer. In some cases, the
protein of interest is separated from impurities (HCPs, protein variants,
etc.) when the impurities preferentially adhere to the column and the
protein of interest less so, i.e., the protein of interest does not
adsorb to the solid phase of a particular column and thus flows through
the column. In some cases, the impurities are separated from the protein
of interest when they fail to adsorb to the column and thus flow through
the column.
[0397] The production process may begin at the separation step after the
recombinant protein has been produced using upstream production methods
described above and/or by alternative production methods conventional in
the art. Once a clarified solution or mixture comprising the protein of
interest, for example, a fusion protein, has been obtained, separation of
the protein of interest from process-related impurities (such as the
other proteins produced by the cell (like HCPs), as well as
product-related substances, such acidic or basic variants) is performed.
A combination of one or more different production techniques, including
affinity, ion exchange (e.g., CEX, AEX), mixed-mode (MM), and/or
hydrophobic interaction chromatography can be employed. Such production
steps separate mixtures of components within a biological sample on the
basis of their, for example, charge, degree of hydrophobicity, and/or
apparent size. Numerous chromatography resins are commercially available
for each of the chromatography techniques mentioned herein allowing
accurate tailoring of the production scheme to a particular protein
involved. Each of the separation methods allow proteins to either
traverse at different rates through a column achieving a physical
separation that increases as they pass further through the column or to
adsorb selectively to a separation resin (or medium). The proteins can
then be differentially collected. In some cases, the protein of interest
is separated from components of a biological sample when other components
specifically adsorb to a column's resin while the protein of interest
does not.
A. Primary Recovery and Virus Inactivation
[0398] In certain embodiments, the initial steps of the production methods
disclosed herein involve the clarification and primary recovery of a
protein of interest from a biological sample. The primary recovery will
include one or more centrifugation steps to separate the protein of
interest from a host cell and attendant cellular debris. Centrifugation
of the sample can be performed at, for example, but not by way of
limitation, 7,000.times.g to approximately 12,750.times.g. In the context
of large-scale production, such centrifugation can occur on-line with a
flow rate set to achieve, for example, a turbidity level of 150 NTU in
the resulting supernatant. Such supernatant can then be collected for
further processing or in-line filtered through one or more depth filters
for further clarification of the sample.
[0399] In certain embodiments, the primary recovery may include the use of
one or more depth filtration steps to clarify the sample and, thereby,
aid in processing the protein of interest. In other embodiments, the
primary recovery may include the use of one or more depth filtration
steps post centrifugation. Non-limiting examples of depth filters that
can be used in the context of the instant invention include the
Millistak+ X0HC, F0HC, D0HC, A1HC, B1HC depth filters (EMD Millipore),
3M.TM. model 30/60ZA, 60/90 ZA, VR05, VR07, delipid depth filters (3M
Corp.). A 0.2 .mu.m filter such as Sartorius's 0.45/0.2 .mu.m
Sartopore.TM. bi-layer or Millipore's Express SHR or SHC filter
cartridges typically follows the depth filters. Other filters well known
to the skilled artisan can also be used.
[0400] In certain embodiments, the primary recovery process can also be a
point to reduce or inactivate viruses that can be present in a biological
sample. Any one or more of a variety of methods of viral
reduction/inactivation can be used during the primary recovery phase of
production including heat inactivation (pasteurization), pH inactivation,
buffer/detergent treatment, UV and .gamma.-ray irradiation and the
addition of certain chemical inactivating agents such as
.beta.-propiolactone or, for example, copper phenanthroline as described
in U.S. Pat. No. 4,534,972, the entire teaching of which is incorporated
herein by reference. In certain exemplary embodiments of the present
invention, the sample is exposed to detergent viral inactivation during
the primary recovery phase. In other embodiments, the sample may be
exposed to low pH inactivation during the primary recovery phase.
[0401] In those embodiments where viral reduction/inactivation is
employed, a biological sample can be adjusted, as needed, for further
production steps. For example, following low pH viral inactivation, the
pH of the sample is typically adjusted to a more neutral pH, for example,
from about 4.5 to about 8.5, prior to continuing the production process.
Additionally, the mixture may be diluted with water for injection (WFI)
to obtain a desired conductivity.
B. Affinity Chromatography
[0402] In certain exemplary embodiments, it may be advantageous to subject
a biological sample to affinity chromatography for production of a
protein of interest. The chromatographic material is capable of
selectively or specifically binding to or interacting with the protein of
interest. Non-limiting examples of such chromatographic material include:
Protein A and Protein G. Also, chromatographic material comprising, for
example, a protein or portion thereof capable of binding to or
interacting with the protein of interest. In one aspect, the protein of
interest is an anti-VEGF protein such as aflibercept, MiniTrap or a
protein related thereto.
[0403] Affinity chromatography can involve subjecting a biological sample
to a column comprising a suitable Protein A resin. When used herein, the
term "Protein A" encompasses Protein A recovered from a native source
thereof, Protein A produced synthetically (e.g., by peptide synthesis or
by recombinant techniques), and variants thereof which retain the ability
to bind proteins which have a C.sub.H2/C.sub.H3 region. In certain
aspects, Protein A resin is useful for affinity-based production and
isolation of a variety of antibody isotypes by interacting specifically
with the Fc portion of a molecule should it possess that region.
[0404] There are several commercial sources for Protein A resin. One
suitable resin is MabSelect.TM. from GE Healthcare. Suitable resins
include, but not limited to, Mab Select SuRe.TM., Mab Select SuRe LX, Mab
Select, Mab Select SuRe pcc, Mab Select Xtra, rProtein A Sepharose from
GE Healthcare, ProSep HC, ProSep Ultra, and ProSep Ultra Plus from EMD
Millipore, MapCapture from Life Technologies. A non-limiting example of a
suitable column packed with MabSelect.TM. is an about 1.0 cm diameter x
about 21.6 cm long column (17 mL bed volume). A suitable column may
comprise a resin such as MabSelect.TM. SuRe or an analogous resin.
Protein A can also be purchased commercially from Repligen, Pharmacia and
Fermatech.
[0405] An affinity column can be equilibrated with a suitable buffer prior
to sample loading. Following loading of the column, the column can be
washed one or multiple times using a suitable wash buffer. The column can
then be eluted using an appropriate elution buffer, for example,
glycine-HCL, acetic acid, or citric acid. The eluate can be monitored
using techniques well known to those skilled in the art such as a UV
detector. The eluated fractions of interest can be collected and then
prepared for further processing.
[0406] In one aspect, the eluate may be subjected to viral inactivation,
for example, either by detergent or low pH. A suitable detergent
concentration or pH (and time) can be selected to obtain a desired viral
inactivation result. After viral inactivation, the eluate is usually pH
and/or conductivity adjusted for subsequent production steps.
[0407] The eluate may be subjected to filtration through a depth filter to
remove turbidity and/or various impurities from the protein of interest
prior to additional chromatographic polishing steps. Examples of suitable
depth filters include, but are not limited to, Millistak+ XOHC, FOHC,
DOHC, AIHC, X0SP, and BIHC Pod filters (EMD Millipore), or Zeta Plus
30ZA/60ZA, 60ZA/90ZA, delipid, VR07, and VR05 filters (3M). The Emphaze
AEX Hybrid Purifier multi-mechanism filter may also be used to clarify
the eluate. The eluate pool may need to be adjusted to a particular pH
and conductivity in order to obtain desired impurity removal and product
recovery from the depth filtration step.
C. Anion Exchange Chromatography
[0408] In certain embodiments, a protein of interest is produced by
subjecting a biological sample to at least one anion exchange separation
step. In one scenario, the anion exchange step can occur following an
affinity chromatography procedure (e.g., Protein A affinity). In other
scenarios, the anion exchange step can occur before the affinity
chromatography step. In yet other protocols, anion exchange can occur
both before and after an affinity chromatography step. In one aspect, the
protein of interest is either aflibercept or MiniTrap.
[0409] The use of an anionic exchange material versus a cationic exchange
material is based, in part, on the local charges of the protein of
interest. Anion exchange chromatography can be used in combination with
other chromatographic procedures such as affinity chromatography, size
exclusion chromatography, hydrophobic interaction chromatography as well
as other modes of chromatography known to the skilled artisan.
[0410] In performing a separation, the initial protein composition
(biological sample) can be placed in contact with an anion exchange
material by using any of a variety of techniques, for example, using a
batch production technique or a chromatographic technique.
[0411] In the context of batch production, anion exchange material is
prepared in, or equilibrated to, a desired starting buffer. Upon
preparation, a slurry of the anion exchange material is obtained. The
biological sample is contacted with the slurry to allow for protein
adsorption to the anion exchange material. A solution comprising acidic
species that do not bind to the AEX material is separated from the slurry
by allowing the slurry to settle and removing the supernatant. The slurry
can be subjected to one or more washing steps and/or elution steps.
[0412] In the context of chromatographic separation, a chromatographic
column is used to house chromatographic support material (resin or solid
phase). A sample comprising a protein of interest is loaded onto a
particular chromatographic column. The column can then be subjected to
one or more wash steps using a suitable wash buffer. Components of a
sample that have not adsorbed onto the resin will likely flow through the
column. Components that have adsorbed to the resin can be differentially
eluted using an appropriate elution buffer.
[0413] A wash step is typically performed in AEX chromatography using
conditions similar to the load conditions or alternatively by decreasing
the pH and/or increasing the ionic strength/conductivity of the wash in a
step wise or linear gradient manner. In one aspect, the aqueous salt
solution used in both the loading and wash buffer has a pH that is at or
near the isoelectric point (pI) of the protein of interest. Typically,
the pH is about 0 to 2 units higher or lower than the pI of the protein
of interest, however it may be in the range of 0 to 0.5 units higher or
lower. It may also be at the pI of the protein of interest.
[0414] The anionic agent may be selected from the group consisting of
acetate, chloride, formate and combinations thereof. The cationic agent
may be selected from the group consisting of Tris, arginine, sodium and
combinations thereof. In a particular example, the buffer solution is a
Tris/formate buffer. The buffer may be selected from the group consisting
of pyridine, piperazine, L-histidine, Bis-tris, Bis-Tris propane,
imidazole, N-ethylmorpholine, TEA (triethanolamine), Tris, morpholine,
N-methyldiethanolamine, AMPD (2-amino-2-methyl-1,3-propanediol),
diethanolamine, ethanolamine, AMP (2-amino-2-methyl-1-propaol),
piperazine, 1,3-diaminopropane and piperidine.
[0415] A packed anion-exchange chromatography column, anion-exchange
membrane device, anion-exchange monolithic device, or depth filter media
can be operated either in bind-elute mode, flowthrough mode, or a hybrid
mode wherein proteins exhibit binding to the chromatographic material and
yet can be washed from such material using a buffer that is the same or
substantially similar to the loading buffer.
[0416] In the bind-elute mode, a column or membrane device is first
conditioned with a buffer with appropriate ionic strength and pH under
conditions where certain proteins will adsorb to the resin-based matrix.
For example, during the feed load, a protein of interest can be adsorbed
to the resin due to electrostatic attraction. After washing the column or
the membrane device with the equilibration buffer or another buffer with
a different pH and/or conductivity, the product recovery is achieved by
increasing the ionic strength (i.e., conductivity) of the elution buffer
to compete with the solute for the charged sites of the anion exchange
matrix. Changing the pH and thereby altering the charge of the solute is
another way to achieve elution of the solute. The change in conductivity
or pH may be gradual (gradient elution) or stepwise (step elution).
[0417] In the flowthrough mode, a column or membrane device is operated at
a selected pH and conductivity such that the protein of interest does not
bind to the resin or the membrane while the acidic species will either be
retained on the column or will have a distinct elution profile as
compared to the protein of interest. In the context of this strategy,
acidic species will interact with or bind to the chromatographic material
under suitable conditions while the protein of interest and certain
aggregates and/or fragments of the protein of interest will flowthrough
the column.
[0418] Non-limiting examples of anionic exchange resins include
diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary
amine (Q) groups. Additional non-limiting examples include: Poros 50PI
and Poros 50HQ, which are a rigid polymeric bead with a backbone
consisting of cross-linked poly[styrene-divinylbenzene]; Capto Q Impres
and Capto DEAE, which are a high flow agarose bead; Toyopearl QAE-550,
Toyopearl DEAE-650, and Toyopearl GigaCap Q-650, which are a polymeric
base bead; Fractogel.RTM. EMD TMAE Hicap, which is a synthetic polymeric
resin with a tentacle ion exchanger; Sartobind STIC.RTM. PA nano, which
is a salt-tolerant chromatographic membrane with a primary amine ligand;
Sartobind Q nano; which is a strong anion exchange chromatographic
membrane; CUNO BioCap; which is a zeta-plus depth filter media
constructed from inorganic filter aids, refined cellulose, and an ion
exchange resin; and XOHC, which is a depth-filter media constructed from
inorganic filter aid, cellulose, and mixed cellulose esters.
[0419] In certain embodiments, the protein load of a sample may be
adjusted to a total protein load to the column of between about 50 g/L
and about 500 g/L, or between about 75 g/L and about 350 g/L, or between
about 200 g/L and about 300 g/L. In other embodiments, the protein
concentration of the load protein mixture is adjusted to a protein
concentration of the material loaded to the column of about 0.5 g/L and
about 50 g/L, between about 1 g/L and about 20 g/L, or between about 3
g/L and about 10 g/L. In yet other embodiments, the protein concentration
of the load protein mixture is adjusted to a protein centration of the
material to the column of about 37 g/L.
[0420] Additives such as polyethylene glycol (PEG), detergents, amino
acids, sugars, chaotropic agents can be added to enhance the performance
of the separation to achieve better separation, recovery and/or product
quality.
[0421] In certain embodiments, including those relating to aflibercept
and/or VEGF MiniTrap, the methods of the instant invention can be used to
selectively remove, significantly reduce, or essentially remove at least
10% of protein variants, thereby producing protein compositions that have
reduced protein variants.
[0422] The protein variants can include modifications of one or more
residues as follows: one or more asparagines are deamidated; one or more
aspartic acids are converted aspartate-glycine and/or Asn-Gly; one or
more methionines are oxidized; one or more tryptophans are converted to
N-formylkynurenin; one or more tryptophans are mono-hydroxyl tryptophan;
one or more tryptophans are di-hydroxyl tryptophan; one or more
tryptophans are tri-hydroxyl tryptophan; one or more arginines are
converted to Arg 3-deoxyglucosone; the C-terminal glycine is not present;
and/or there are one or more non-glycosylated glycosites. The use of AEX
was also observed to reduce oxidized and acidic species of anti-VEGF
variants in said affinity eluate. Compared to the affinity eluate,
following use of AEX, the flowthrough fraction may show a reduction of at
least about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,
8%, 7%, 6%, or 5% in oxidized and/or acidic species of anti-VEGF
variants.
[0423] Protein variants of aflibercept and/or VEGF MiniTrap can include
one or more of (i) oxidated histidines from the histidine residues
selected from His86, His110, His145, His209, His95, His19 and/or His203;
(ii) oxidated tryptophan residues selected from tryptophan residues at
Trp58 and/or Trp138; (iii) oxidated tyrosine residue at Tyr64; (iv)
oxidated phenylalanine residues selected from Phe44 and/or Phe166; and/or
(v) oxidated methionine residues selected from Met10, Met 20, Met163
and/or Met192.
D. Cation Exchange Chromatography
[0424] The compositions of the present invention can be produced by
subjecting a biological sample comprising a protein of interest to at
least one cation exchange (CEX) step. In certain exemplary embodiments,
the CEX step will be in addition to an AEX step and occur either before
or after the AEX step. In one aspect, the protein of interest is either
aflibercept, MiniTrap or a molecule related thereto.
[0425] The use of a cationic exchange material versus an anionic exchange
material, such as those anionic exchange materials discussed supra, is
based, in part, on the local charges of the protein of interest in a
given solution and the separation conditions desired. It is within the
scope of this invention to employ a cationic exchange step prior to the
use of an anionic exchange step, or an anionic exchange step prior to the
use of a cationic exchange step. Furthermore, it is within the scope of
this invention to employ only a cationic exchange step in combination
with other chromatography procedures.
[0426] In performing cation exchange, a sample comprising a protein of
interest can be contacted with a cation exchange material by using any of
a variety of techniques, for example, using a batch production technique
or a chromatographic technique, as described above for AEX.
[0427] An aqueous salt solution may be used as both a loading and wash
buffer having a pH that is lower than the isoelectric point (pI) of the
protein of interest. In one aspect, the pH is about 0 to 5 units lower
than the pI of the protein. In another aspect, it is in the range of 1 to
2 units lower than the pI of the protein. In yet another aspect, it is in
the range of 1 to 1.5 units lower than the pI of the protein.
[0428] In certain embodiments, the concentration of the anionic agent in
aqueous salt solution is increased or decreased to achieve a pH of
between about 3.5 and about 10.5, or between about 4 and about 10, or
between about 4.5 and about 9.5, or between about 5 and about 9, or
between about 5.5 and about 8.5, or between about 6 and about 8, or
between about 6.5 and about 7.5. In one aspect, the concentration of
anionic agent is increased or decreased in the aqueous salt solution in
order to achieve a pH of 5, or 5.5, or 6, or 6.5, or 6.8, or 7.5. Buffer
systems suitable for use in the CEX methods include, but are not limited
to, Tris formate, Tris acetate, ammonium sulfate, sodium chloride, and
sodium sulfate.
[0429] In certain embodiments, the conductivity and pH of the aqueous salt
solution is adjusted by increasing or decreasing the concentration of a
cationic agent. In one aspect, the cationic agent is maintained at a
concentration ranging from about 20 mM to about 500 mM, about 50 mM to
about 350 mM, about 100 mM to about 300 mM, or about 100 mM to about 200
mM. Non-limiting examples of the cationic agent is selected from the
group consisting of sodium, Tris, triethylamine, ammonium, arginine, and
combinations thereof.
[0430] A packed cation-exchange chromatography column or a cation-exchange
membrane device can be operated either in bind-elute mode, flowthrough
mode, or a hybrid mode wherein the product exhibits binding to or
interacting with a chromatographic material yet can be washed from such
material using a buffer that is the same or substantially similar to the
loading buffer (details of these modes are outlined above).
[0431] Cationic substituents include carboxymethyl (CM), sulfoethyl (SE),
sulfopropyl (SP), phosphate (P) and sulfonate (S). Additional cationic
materials include, but are not limited to: Capto SP ImpRes, which is a
high flow agarose bead; CM Hyper D grade F; which is a ceramic bead
coated and permeated with a functionalized hydrogel, 250-400 ionic groups
.mu.eq/mL; Eshmuno S, which is a hydrophilic polyvinyl ether base matrix
with 50-100 .mu.eq/mL ionic capacity; Nuvia C Prime, which is a
hydrophobic cation exchange media composed of a macroporous highly
crosslinked hydrophilic polymer matrix 55-75 .mu..epsilon./mL; Nuvia S,
which has a UNOsphere base matrix with 90-150 .mu..epsilon./mL ionic
groups; Poros HS; which is a rigid polymeric bead with a backbone
consisting of cross-linked poly[styrene-divinylbenzene]; Poros XS; which
is a rigid polymetic bead with a backbone consisting of cross-linked
poly[styrene divinyl-benzene]; Toyo Pearl Giga Cap CM 650M, which is a
polymeric base bead with 0.225 meq/mL ionic capacity; Toyo Pearl Giga Cap
S 650M which is a polymeric base bead; Toyo Pearl MX TRP, which is a
polymeric base bead. It is noted that CEX chromatography can be used with
MM resins, described herein.
[0432] The protein load of a sample comprising a protein of interest is
adjusted to a total protein load to the column of between about 5 g/L and
about 150 g/L, or between about 10 g/L and about 100 g/L, between about
20 g/L and about 80 g/L, between about 30 g/L and about 50 g/L, or
between about 40 g/L and about 50 g/L. In certain embodiments, the
protein concentration of the load protein mixture is adjusted to a
protein concentration of the material to be loaded onto the column of
about 0.5 g/L and about 50 g/L, or between about 1 g/L and about 20 g/L.
[0433] Additives such as polyethylene glycol, detergents, amino acids,
sugars, chaotropic agents can be added to enhance the performance of the
separation so as to achieve better separation, recovery and/or product
quality.
[0434] In certain embodiments, including those relating to aflibercept or
anti-VEGF antibody or VEGF MiniTrap, the methods of the instant invention
can be used to selectively remove, significantly reduce, or essentially
remove all of the oxo-variants in a sample where the protein of interest
will essentially be in the flowthrough of a CEX procedure while the
oxo-variants will be substantially captured by the column media.
E. Mixed Mode Chromatography
[0435] Mixed mode ("MM") chromatography may also be used to prepare the
compositions of the invention. MM chromatography, also referred to herein
as "multimodal chromatography", is a chromatographic strategy that
utilizes a support comprising a ligand that is capable of providing at
least two different interactions with an analyte or protein of interest
from a sample. One of these sites provides an attractive type of
charge-charge interaction between the ligand and the protein of interest
and the other site provides for electron acceptor-donor interaction
and/or hydrophobic and/or hydrophilic interactions. Electron
donor-acceptor interactions include interactions such as
hydrogen-bonding, .pi.-.pi., cation-.pi., charge transfer, dipole-dipole,
induced dipole etc.
[0436] The column resin employed for a mixed mode separation can be Capto
Adhere. Capto Adhere is a strong anion exchanger with multimodal
functionality. Its base matrix is a highly cross-linked agarose with a
ligand (N-benzyl-N-methyl ethanol amine) that exhibits different
functionalities for interaction, such as ionic interaction, hydrogen
bonding and hydrophobic interaction. In certain aspects, the resin
employed for a mixed mode separation is selected from PPA-HyperCel and
HEA-HyperCel. The base matrices of PPA-HyperCel and HEA-HyperCel are high
porosity cross-linked cellulose. Their ligands are phenylpropylamine and
hexylamine, respectively. Phenylpropylamine and hexylamine offer
different selectivity and hydrophobicity options for protein separations.
Additional mixed mode chromatographic supports include, but are not
limited to, Nuvia C Prime, Toyo Pearl MX Trp 650M, and Eshmuno.RTM. HCX.
In certain aspects, the mixed mode chromatography resin is comprised of
ligands coupled to an organic or inorganic support, sometimes denoted a
base matrix, directly or via a spacer. The support may be in the form of
particles, such as essentially spherical particles, a monolith, filter,
membrane, surface, capillaries, and the like. In certain aspects, the
support is prepared from a native polymer, such as cross-linked
carbohydrate material, such as agarose, agar, cellulose, dextran,
chitosan, konjac, carrageenan, gellan, alginate and the like. To obtain
high adsorption capacities, the support can be porous, and ligands are
then coupled to the external surfaces as well as to the pore surfaces.
Such native polymer supports can be prepared according to standard
methods, such as inverse suspension gelation (S Hjerten: Biochim Biophys
Acta 79(2), 393-398 (1964), the entire teaching of which is incorporated
herein by reference). Alternatively, the support can be prepared from a
synthetic polymer, such as cross-linked synthetic polymers, for example,
styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate
esters, methacrylate esters, vinyl esters, vinyl amides, and the like.
Such synthetic polymers can be produced according to standard methods,
see "Styrene based polymer supports developed by suspension
polymerization" (R Arshady: Chimica e L'Industria 70(9), 70-75 (1988),
the entire teaching of which is incorporated herein by reference). Porous
native or synthetic polymer supports are also available from commercial
sources, such as GE Healthcare, Uppsala, Sweden.
[0437] The protein load of a biological sample mixture comprising a
protein of interest can be adjusted to a total protein load to the column
of between about 25 g/L and about 750 g/L, or between about 75 g/L and
about 500 g/L, or between about 100 g/L and about 300 g/L. In certain
exemplary embodiments, the protein concentration of the load protein
mixture is adjusted to a protein concentration of the material loaded to
the column of about 1 g/L and about 50 g/L, or between about 9 g/L and
about 25 g/L.
[0438] Additives such as polyethylene glycol, detergents, amino acids,
sugars, chaotropic agents can be added to enhance the performance of the
separation, so as to achieve better separation, recovery and/or product
quality.
[0439] In certain embodiments, including those relating to aflibercept
and/or MiniTrap, the methods of the instant invention can be used to
selectively remove, significantly reduce, or essentially remove all of
the PTMs, including oxo-variants.
[0440] The methods for producing the composition of the invention can also
be implemented in a continuous chromatography mode. In this mode, at
least two columns are employed (referred to as a "first" column and a
"second" column). In certain embodiments, this continuous chromatography
mode can be performed such that the eluted fractions and/or stripped
fractions comprising PTMs, for example, oxo-variants, can then be loaded
subsequently or concurrently onto the second column (with or without
dilution).
[0441] In one embodiment, the media choice for continuous mode can be one
of many chromatographic resins with pendant hydrophobic and anion
exchange functional groups, monolithic media, membrane adsorbent media or
depth filtration media.
F. Hydrophobic Interaction Chromatography
[0442] The compositions of the invention may also be prepared using
hydrophobic interaction chromatography (HIC).
[0443] In performing the separation, a biological sample is contacted with
a HIC material, for example, using a batch production technique or using
a column or membrane chromatography. Prior to HIC processing it may be
desirable to adjust the concentration of the salt buffer to achieve
desired protein binding/interaction to the resin or the membrane.
[0444] Whereas ion exchange chromatography relies on the local charge of
the protein of interest for selective separation, hydrophobic interaction
chromatography exploits the hydrophobic properties of proteins to achieve
selective separation. Hydrophobic groups on or within a protein interact
with hydrophobic groups of chromatography resin or a membrane. Typically,
under suitable conditions, the more hydrophobic a protein is (or portions
of a protein) the stronger it will interact with the column or the
membrane. Thus, under suitable conditions, HIC can be used to facilitate
the separation of process-related impurities (e.g., HCPs) as well as
product-related substances (e.g., aggregates and fragments) from a
protein of interest in a sample.
[0445] Like ion exchange chromatography, a HIC column or a HIC membrane
device can also be operated in an elution mode, a flowthrough, or a
hybrid mode wherein the product exhibits binding to or interacting with a
chromatographic material yet can be washed from such material using a
buffer that is the same or substantially similar to the loading buffer.
(The details of these modes are outlined above in connection with AEX
processing.) As hydrophobic interactions are strongest at high ionic
strength, this form of separation is conveniently performed following a
salt elution step such as those typically used in connection with ion
exchange chromatography. Alternatively, salts can be added to a sample
before employing a HIC step. Adsorption of a protein to a HIC column is
favored by high salt concentrations, but the actual concentrations can
vary over a wide range depending on the nature of the protein of
interest, salt type and the particular HIC ligand chosen. Various ions
can be arranged in a so-called soluphobic series depending on whether
they promote hydrophobic interactions (salting-out effects) or disrupt
the structure of water (chaotropic effect) and lead to the weakening of
the hydrophobic interaction. Cations are ranked in terms of increasing
salting out effect as Ba.sup.2+; Ca.sup.2+; Mg.sup.2+; Li.sup.+;
Cs.sup.+; Na.sup.+; K.sup.+; Rb.sup.+; NH4.sup.+, while anions may be
ranked in terms of increasing chaotropic effect as PO.sub.4.sup.3-;
SO.sub.4.sup.2-; CH.sub.3CO.sub.3.sup.-; CI.sup.-; Br.sup.-;
NO.sub.3.sup.-; ClO.sub.4.sup.-; I.sup.-; SCN.sup.-.
[0446] In general, Na.sup.+, K.sup.+ or NH.sub.4.sup.+ sulfates
effectively promote ligand-protein interaction using HIC. Salts may be
formulated that influence the strength of the interaction as given by the
following relationship:
(NH.sub.4).sub.2SO.sub.4>Na.sub.2SO.sub.4>NaCl>NH.sub.4Cl>NaB-
r>NaSCN. In general, salt concentrations of between about 0.75 M and
about 2 M ammonium sulfate or between about 1 M and about 4 M NaCl are
useful.
[0447] HIC media normally comprise a base matrix (e.g., cross-linked
agarose or synthetic copolymer material) to which hydrophobic ligands
(e.g., alkyl or aryl groups) are coupled. A suitable HIC media comprises
an agarose resin or a membrane functionalized with phenyl groups (e.g., a
Phenyl Sepharose.TM. from GE Healthcare or a Phenyl Membrane from
Sartorius). Many HIC resins are available commercially. Examples include,
but are not limited to, Capto Phenyl, Phenyl Sepharose.TM. 6 Fast Flow
with low or high substitution, Phenyl Sepharose.TM. High Performance,
Octyl Sepharose.TM. High Performance (GE Healthcare); Fractogel.TM. EMD
Propyl or Fractogel.TM. EMD Phenyl (E. Merck, Germany); Macro-Prep.TM.
Methyl or Macro-Prep.TM. t-Butyl columns (Bio-Rad, California); WP
HI-Propyl (C3).TM. (J. T. Baker, New Jersey); and Toyopearl.TM. ether,
phenyl or butyl (TosoHaas, PA); ToyoScreen PPG; ToyoScreen Phenyl;
ToyoScreen Butyl; ToyoScreen Hexyl; GE HiScreen and Butyl FF HiScreen
Octyl FF.
[0448] The protein load of a sample comprising a protein of interest is
adjusted to a total protein load to the column of between about 50 g/L to
about 1000 g/L; about 5 g/L and about 150 g/L, between about 10 g/L and
about 100 g/L, between about 20 g/L and about 80 g/L, between about 30
g/L and about 50 g/L, or between about 40 g/L and about 50 g/L. In
certain embodiments, the protein concentration of the load protein
mixture is adjusted to a protein concentration of the material to be
loaded onto the column of about 0.5 g/L and about 50 g/L, or between
about 1 g/L and about 20 g/L.
[0449] Because the pH selected for any particular production process must
be compatible with protein stability and activity, thus particular pH
conditions may be specific for each application. However, because at pH
5.0-8.5, particular pH values have very little significance on the final
selectivity and resolution of a HIC separation, such conditions may be
favored. An increase in pH weakens hydrophobic interactions and retention
of proteins changes more drastically at pH values above 8.5 or below 5.0.
In addition, changes in ionic strength, the presence of organic solvents,
temperature and pH (especially at the isoelectric point, pI, when there
is no net surface charge) can impact protein structure and solubility
and, consequently, the interaction with other hydrophobic surfaces, such
as those in HIC media and hence, in certain embodiments, the present
invention incorporates production strategies wherein one or more of the
foregoing are adjusted to achieve the desired reduction in
process-related impurities and/or product-related substances.
[0450] In certain embodiments, spectroscopy methods such as UV, NIR, FTIR,
Fluorescence, Raman may be used to monitor the protein of interest and
impurities in an on-line, at-line or in-line mode, which can then be used
to control the level of aggregates in the pooled material collected from
the HIC adsorbent effluent. In certain embodiments, on-line, at-line or
in-line monitoring methods can be used either on the effluent line of the
chromatography step or in the collection vessel, to enable achievement of
the desired product quality/recovery. In certain embodiments, the UV
signal can be used as a surrogate to achieve an appropriate product
quality/recovery, wherein the UV signal can be processed appropriately,
including, but not limited to, such processing techniques as integration,
differentiation, moving average, such that normal process variability can
be addressed and the target product quality can be achieved. In certain
embodiments, such measurements can be combined with in-line dilution
methods such that ion concentration/conductivity of the load/wash can be
controlled by feedback and hence facilitate product quality control.
G. Size Exclusion Chromatography
[0451] Size exclusion chromatography or gel filtration relies on the
separation of components as a function of their molecular size.
Separation depends on the amount of time that the substances spend in the
porous stationary phase as compared to time in the fluid. The probability
that a molecule will reside in a pore depends on the size of the molecule
and the pore. In addition, the ability of a substance to permeate into
pores is determined by the diffusion mobility of macromolecules which is
higher for small macromolecules. Very large macromolecules may not
penetrate the pores of the stationary phase at all; and, for very small
macromolecules the probability of penetration is close to unity. While
components of larger molecular size move more quickly past the stationary
phase, components of small molecular size have a longer path length
through the pores of the stationary phase and are thus retained longer in
the stationary, phase.
[0452] The chromatographic material can comprise a size exclusion material
wherein the size exclusion material is a resin or membrane. The matrix
used for size exclusion is preferably an inert gel medium which can be a
composite of cross-linked polysaccharides; for example, cross-linked
agarose and/or dextran in the form of spherical beads. The degree of
cross-linking determines the size of pores that are present in the
swollen gel beads. Molecules greater than a certain size do not enter the
gel beads and thus move through the chromatographic bed the fastest.
Smaller molecules, such as detergent, protein, DNA and the like, which
enter the gel beads to varying extent depending on their size and shape,
are retarded in their passage through the bed. Molecules are thus
generally eluted in the order of decreasing molecular size.
[0453] Porous chromatographic resins appropriate for size-exclusion.
chromatography of viruses may be made of dextrose, agarose,
polyacrylamide, or silica which have different physical characteristics.
Polymer combinations can also be also used. Most commonly used are those
under the tradename, "SEPHADEX" available from Amersham Biosciences.
Other size exclusion supports from different materials of construction
are also appropriate, for example Toyopearl 55F (polymethacrylate, from
Tosoh Bioscience, Montgomery Pa.) and Bio-Gel P-30 Fine (BioRad
Laboratories, Hercules, Calif.).
[0454] The protein load of a sample comprising a protein of interest can
be adjusted to a total protein load to the column of between about 50 g/L
to about 1000 g/L; about 5 g/L and about 150 g/L, between about 10 g/L
and about 100 g/L, between about 20 g/L and about 80 g/L, between about
30 g/L and about 50 g/L, or between about 40 g/L and about 50 g/L. In
certain embodiments, the protein concentration of the load protein
mixture is adjusted to a protein concentration of the material to be
loaded onto the column of about 0.5 g/L and about 50 g/L, or between
about 1 g/L and about 20 g/L.
H. Viral Filtration
[0455] Viral filtration is a dedicated viral reduction step in a
production process. This step is usually performed post chromatographic
polishing. Viral reduction can be achieved via the use of suitable
filters including, but not limited to, Planova 20N.TM., 50 N or BioEx
from Asahi Kasei Pharma, Viresolve.TM. filters from EMD Millipore,
ViroSart CPV from Sartorius, or Ultipor DV20 or DV50.TM. filter from Pall
Corporation. It will be apparent to one of ordinary skill in the art to
select a suitable filter to obtain desired filtration performance.
I. Ultrafiltration/Diafiltration
[0456] Certain embodiments of the present invention employ ultrafiltration
and diafiltration to further concentrate and formulate a protein of
interest. Ultrafiltration is described in detail in: Microfiltration and
Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney
(Marcel Dekker, Inc., New York, N.Y., 1996); and in: Ultrafiltration
Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No.
87762-456-9); the entire teachings of which are incorporated herein by
reference. One filtration process is Tangential Flow Filtration as
described in the Millipore catalogue entitled "Pharmaceutical Process
Filtration Catalogue" pp. 177-202 (Bedford, Mass., 1995/96), the entire
teaching of which is incorporated herein by reference. Ultrafiltration is
generally considered to mean filtration using filters with a pore size of
smaller than 0.1 .mu.m. By employing filters having such a small pore
size, the volume of sample can be reduced through permeation of the
sample buffer through the filter membrane pores while proteins are
retained above the membrane surface.
[0457] One of ordinary skill in the art can select an appropriate membrane
filter device for the UF/DF operation. Examples of membrane cassettes
suitable for the present invention include, but not limited to, Pellicon
2 or Pellicon 3 cassettes with 10 kD, 30 kD or 50 kD membranes from EMD
Millipore, Kvick 10 kD, 30 kD or 50 kD membrane cassettes from GE
Healthcare, and Centramate or Centrasette 10 kD, 30 kD or 50 kD cassettes
from Pall Corporation.
J. Exemplary Production Strategies
[0458] Primary recovery can proceed by sequentially employing pH
reduction, centrifugation, and filtration to remove cells and cellular
debris (including HCPs) from a production bioreactor harvest. The present
invention is directed to subjecting a biological sample comprising a
protein of interest from the primary recovery to one or more production
steps, including (in no particular order) AEX, CEX, SEC, HIC and/or MM.
Certain aspects of the present invention include further processing
steps. Examples of additional processing procedures include ethanol
precipitation, isoelectric focusing, reverse phase HPLC, chromatography
on silica, chromatography on heparin Sepharose.TM., further anion
exchange chromatography and/or further cation exchange chromatography,
chromatofocusing, SDS-PAGE, ammonium sulfate precipitation,
hydroxyapatite chromatography, gel electrophoresis, dialysis, and
affinity chromatography (e.g., using Protein A or G, an antibody, a
specific substrate, ligand or antigen as the capture reagent). In certain
aspects, the column temperature (as well as other parameters) can be
independently varied to improve the separation efficiency and/or yield of
any particular production step.
[0459] In certain embodiments, unbound flowthrough and wash fractions can
be further fractionated and a combination of fractions providing a target
product purity can be pooled.
[0460] Column loading and washing steps can be controlled by in-line,
at-line or off-line measurement of the product related impurity/substance
levels, either in the column effluent, or the collected pool or both, so
as to achieve a particular target product quality and/or yield. In
certain embodiments, the loading concentration can be dynamically
controlled by in-line or batch or continuous dilutions with buffers or
other solutions to achieve the partitioning necessary to improve the
separation efficiency and/or yield.
[0461] Examples of such production procedures are depicted in FIGS. 5-8.
[0462] FIG. 5 represents one exemplary embodiment used for the production
of aflibercept. Referring to FIG. 5, the method comprises: (a) expressing
aflibercept in a host cell cultured in a CDM; (b) capturing aflibercept
using a first chromatography support, which can include affinity capture
resin; and (c) contacting at least a portion of aflibercept with a second
chromatography support, which can include anion-exchange chromatography.
Step (c) can further comprise washing an AEX column and collecting
flowthrough fraction(s) of a sample comprising aflibercept. Optionally,
step (c) can comprise stripping the second chromatographic support and
collecting stripped fractions. The steps can be carried out by routine
methodology in conjunction with methodology mentioned supra. It should be
understood that one skilled in the art might opt to employ CEX rather
than or in addition to AEX. In no particular order, additional
chromatographic steps may be employed as well including, but not limited
to, HIC and SEC.
[0463] In addition to the exemplary embodiment in FIG. 5, other additional
exemplary embodiments can include (d) contacting at least a portion of
said aflibercept of step (c) with a third chromatography support. In one
aspect, the protocol can include (e) contacting at least a portion
aflibercept of step (d) with a fourth chromatography support. In one
aspect of this embodiment, the protocol can optionally comprise
subjecting the sample comprising aflibercept of step (c) to a pH less
than 5.5. In one aspect, the present method comprises a clarification
step prior to a step (a).
[0464] FIG. 6 represents one exemplary embodiment used for the production
of VEGF MiniTrap. This method comprises: (a) expressing aflibercept in a
host cell cultured in a CDM; (b) capturing aflibercept using a first
chromatography support which can include affinity chromatography resin;
(c) cleaving the aflibercept thereby removing the Fc domain and forming a
sample comprising VEGF MiniTrap; (d) contacting the sample of step (c)
with a second chromatographic support which can be affinity
chromatography and (e) contacting the flowthrough of step (d) to a third
chromatography support which can include an anion-exchange
chromatography. Step (d) comprises the collection of flowthrough
fraction(s) where due to the absence of an Fc domain, the MiniTrap should
reside while the aflibercept or any other protein having an Fc domain
should essentially interact with the affinity column of step (d).
Optionally, step (d) can comprise stripping the third chromatographic
support and collecting stripped fractions. The steps can be carried out
by routine methodology in conjunction with methodology outlined above. In
no particular order, additional chromatographic steps can be included,
but not limited to, such as HIC and SEC.
[0465] FIG. 7 represents one exemplary embodiment for the production of
aflibercept. This method comprises: (a) expressing aflibercept in a host
cell cultured in a CDM; (b) capturing aflibercept using a first
chromatography support, which can include cation exchange chromatography;
and (c) contacting a flowthrough of step (b) to a second chromatography
support which can include an anion-exchange chromatography. Optionally,
step (c) can comprise stripping the second chromatographic support and
collecting stripped fractions. The steps can be carried out by routine
methodology in conjunction with protocols alluded to above. In no
particular order, other chromatographic procedures may be employed
including, but not limited to, HIC and SEC.
[0466] FIG. 8 represents one exemplary embodiment for producing VEGF
MiniTrap. This method comprises: (a) expressing aflibercept in a host
cell cultured in a CDM; (b) capturing aflibercept using a first
chromatography support which can include an ion exchange chromatography;
(c) subjecting a flowthrough fraction of (b) comprising aflibercept to
affinity chromatography; eluting, wherein the elution comprises
aflibercept; (d) subjecting the aflibercept of (c) to a cleavage
activity, whereby the Fc domain is cleaved thus forming VEGF MiniTrap. In
one aspect, the ion exchange of step (b) comprises AEX. Alternatively,
step (b) may comprise CEX. In no particular order, additional
chromatographic steps may be included such as further ion exchange
chromatography steps following step (d), the addition of HIC and/or SEC.
VII. Pharmaceutical Formulations Comprising the Compositions
[0467] The invention also discloses formulations comprising anti-VEGF
compositions (as described above). Suitable formulations for anti-VEGF
proteins include, but are not limited to, formulations described in U.S.
Pat. Nos. 7,608,261, 7,807,164, 8,092,803, 8,481,046, 8,802,107,
9,340,594, 9,914,763, 9,580,489, 10,400,025, 8,110,546, 8,404,638,
8,710,004, 8,921,316, 9,416,167, 9,511,140, 9,636,400, and 10,406,226,
which are all incorporated herein by reference in their entirety.
[0468] The upstream process technologies (described in Section IV, supra),
downstream process technologies (described in Section V, supra) may be
used alone or in combination with each other to effect formulation
production.
[0469] The present invention discloses formulations comprising anti-VEGF
compositions in association with one or more ingredients/excipients as
well as methods of use thereof and methods of making such compositions.
In an embodiment of the invention, a pharmaceutical formulation of the
present invention has a pH of approximately 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,
6.1 or 6.2.
[0470] To prepare pharmaceutical formulations for anti-VEGF compositions,
an anti-VEGF composition is admixed with a pharmaceutically acceptable
carrier or excipient. See, for example, Remington's Pharmaceutical
Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing
Company, Easton, Pa. (1984); Hardman, et al. (2001) Goodman and Gilman's
The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;
Gennaro (2000) Remington: The Science and Practice of Pharmacy,
Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)
(1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel
Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:
Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)
Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, N.Y.;
Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker,
Inc., New York, N.Y.; the entire teachings of which are incorporated
herein by reference. In an embodiment of the invention, the
pharmaceutical formulation is sterile.
[0471] Pharmaceutical formulations of the present invention include an
anti-VEGF composition and a pharmaceutically acceptable carrier
including, for example, water, buffering agents, preservatives and/or
detergents.
[0472] The present invention provides a pharmaceutical formulation
comprising any of the anti-VEGF compositions set forth herein and a
pharmaceutically acceptable carrier, for example, wherein the
concentration of polypeptide is about 40 mg/mL, about 60 mg/mL, about 80
mg/mL, 90 mg/mL, about 100 mg/mL, about 110 mg/mL, about 120 mg/mL, about
130 mg/mL, about 140 mg/mL, about 150 mg/mL, about 200 mg/mL or about 250
mg/mL.
[0473] The scope of the present invention includes desiccated, for
example, freeze-dried, compositions comprising an anti-VEGF protein and a
pharmaceutically acceptable carrier substantially (about 85% to about 99%
or greater) lacking water.
[0474] In one embodiment, a further therapeutic agent that is administered
to a subject in association with an anti-VEGF composition disclosed
herein is administered to the subject in accordance with the Physicians'
Desk Reference 2003 (Thomson Healthcare; 57th edition (Nov. 1, 2002), the
teaching of which is incorporated herein by reference).
[0475] The present invention provides a vessel (e.g., a plastic or glass
vial with a cap or a chromatography column, hollow bore needle or a
syringe cylinder) comprising any of the anti-VEGF compositions or a
pharmaceutical formulation comprising a pharmaceutically acceptable
carrier described herein. The present invention also provides an
injection device comprising the anti-VEGF composition or formulation set
forth herein, for example, a syringe, a pre-filled syringe or an
autoinjector. In one aspect, the vessel is tinted (e.g., brown) to block
out light, natural or otherwise.
[0476] The present invention includes combinations including anti-VEGF
compositions in association with one or more further therapeutic agents.
The anti-VEGF composition and the further therapeutic agent can be in a
single composition or in separate compositions. For example, the
therapeutic agent is an Ang-2 inhibitor (e.g., nesvacumab), a Tie-2
receptor activator, an anti-PDGF antibody or antigen-binding fragment
thereof, an anti-PDGF receptor or PDGF receptor beta antibody or
antigen-binding fragment thereof and/or an additional VEGF antagonist
such as aflibercept, conbercept, bevacizumab, ranibizumab, an anti-VEGF
aptamer such as pegaptanib (e.g., pegaptanib sodium), a single chain
(e.g., VL-VH) anti-VEGF antibody such as brolucizumab, an anti-VEGF
DARPin such as the Abicipar Pegol DARPin, a bispecific anti-VEGF
antibody, for example, which also binds to ANG2, such as RG7716, or a
soluble form of human vascular endothelial growth factor receptor-3
(VEGFR-3) comprising extracellular domains 1-3, expressed as an Fc-fusion
protein.
VIII. Methods of Treatment
[0477] The present invention provides methods for treating or preventing a
cancer (e.g., whose growth and/or metastasis is mediated, at least in
part, by VEGF, for example, VEGF-mediated angiogenesis) or an angiogenic
eye disorder, in a subject, comprising administering a therapeutically
effective amount of compositions as disclosed herein (Section III supra).
[0478] Upstream process technologies (Section IV supra), downstream
process technologies (Sections V and VI supra) may be used alone or in
combination with the each other to produce the compositions as described
in Section III and/or the formulations as described in Section VII which
can be used for treating or preventing a variety of disorders including
ophthalmological and oncological disease.
[0479] The present invention also provides a method for administering
compositions set forth herein (Section III and Section VII) to a subject
(e.g., a human) comprising introducing the compositions with about 0.5
mg, 2 mg, 4 mg, 6 mg, 8 mg, 10 mg, 12 mg, 14 mg, 16 mg, 18 mg or 20 mg of
the protein of interest (e.g., aflibercept or MiniTrap) in no more than
about 100 .mu.L, for example, about 50 .mu.L, about 70.mu..L or about 100
.mu.L, and optionally a further therapeutic agent, into the body of the
subject by, for example, intraocular injection such as by intravitreal
injection.
[0480] The present invention provides a method for treating cancer whose
growth and/or metastasis is mediated, at least in part, by VEGF, for
example, VEGF-mediated angiogenesis or an angiogenic eye disorder in a
subject in need thereof, the method comprising administering a
therapeutically effective amount of the compositions set forth herein
(Section III and Section VII above), for example, 2 mg, 4 mg, 6 mg, 8 mg
or 10 mg of the protein of interest, in no more than about 100 and
optionally a further therapeutic agent, to a subject. In one embodiment
of the invention, administration is done by intravitreal injection.
Non-limiting examples of angiogenic eye disorders that are treatable or
preventable using the methods herein, include: [0481] age-related
macular degeneration (e.g., wet or dry), [0482] macular edema, [0483]
macular edema following retinal vein occlusion, [0484] retinal vein
occlusion (RVO), [0485] central retinal vein occlusion (CRVO), [0486]
branch retinal vein occlusion (BRVO), [0487] diabetic macular edema
(DME), [0488] choroidal neovascularization (CNV), [0489] iris
neovascularization, [0490] neovascular glaucoma, [0491] post-surgical
fibrosis in glaucoma, [0492] proliferative vitreoretinopathy (PVR),
[0493] optic disc neovascularization, [0494] corneal neovascularization,
[0495] retinal neovascularization, [0496] vitreal neovascularization,
[0497] pannus, [0498] pterygium, [0499] vascular retinopathy, [0500]
diabetic retinopathy in a subject with diabetic macular edema; and [0501]
diabetic retinopathies (e.g., non-proliferative diabetic retinopathy
(e.g., characterized by a Diabetic Retinopathy Severity Scale (DRSS)
level of about 47 or 53) or proliferative diabetic retinopathy; e.g., in
a subject that does not suffer from DME).
[0502] The mode of administration of such compositions or formulations
(Section III and Section VII) can vary and determined by a skilled
practitioner. Routes of administration include parenteral,
non-parenteral, oral, rectal, transmucosal, intestinal, parenteral;
intramuscular, subcutaneous, intradermal, intramedullary, intrathecal,
direct intraventricular, intravenous, intraperitoneal, intranasal,
intraocular, inhalation, insufflation, topical, cutaneous, intraocular,
intravitreal, transdermal or intra-arterial.
[0503] In one embodiment of the invention, intravitreal injection of a
pharmaceutical formulation of the present invention (which includes a
compositions or formulations of the present invention) includes the step
of piercing the eye with a syringe and needle (e.g., 30-gauge injection
needle) comprising the formulation and injecting the formulation (e.g.,
less than or equal to about 100 microliters; about 40, 50, 55, 56, 57,
57.1, 58, 60 or 70 microliters) into the vitreous of the eye with a
sufficient volume as to deliver a therapeutically effective amount as set
forth herein, for example, of about 2, 4, 6, 8.0, 8.1, 8.2, 8.3, 8.4,
8.5, 8.6, 8.7, 8.8 or 8.9, 10 or 20 mg of the protein of interest.
Optionally, the method includes the steps of administering a local
anesthetic (e.g., proparacaine, lidocaine or tetracaine), an antibiotic
(e.g., a fluoroquinolone), antiseptic (e.g., povidone-iodine) and/or a
pupil dilating agent to the eye being injected. In one aspect, a sterile
field around the eye to be injected is established before the injection.
Following intravitreal injection, the subject is monitored for elevations
in intraocular pressure, inflammation and/or blood pressure.
[0504] An effective or therapeutically effective amount of protein of
interest for an angiogenic eye disorder refers to the amount of the
protein of interest sufficient to cause the regression, stabilization or
elimination of the cancer or angiogenic eye disorder, for example, by
regressing, stabilizing or eliminating one or more symptoms or indicia of
the cancer or angiogenic eye disorder by any clinically measurable
degree, for example, with regard to an angiogenic eye disorder, by
causing a reduction in or maintenance of diabetic retinopathy severity
score (DRSS), by improving or maintaining vision (e.g., in best corrected
visual acuity as measured by an increase in ETDRS letters), increasing or
maintaining visual field and/or reducing or maintaining central retinal
thickness and, with respect to cancer, stopping or reversing the growth,
survival and/or metastasis of cancer cells in the subject.
[0505] In one embodiment of the invention, an effective or therapeutically
effective amount of a protein of interest such as aflibercept for
treating or preventing an angiogenic eye disorder is about 0.5 mg, 2 mg,
3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 7.25 mg, 7.7 mg, 7.9 mg, 8.0 mg, 8.1 mg,
8.2 mg, 8.3 mg, 8.4 mg, 8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9 mg, 10
mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg or 20
mg, e.g., in no more than about 100 .mu.L. The amount may vary depending
upon the age and the size of a subject to be administered, target
disease, conditions, route of administration, and the like. In certain
exemplary embodiments, the initial dose may be followed by administration
of a second or a plurality of subsequent doses of the protein of interest
in an amount that can be approximately the same or less or more than that
of the initial dose, wherein the subsequent doses are separated by at
least 1 day to 3 days; at least one week, at least 2 weeks; at least 3
weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7
weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12
weeks; or at least 14 weeks.
[0506] It is to be noted that dosage values may vary with the type and
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage regimens
should be adjusted over time according to the individual need and the
professional judgment of the person administering or supervising the
administration of the compositions, and that dosage ranges set forth
herein are exemplary only and are not intended to limit the scope or
practice of the claimed composition.
IX. Method of Assaying Protein Variants
[0507] The levels of protein variants in a chromatographic sample produced
using the techniques described herein may be analyzed as described in the
Examples below. In certain embodiments, a cIEF method is employed using
an iCE3 analyzer (ProteinSimple) with a fluorocarbon coated capillary
cartridge (100 .mu.m.times.5 cm). The ampholyte solution consists of a
mixture of 0.35% methyl cellulose (MC), 4% Pharmalyte 3-10 carrier
ampholytes, 4% Pharmalyte 5-8 carrier ampholytes, 10 mM L-Arginine HCl,
24% formamide, and pI markers 5.12 and 9.77 in purified water. The
anolyte was 80 mM phosphoric acid, and the catholyte was 100 mM sodium
hydroxide, both in 0.10% methylcellulose. Samples were diluted in
purified water to 10 mg/mL. Samples were mixed with the ampholyte
solution and then focused by introducing a potential of 1500 V for one
minute, followed by a potential of 3000 V for 7 minutes. An image of the
focused variants was obtained by passing 280 nm ultraviolet light through
the capillary and into the lens of a charge coupled device digital
camera. This image was then analyzed to determine the distribution of the
various charge variants. Persons of skill in the art may vary the precise
parameters while still achieving the desired outcome.
[0508] Various publications, including patents, patent applications,
published patent applications, accession numbers, technical articles and
scholarly articles are cited throughout the specification. Each of these
cited references is incorporated by reference, in its entirety.
[0509] The present invention will be more fully understood by reference to
the following Examples. They should not, however, be construed as
limiting the scope of the invention.
EXAMPLES
[0510] The MiniTraps (MT) 1-6 discussed in the Examples are as follows:
MT1: VEGF MiniTrap obtained by cleavage of aflibercept produced using
CDM1. MT2: VEGF MiniTrap obtained by cleavage of aflibercept produced
using CDM2. MT3: VEGF MiniTrap obtained by cleavage of aflibercept
produced using CDM3. MT4: VEGF MiniTrap obtained by cleavage of
aflibercept produced using soy hydrolysate. MT5: recombinant VEGF
MiniTrap (dimer). MT6: recombinant VEGF MiniTrap (scFv). Characterization
of MT1, MT5 and MT6 are described below in Example 8.
Color Assessment of Samples
[0511] The spectrophotometric assay method of measuring the b* value
(CIELAB) was found suitable for performing color assessment.
[0512] The absorbance of a 1 mL protein sample was quantified over the
visible light spectrum (380 to 780 nm) and the absorbance curve was
transformed into the CIELAB color space using a set of matrix operations.
The instrument can process approximately 6 samples an hour. The high
throughput format of the assay used a CLARIOstar plate reader (BMG
Labtech). Up to 96 samples can be analyzed using a 96-well plate
requiring 0.3 mL of sample.
[0513] To convert the BY standards into the b* values, BY reference
standards (BY1 to BY7) were quantified using the high throughput assay
format.
[0514] The solutions were prepared as per the BY standards discussed
above. The b* value for each of the standards are as shown in FIG. 9.
This method provided a faster assay with a smaller sample requirement and
shorter run times as shown in Table 3 below. For all the samples
evaluated using this method, the protein concentration of the test
samples was standardized to either 5 g/L or 10 g/L.
TABLE-US-00012
TABLE 3
Original High-throughput
Amount/Sample 1 mL 0.3 mL
Measurement Format cuvette (individual) 96-well-plate (bulk)
Run Time 6 samples per hour 96 samples per 5 minutes
Data Entry manual automated
Data Storage Excel LIMS
Example 1: Production of a Protein Using a Chemically Defined Medium
1.1 Cell Source and Harvest
[0515] An aflibercept producing cell line was employed in the present
study. Aflibercept producing cell lines were cultured and harvested using
chemically defined media (CDM).
1.2 Proteolytic Cleavage of Aflibercept
[0516] A column with an immobilized IdeS enzyme (FabRICATOR.RTM. obtained
from Genovis (Cambridge, Mass.)) was used to generate MT1. Aflibercept
obtained from a cell culture harvest (20 mg in 1.0 mL cleavage buffer)
was added to the column and incubated for 30 min at 18.degree. C. After
30 min, the column was washed with the cleavage buffer (1.0 mL). The
digestion mixture and washing solutions were combined. The mixture was
loaded onto and eluted from an analytical Protein A affinity column
(Applied Biosystems.TM., POROS.TM. 20 .mu.M Protein A Cartridge
2.1.times.30 mm, 0.1 mL (Cat #2-1001-00). The processing was carried out
according to Applied Biosystems'.TM. protocol for POROS.TM. 20 .mu.M
Protein A Cartridge 2.1.times.30 mm, 0.1 mL (Cat #2-1001-00). The column
height was 20.+-.1.0 cm, residence time was 15 minutes and
equilibration/wash was performed using 40 mM Tris, 54 mM Acetate pH
7.0.+-.0.1.
Example 2. Anion Exchange Chromatography (AEX) for Color Minimization
(A) AEX was Employed to Reduce Color Formation
[0517] AEX chromatography was performed to remove the coloration obtained
during production of aflibercept expressed using CDM1.
2.1 Design
[0518] Five AEX separations were performed for this study as detailed in
Table 2-1 with the AEX protocol as described in Table 2-2. A 15.7 mL Q
Sepharose Fast Flow column (19.5 cm bed height, 1.0 cm I.D.) and a 14.1
mL POROS 50 HQ column (18.0 cm bed height, 1.0 cm I.D.) were integrated
into an AKTA Avant benchtop liquid chromatography controller.
[0519] AEX load pH was adjusted to target .+-.0.05 pH units using 2 M Tris
base or 2 M acetic acid. AEX load conductivity was adjusted to target
.+-.0.1 mS/cm using 5 M sodium chloride or deionized water. All pool
samples were analyzed for high molecular weight (HMW), color and yield.
TABLE-US-00013
TABLE 2-1
Summary of the Study Design for AEX Color Reduction
AEX Separation Condition Evaluated Resin
1 pH 8.30-8.50, 1.90-2.10 mS/cm POROS 50 HQ
2 pH 7.90-8.10, 2.40-2.60 mS/cm Q Sepharose FF
3 pH 7.90-8.10, 2.40-2.60 mS/cm POROS 50 HQ
4 pH 7.70-7.90, 3.90-4.10 mS/cm Q Sepharose FF
5 pH 7.70-7.90, 3.90-4.10 mS/cm POROS 50 HQ
TABLE-US-00014
TABLE 2-2
AEX Protocol for Color Reduction
Column Linear
Vol- Vel-
umes ocity
Step Description Mobile Phase (CVs) (cm/h)
1 Pre- 2M Sodium Chloride (NaCl) 2 200
Equilibration
2 Equilibration 50 mM Tris, Variable mM 2 200
NaCl Variable pH and
Conductivity
3 Load AEX Load 40 g/L- 200
Variable pH and Conductivity resin
4 FT/Wash 50 mM Tris, Variable mM 2 200
NaCl Variable pH and
Conductivity
5 Strip 1 2M Sodium Chloride (NaCl) 2 200
6 Strip 2 1N Sodium Hydroxide (NaOH) 2 200
2.2 Results
[0520] Employing AEX separations for production exhibited a significant
reduction in color. (Table 2-3). For example, as seen in Table 2-3, the
color observed in the flowthrough (FT) and wash in AEX separation 1 (pH
8.30-8.50, 1.90-2.10 mS/cm) had a b* value of 1.05, as compared to the
color of the Load for AEX ("AEX Load") with a b* value of 3.06. The
increase in b* value reflects the intensity of yellow-brown coloration of
a sample.
[0521] Five AEX separations were performed to evaluate the impact of resin
(Q Sepharose FF or POROS 50 HQ) and pH and conductivity setpoint (pH 8.40
and 2.00 mS/cm, pH 8.00 and 2.50 mS/cm, or pH 7.80 and 4.00 mS/cm) on
color reduction. For POROS 50 HQ, yields (64.4, 81.9, and 91.4%) and pool
HMW levels (1.02, 1.29, and 1.83%) increased as the setpoint was changed
to a lower pH and higher conductivity. Color (b* values) also increased
(1.05, 1.33, and 1.55) as the setpoint was changed to a lower pH and
higher conductivity. The higher pH levels and lower conductivities
provided the most reduction in color over the AEX separation for POROS 50
HQ.
[0522] For Q Sepharose Fast Flow, yields (49.5 and 77.7%) and pool HMW
levels (0.59 and 1.25%) also increased as the setpoint was changed to a
lower pH and higher conductivity. Color (b* values) also increased (0.96
and 1.35) as the setpoint was changed to a lower pH and higher
conductivity.
[0523] The use of AEX reduces yellow-brown coloration--see Table 2-3.
Additionally, it was determined that Q Sepharose Fast Flow reduced color
more than POROS 50 HQ for the two set points evaluated on both resins. At
pH 8.00 and 2.50 mS/cm setpoint, POROS 50 HQ pool had a b* value of 1.33
while Q Sepharose Fast Flow pool had a b* value of 0.96. Similarly, at pH
7.80 and 4.00 mS/cm setpoint, POROS 50 HQ pool had a b* value of 1.55
while Q Sepharose Fast Flow pool had a b* value of 1.35 (Table 2-3).
TABLE-US-00015
TABLE 2-3
Summary of Experimental Results
of the AEX Color Reduction Study
AEX Yield HMW Color Color Color
Separation Fraction (%) (%) (L*) (a*) (b*)
1 FT/wash 64.4 1.02 98.89 0.01 1.05
2 FT/wash 49.5 0.59 98.30 -0.03 0.96
3 FT/wash 81.9 1.29 99.07 -0.07 1.33
4 FT/wash 77.7 1.25 99.42 -0.04 1.35
5 FT/wash 91.4 1.83 99.19 -0.09 1.55
-- filtered pool -- 3.66-3.98 98.73 -0.21 3.06
(AEX Load)
AEX, anion exchange chromatography;
HMW, high molecular weight species;
N/A, not applicable
The fractions were adjusted to a protein concentration of 10 g/L for color
measurements.
2.3 Conclusion
[0524] Use of AEX was found to reduce the yellow-brown coloration, see
Table 2-3. Referring to Table 2-3, the AEX Load has a b* value of 3.06,
but when subjected to AEX chromatography (AEX Separation 1-5), the b*
value decreases indicating a decrease in yellow-brown coloration. Again,
as the b* value decreases so does the coloration; as the b* value
increases it is reflective of the yellow-brown color increasing in a
given sample.
[0525] Color reduction was evaluated using two AEX resins (POROS 50 HQ and
Q Sepharose Fast Flow) and three set points (pH 8.40 and 2.00 mS/cm, pH
8.00 and 2.50 mS/cm, and pH 7.80 and 4.00 mS/cm). For both resins, color
reduction was higher for the higher pH and lower conductivity set points.
In addition, Q Sepharose Fast Flow provided more color reduction than
POROS 50 HQ at the two set points evaluated on both resins (pH 8.00 and
2.50 mS/cm and pH 7.80 and 4.00 mS/cm). However, all the five AEX
separation methods led to a significant color reduction when compared to
the loading solution for AEX ("AEX Load"), demonstrating the importance
of AEX production in the process of aflibercept production expressed
using a CDM. The initial b* value of the AEX Load (at a concentration of
10 g/L) may range from about 0.5 to about 30, more particularly from
about 1.0 to about 25.0, and even more particularly from about 2.0 to
about 20.0. Following use of AEX, the b* value for the flowthrough (at a
concentration of 10 g/L) may range from 0.5 to about 10.0, more
particularly from about 0.5 to about 7.0, and even more particularly from
about 0.5 to about 5.0.
2.4 Peptide Mapping
[0526] Sample preparation. Tryptic mapping of reduced and alkylated
aflibercept samples obtained from AEX Load and flowthrough of the above
experiment (Table 2-3) were performed to identify and quantify
post-translational modification (PTM). An aliquot of each sample (Load
and flowthrough) was denatured using 8.0 M Urea, 0.1 M Tris-HCl, pH 7.5,
reduced with DTT and then alkylated with iodoacetamide. The denatured,
reduced and alkylated sample was first digested with recombinant
Lys-C(rLys-C) at an enzyme to substrate ratio of 1:100 (w/w) at
37.degree. C. for 30 minutes, diluted with 0.1 M Tris-HCl, pH 7.5 such
that a final urea concentration was 1.8 M, subsequently digested with
trypsin at an enzyme to substance ratio of 1:20 (w/w) at 37.degree. C.
for 2 hours and then deglycosylated with PNGase F at an enzyme substrate
ratio of 1:5 (w/w) for 37.degree. C. for 1 hour. The digestion was
stopped by bringing the pH below 2.0 using formic acid (FA).
[0527] LC-MS analysis. A 20 .mu.g aliquot of resulting rLys-C/tryptic
peptides from each sample was separated and analyzed by reverse-phase
ultra-performance liquid chromatography (UPLC) using Waters ACQUITY UPLC
CSH C18 column (130 .ANG., 1.7 .mu.m, 2.1.times.150 mm) followed by
on-line PDA detection (at wavelengths of 280 nm, 320 nm and 350 nm) and
mass spectrometry analysis. Mobile phase A was 0.1% FA in water, and
mobile phase B was 0.1% FA in acetonitrile. After sample injection, a
gradient was initiated with a 5 min hold at 0.1% B followed by a linear
increase to 35% B over 75 minutes for optimum peptide separation. MS and
MS/MS experiments were conducted using a Thermo Scientific Q Exactive
Hybrid Quadrupole-Orbitrap mass spectrometer with higher-energy
collisional dissociation (HCD) employed for peptide fragmentation for
MS/MS experiments. Peptide identity assignments were based on the
experimentally determined accurate mass of a given peptide in the full MS
spectrum as well as the b and y fragment ions in the corresponding HCD
MS/MS spectrum. Extracted ion chromatograms of the peptides from the Load
and flowthrough were generated (see FIG. 10). As seen in the extracted
ion chromatogram in FIG. 10, the peptide fragments identified in "AEX
Load" and "AEX FT/wash" from AEX separations 1-5 (as identified in Table
2-3) are shown. The relative abundance of some of these peptides
identified in FIG. 10 from the peptide mapping analysis are shown in FIG.
11.
[0528] Referring to FIG. 11, this figure identifies various peptide
fragments analyzed and their relative levels of oxidation. In particular,
the third column identifies the amino acid residues ("Peptide Sequence")
of peptide fragments that were isolated and analyzed. Each Peptide
Sequence has an amino acid residue that is underscored. The underscored
amino acid residue identifies the amino acid in the Peptide Sequence that
is oxidized. The oxidized amino acids correspond to either histidine (H)
oxidation or tryptophan (W) oxidation. There is also depicted in this
figure rows to the right of each of the Peptide Sequences showing the
abundance of oxidized species. This shading in the rows indicates
differences in the relative amount of oxidized residues in a particular
sample using different AEX separations identified in the respective
column headings. For example, referring to the second peptide
(EIGLLTCEATVNGHLYK) in FIG. 11, when read across along in a horizontal
manner, the relative total population of this peptide in a particular
sample ("aflibercept AEX Load") that is oxidized is approximately 0.013%
oxidized. As progression is made across the same row, there is a shift in
the shading, indicating a change in the relative abundance of oxidized
species. For example, using this same Peptide Sequence, the relative
abundance of oxidized species for AEX separation are 0.006% to 0.010%
when following different AEX separation protocols. Thus, it can be
appreciated that AEX chromatography decreases the abundance of oxidized
species.
(B) AEX was Employed to Reduce the Color Formation in MiniTrap Production
[0529] AEX chromatography was performed to remove the coloration obtained
during production of MT1 which was obtained on performing cleavage of
full-length aflibercept expressed using CDM1.
2.5 Design
[0530] Four AEX separations were performed for this study as described in
Table 2-4. The AEX Load was obtained from a filtration sample of MT1
("MT1 filtered pool"). A 15.7 mL Capto Q column (20.0 cm bed height, 1.0
cm I.D.), a 14.1 mL POROS 50 HQ column (18.0 cm bed height, 1.0 cm I.D.),
and a 16.5 mL Q Sepharose FF column (21.0 cm bed height, 1.0 cm I.D.)
were integrated into an AKTA Avant benchtop liquid chromatography
controller for this experiment.
[0531] AEX load pH was adjusted to target .+-.0.05 pH units using 2 M tris
base or 2 M acetic acid. AEX load conductivity was adjusted to target
.+-.0.1 mS/cm using 5 M sodium chloride or deionized water. All pool
samples were analyzed for HMW, color and yield.
TABLE-US-00016
TABLE 2-4
Summary of the Study Design for the AEX Color Reduction Study
AEX Separation Resin AEX Protocol
1 Capto Q Table 2-6
2 POROS 50 HQ Table 2-6
3 Q Sepharose FF Table 2-6
4 POROS 50 HQ Table 2-5
TABLE-US-00017
TABLE 2-5
Flowthrough AEX Protocol Used for the Color Reduction Study
Column Linear
Vol- Vel-
umes ocity
Step Description Mobile Phase (CVs) (cm/h)
1 Pre- 2M Sodium Chloride (NaCl) 2 200
Equilibration
2 Equilibration 50 mM Tris, 40 mM NaCl 2 200
pH 7.90-8.10, 6.50-7.50 mS/cm
3 Load AEX Load 30 g/L- 200
pH 7.90-8.10, 6.50-7.50 mS/cm resin
4 Wash 50 mM Tris, 40 mM NaCl 2 200
pH 7.90-8.10, 6.50-7.50 mS/cm
5 Strip 1 2M Sodium Chloride (NaCl) 2 200
6 Strip 2 1N Sodium Hydroxide (NaOH) 2 200
AEX, anion exchange chromatography;
CV, column volume
TABLE-US-00018
TABLE 2-6
Bind and Elute AEX Protocol Used for the Color Reduction Study
Column Linear
Vol- Vel-
umes ocity
Step Description Mobile Phase (CVs) (cm/h)
1 Pre- 2M Sodium Chloride (NaCl) 2 200
Equilibration
2 Equilibration 50 mM Tris 2 200
pH 8.30-8.50, 1.90-2.10 mS/cm
3 Load AEX Load 30 g/L- 200
pH 8.30-8.50, 1.90-2.10 mS/cm resin
4 Wash 50 mM Tris 2 200
pH 8.30-8.50, 1.90-2.10 mS/cm
5 Elution 50 mM Tris, 70 mM NaCl 2 200
pH 8.30-8.50, 8.50-9.50 mS/cm
6 Strip 1 2M Sodium Chloride (NaCl) 2 200
7 Strip 2 1N Sodium Hydroxide (NaOH) 2 200
AEX, anion exchange chromatography;
CV, column volume
2.6 Results
[0532] All four AEX separations led to reduction in color as seen for
coloration of the flowthrough and wash for AEX separations 1-4 (Table
2-7). While the first three AEX separations were evaluated in a bind and
elute mode (Table 2-6), it was observed that the majority of the product
was present in the load and wash blocks (62%-94%).
[0533] The first three separations evaluated the pH 8.4 and 2.0 mS/cm
setpoint for Capto Q, POROS 50 HQ, and Q Sepharose FF resins. All three
separations had a good yield (>80%). The POROS 50 HQ AEX pool showed
the lowest yellow color in AEX pool (b* value of 2.09) followed by the Q
Sepharose FF AEX pool (b* value of 2.22) and the Capto Q AEX pool (b*
value of 2.55).
TABLE-US-00019
TABLE 2-7
Summary of Experimental Results
of the AEX Color Reduction Study
AEX Yield HMW Color Color Color
Separation Fraction (%) (%) (L*) (a*) (b*)
1 FT/wash 90.7 0.49 99.11 -0.27 2.55
2 FT/wash 93.8 0.33 99.20 -0.28 2.09
3 FT/wash 86.7 0.23 98.88 -0.23 2.22
4 FT/wash 99.5 1.13 98.90 -0.39 3.40
-- MT1 Filtered Pool -- 0.65 98.18 -0.37 4.17
(AEX Load)
AEX, anion exchange chromatography; HMW, high molecular weight species.
The fractions were adjusted to a protein concentration of 5 g/L for color
measurements.
2.7 Conclusion
[0534] As seen for aflibercept (see Section 2.3 above), use of AEX was
found to reduce yellow-brown coloration (Table 2-7) for MiniTrap
production. Referring to Table 2-7, the AEX Load has a b* value of 4.17,
but when subjected to AEX chromatography (AEX Separation 1-4), the b*
value decreases indicating a decrease in yellow-brown coloration. Again,
as the b* value decreases so too does the coloration. The initial b*
value of the AEX Load (at a concentration of 5 g/L) may range from about
0.5 to about 25, more particularly from about 1.0 to about 20.0, and even
more particularly from about 1.5 to about 15.0. Following use of AEX, the
b* value of the flowthrough (at a concentration of 5 g/L) may range from
0.5 to about 10.0, more particularly from about 0.5 to about 7.0, and
even more particularly from about 0.5 to about 5.0.
Example 3. Oxidized Peptide Study
[0535] 3.1 Peptide Mappings
[0536] Sample Preparation. Tryptic mapping of reduced and alkylated
MiniTrap (MT1) and MT4 (MiniTrap similar to MT1 using a different
full-length aflibercept one produced using soy hydrolysate cell culture)
samples were performed to identify and quantify post-translational
modification. An aliquot of sample was denatured using 8.0 M Urea in 0.1
M Tris-HCl, pH 7.5, reduced with DTT and then alkylated with
iodoacetamide. The denatured, reduced and alkylated drug substance was
first digested with recombinant Lys-C(rLys-C) at an enzyme to substrate
ratio of 1:100 (w/w) at 37.degree. C. for 30 minutes, diluted with 0.1 M
Tris-HCl, pH 7.5 such that the final urea concentration was 1.8 M,
subsequently digested with trypsin at an enzyme to substance ratio of
1:20 (w/w) at 37.degree. C. for 2 hours and then deglycosylated with
PNGase F at an enzyme substrate ratio of 1:5 (w/w) for 37.degree. C. for
1 hour. The digestion was stopped by bringing the pH below 2.0 using
formic acid (FA).
[0537] LC-MS Analysis. A 20 .mu.g aliquot of resulting rLys-C/tryptic
peptides from each sample was separated and analyzed by reverse-phase
ultra-performance liquid chromatography (UPLC) using Waters ACQUITY UPLC
CSH C18 column (130 .ANG., 1.7 .mu.m, 2.1.times.150 mm) followed by
on-line PDA detection (at wavelengths of 280 nm, 320 nm and 350 nm) and
mass spectrometry analysis. Mobile phase A was 0.1% FA in water and
mobile phase B was 0.1% FA in acetonitrile. After sample injection, a
gradient started with a 5 min hold at 0.1% B followed by a linear
increase to 35% B over 75 minutes for optimum peptide separation. MS and
MS/MS experiments were conducted on a Thermo Scientific Q Exactive Hybrid
Quadrupole-Orbitrap mass spectrometer with higher-energy collisional
dissociation (HCD) employed for peptide fragmentation for MS/MS
experiments. Peptide identity assignments were based on the
experimentally determined accurate mass of a given peptide in the full MS
spectrum as well as the b and y fragment ions in the corresponding HCD
MS/MS spectrum. Extracted ion chromatograms of oxidized peptides and
corresponding native peptide were generated with the peak areas
integrated to calculate the site-specific percentage of oxidized amino
acid residue(s) within the MT1 sample.
Peptide Fragments Linked to Increased Absorbance at 350 nm
[0538] The PTMs on MT1 were observed upon comparing the tryptic peptide
maps for MT1 and MT4 (FIG. 12A shows the absorbance of peptides eluted
from 20.0 to 75 minutes). The peptides with varying UV peaks are
highlighted. The expanded view of the chromatogram is shown in FIG. 12B
which shows the absorbance of peptides eluted from 16 to 30 minutes. The
peptides with sharp contrast in UV absorbance between MT1 and MT4 were
TNYLTH*R, IIW(+4)DSR and IIIW(+132)DSR (* or underscoring represents
oxidation of the residue). Further, the expanded view of the chromatogram
is shown in FIG. 12C, which shows the absorbance of peptides eluted from
30 to 75 minutes. The peptides with sharp contrast in UV absorbance
between MT1 and MT4 were DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17),
TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20), EIGLLTCEATVNGH*LYK (SEQ ID NO.:
18) and QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) (* represents oxidation
of the residue). The peptide mapping revealed identity of peptides that
are significantly different in abundance between the VEGF MiniTraps. The
relative abundance of the peptides identified from the peptide mapping
analysis is shown in Table 3-1. The amount of 2-oxo-histidines in MT1
(produced in a CDM) were higher than MT4 (produced in soy hydrolysate),
suggesting that the media used to express aflibercept can have a
significant effect on the relative abundance of peptides with oxidized
histidines or oxidized tryptophans. For example, for the peptide
QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), the percent relative abundance
of the peptide in MT1 (CDM produced) was 0.015% compared to percent
relative abundance of the peptide in MT4 (soy hydrolysate produced; which
is about 15-fold less as compared to MT1).
TABLE-US-00020
TABLE 3-1
Peptide Modified Fold change
Peptide Sequence MT1 MT4 MT1/MT4
EIGLLTCEATVNGH EIGLLTC[+57]EATVN 0.011% 0.004% 2.75
LYK (SEQ ID NO.: GH[+14]LYK (SEQ ID
57) NO.: 18)
QTNTIIDVVLSPSH QTNTIIDVVLSPSH[+14] 0.015% 0.001% 15.00
GIELSVGEK (SEQ GIELSVGEK (SEQ
ID NO.: 58) ID NO.: 19)
TELNVGIDFNWEYP TELNVGIDFNWEYPS 0.204% 0.026% 7.85
SSKHQHK (SEQ ID SKH[+14]QHK (SEQ ID
NO.: 59) NO.: 20)
DKTHTCPPCPAPEL DKTH[+14]TC[+57]PP 0.115% 0.018% 6.39
LG (SEQ ID NO.: C[+57]PAPELLG (SEQ
60) ID NO.: 17)
TNYLTHR (SEQ ID TNYLTH[+14]R (SEQ 0.130% 0.020% 6.50
NO.: 61) ID NO.: 21)
[0539] Color and 2-oxo-Histidine Quantitation. The percentage of
2-oxo-histidines in the oligopeptides that were generated by protease
digestion, as measured by mass spectrometry, are also shown (Table 3-2).
(Values were normalized against unmodified peptides.) Table 3-2 (I) shows
the percent of oxidized histidines/tryptophans observed for AEX
flowthrough: MT1 lot 1, AEX flow through for MT1 lot 2, and AEX flow
through for MT1 lot 3. Table 3-2 (II) shows the percent of oxidized
histidines/tryptophans observed for acidic fraction 1, acidic fraction 2,
and main fraction obtained on performing CEX separation for MT1 lot 3.
From this Table, it is clear that the acidic variants are comprised of
oxidized species. From Table 3-2(I), it is clear that the % of
2-oxo-histidines and tryptophan dioxidation comprising peptides/protein
is reduced in the D9 AEX flowthrough compared to the AEX Strip. It is
evident that stripping the AEX column enriches for the percentage of such
modified peptides. For example, the % of the modified peptide
"EIGLLTC[+57]EATVNGH[+14]LYK (SEQ ID NO.: 18)" in the AEX Flowthrough
(MT1 lot 1) was 0.013% and in the "AEX Strip" was 0.080%. This also
corroborates that the AEX column captures modified peptides, thus
reducing the percentage of modified peptides in the AEX flowthrough.
TABLE-US-00021
TABLE 3-2 (I)
Percentage of 2-oxo-Histidines/Tryptophans
AEX Flowthrough
AEX Strip MT1 lot 1 MT1 lot 2 MT1 lot 3
Intense BY1, 110 .ltoreq.BY3, 110 .ltoreq.BY3, 110
Modified Peptides yellow mg/mL mg/mL mg/mL
EIGLLTC[+57]EATVNGH[+14]LYK 0.080% 0.013% 0.008% 0.006%
(SEQ ID NO.: 18)
QTNTIIDVVLSPSH[+14]GIELSVGEK 0.054% 0.028% 0.023% 0.019%
(SEQ ID NO.: 19)
TELNVGIDFNWEYPSSKH[+14]QHK 0.235% 0.085% 0.049% 0.049%
(SEQ ID NO.: 20)
DKTH[+14]TC[+57]PPC[+57] 0.544% 0.092% 0.077% 0.057%
PAPELLG (SEQ ID NO.: 17)
TNYLTH[+14]R (SEQ ID NO.: 21) 0.089% 0.022% 0.011% 0.010%
IIW[+32]DSR (SEQ ID NO.: 28) 0.738% 0.252% 0.198% 0.298%
TABLE-US-00022
TABLE 3-2 (II)
Percentage of 2-oxo-Histidines/Tryptophans
CEX flowthrough
Acidic fraction 1 Acidic fraction 2 Main fraction
from MT1 lot 3 from MT1 lot 3 from MT1 lot 3
Modified Peptides Yellow Yellow No Color
EIGLLTC[+57]EATVNGH[+14]LYK 0.009% 0.008% 0.004%
(SEQ ID NO.: 18)
QTNTIIDVVLSPSH[+14]GIELSVGEK 0.013% 0.015% 0.006%
(SEQ ID NO.: 19)
TELNVGIDFNWEYPSSKH[+14]QHK 0.131% 0.151% 0.049%
(SEQ ID NO.: 20)
DKTH[+14]TC[+57]PPC[+57]PAPELLG 0.117% 0.132% 0.068%
(SEQ ID NO.: 17)
TNYLTH[+14]R (SEQ ID NO.: 21) 0.014% 0.008% 0.008%
IIW[+32]DSR (SEQ ID NO.: 28) 0.458% 0.269% 0.185%
[0540] In Table 3-2(11), [+57] represent alkylation of cysteine by
iodoactamide adds a carboxymethyl amine moiety on the cysteine, which is
a net mass increase of about +57 over unmodified cysteine:
##STR00002##
[0541] In Table 3-2(11), [+14] represent conversion from His to 2-oxo-His,
one oxygen atom is added on carbon 2, but two hydrogen atoms are lost
(one from Carbon 2, the other from nitrogen 3), which is a net mass
increase of about +14 over unmodified histidine.
##STR00003##
[0542] In Table 3-2(11), [+32] represents tryptophan dioxidation resulting
in the formation of N-formylkynurenine, which is a net mass increase of
about +32 over unmodified tryptophan (FIG. 4).
[0543] A second set of experiments were performed to evaluate the
percentage of 2-oxo-histidines (and tryptophan dioxidation) in
oligopeptides from protease digested FabRICATOR-cleaved aflibercept (MT4)
which was processed by AEX chromatography (FIG. 13 and Table 3-3 below).
The percent of 2-oxo-histidines and tryptophan dioxidation in AEX strip
for oligopeptides from protease digested FabRICATOR-cleaved aflibercept
(MT4) was significantly more than the percent of 2-oxo-histidines and
tryptophan dioxidation in the AEX flowthrough (referring to "MT1" in
Table 3-3 below).
TABLE-US-00023
TABLE 3-3
Percentage of 2-oxo-Histidines
full length AEX Strip fold change
Modified peptides aflibercept MT1 from MT1 AEX Strip/MT1
IIW (SEQ ID NO.: 28) 0.22% 0.34% 0.81% 2.4
EIGLLTC[+57]EATVNGH[+14]LYK 0.00% 0.02% 0.08% 4.0
(SEQ ID NO.: 18)
QTNTIIDVVLSPSH[+14]GIELSVGEK 0.01% 0.04% 0.07% 1.8
(SEQ ID NO.: 19)
TELNVGIDFNWEYPSSKH[+14]QHK 0.01% 0.19% 0.42% 2.2
(SEQ ID NO.: 20)
DKTH[+14]TC[+57]PPC[+57]PAPELLG 0.01%.sup.a 0.11% 0.63% 5.7
(SEQ ID NO.: 17)
TNYLTH[+14]R (SEQ ID NO.: 21) 0.00% 0.03% 0.10% 3.3
.sup.avalue calculated using a different peptide for full-length
aflibercept, as the C-terminal peptide is different from MiniTrap.
[0544] The percent of 2-oxo-histidines and tryptophan dioxidation in AEX
strip was significantly more than the percent of 2-oxo-histidines and
tryptophan dioxidation in the AEX flowthroughs during the MT1 productions
(referring to "MT1" in Table 3-3 above). Compared to Table 3-2, Table 3-3
shows similar results that stripping the AEX column produced a sample
with a significantly higher percent of 2-oxo-histidines and tryptophan
dioxidation compared to the percent of 2-oxo-histidines and tryptophan
dioxidation in AEX flowthrough suggesting that the 2-oxo-histidines and
tryptophan dioxidation species are bound to the AEX column during the
separation and are removed upon stripping the AEX column. This is further
evident in the extracted ion chromatogram as seen in FIG. 14.
Strong Cation Exchange Chromatogram (CEX)
[0545] A series of experiments were conducted in order to identify acidic
species and other variants present in samples comprising anti-VEGF
proteins.
[0546] Strong cation exchange chromatography was performed using a MonoS
(10/100) GL column (GE Life Sciences, Marlborough, Mass.). For the sample
separations, the mobile phases used were 20 mM
2-(N-morpholino)ethanesulfonic acid (MES), pH 5.7 (Mobile phase A) and 40
mM sodium phosphate, 100 mM sodium chloride pH 9.0 (Mobile phase B). A
non-linear pH gradient was used to elute charge variants of MT1 with
detection at 280 nm. Peaks that elute at a relative residence time
earlier than the main peak are designated herein as acidic species.
[0547] A sample from the MT1 lot 2 (<BY3), prior to any enrichment, was
subjected to CEX using the method as depicted in FIG. 15. Desialylation
was applied to the sample in order to reduce the complexity of variants
of MT1. This was followed by preparative SEC processing (Superdex 200
prep grade XK26/100) using 1.times.DPBS, pH 7.2.+-.0.2, as the mobile
phase. The fractions obtained from the preparative SEC column comprising
desialylated MiniTrap (dsMT1) were combined and further subjected to
strong cation exchange (SCX) chromatography to enrich for charge variants
of MT1 using a dual salt-pH gradient. The procedure resulted in a total
of 7 fractions (F1-F7; MC represents the method control, FIG. 16 and FIG.
17).
[0548] On performing CEX, the acidic species elute earlier than the main
peaks and basic species elute after the main peaks. As observed in FIG.
17, peaks 3-5 are the main peaks. Peaks 1 and 2 are eluted before elution
of the main species of MT1 (peaks 3-5), and thus, comprise the acidic
species. Peak 6 is eluted after the elution of the main species of MT1
(peaks 3-5), and thus, comprises the basic species. Table 3-4 shows the
relative abundance of the peaks in MC (as identified in FIG. 16). For
example, row two of Table 3-4 (labeled MC) shows that the total relative
amount of acidic species in MC is about 19.8% (i.e., peak 1+peak 2).
Table-3-4 also shows the relative abundance of the peaks for each
individual fraction. While there are overlapping species in the different
fractions (as reflected in FIG. 16 and FIG. 17), the majority of
fractions F1 and F2 are acidic species (i.e., peak 1 and peak 2). For
example, fraction F1 is comprised of 63.7% peak 1 and 19.2% peak 2 (for a
total of 82.9% acidic species). Fraction F2 is comprised of 9.6% peak 1
and 75.9%. peak 2 (for a total of 85.5% acidic species). The majority of
fractions F3-F5 are the main species of MT1 (peaks 3-5). Lastly, the
majority of fractions F6-F7 are the basic species (peak 6) but do include
some portions of the main species (e.g., peaks 4 and 5).
[0549] It was also observed that fractions F1 and F2 (which comprises the
acidic species) had an intense yellow-brown coloration compared to the
fractions F3-F5 (which comprises the main species or "MT1"). All the
fractions were inspected for color at concentrations >13 mg/mL. As
evident from this Example, the presence of acidic species in the sample
tracked with the appearance of yellow-brown coloration, removal (or
minimization) of which can be accomplished by removing (or minimizing)
the acidic species from MT1.
[0550] The 3D chromatograms for MT1 lot 2 and fractions F1-F7 are shown in
FIGS. 18A-H. MT1 lot 2 did not exhibit any significant spectral features
(FIG. 18A). Fractions 1 and 2 (comprising the acidic species) exhibited a
spectral signature between 320-360 nm (see the circle in FIG. 18B). This
feature was more prominent in fraction 1 compared to fraction 2 (FIGS.
18B and 18C) and was absent in fraction 3 and fractions 4-7 (main
species, MT1) (FIGS. 18D and 1811), which did not exhibit yellow-brown
coloration.
[0551] Thus, as observed above, CEX led to identification of acidic
species/acidic fractions (fractions 1 and 2) which shows an intense
yellow-brown coloration as compared to the main species/fractions
(fractions 3-6). This result was also observed in the form of a distinct
spectral signature present in the 3D chromatograms of the fractions F1-F2
and absent in fractions F3-F7.
[0552] The distribution of variants in fractions F1-7 and MC (from
MT1--lot 2 after CEX) was further assessed by icIEF (FIG. 19).
[0553] The distribution of variants in fractions F1-7 and MC (from
MT1--lot 2 after CEX) was further assessed by icIEF using an iCE3
analyzer (ProteinSimple) with a fluorocarbon coated capillary cartridge
(100 .mu.m.times.5 cm). The ampholyte solution consisted of a mixture of
0.35% methyl cellulose (MC), 0.75% Pharmalyte 3-10 carrier ampholytes,
4.2% Pharmalyte 8-10.5 carrier ampholytes, and 0.2% pI marker 7.40 and
0.15% pI marker 9.77 in purified water. The anolyte was 80 mM phosphoric
acid, and the catholyte was 100 mM sodium hydroxide, both in 0.10%
methylcellulose. Samples were diluted in purified water and sialidase A
was added to each diluted sample at an enzyme to substrate ratio of 1:200
(units of sialidase A per milligram of MT1) followed by incubation at
ambient temperature for approximately 16 hours. The sialidase A treated
samples were mixed with the ampholyte solution and then focused by
introducing a potential of 1500 V for one minute followed by a potential
of 3000 V for 7 minutes. An image of the focused MT1 variants was
obtained by passing 280 nm ultraviolet light through the capillary and
into the lens of a charge coupled device digital camera. This image was
then analyzed to determine the distribution of the various charge
variants. (FIG. 19). Referring to FIG. 19, fractions F1 and F2 (or the
acidic fractions) showed an absence of the peak for MT1, which is clearly
observed for MC and fractions F3-F7 (main species, MT1). Thus, icIEF
electropherograms were considered able to detect and determine the
distribution of the different charge variants of the protein under
consideration, MT1 in this case. Thus, it was evident that acidic
fractions on performing CEX analysis showed (a) increased relative
abundance of percent of 2-oxo-histidine or dioxo-tryptophan (Table 3-2
(II)); (b) increased yellow-brown coloration (data not shown); and (c)
presence of a spectral signature as seen on the 3D chromatograms for
fractions 1 and 2 (FIG. 18B and FIG. 18C).
Example 4. Photo-Induction Study
[0554] In this Example, photo-induction of VEGF MiniTrap (MT), for example
MT1, was performed by exposure of a protein sample to varying amounts of
cool white (CW) fluorescent light or ultra-violet A (UVA) light. The
color and oxidized amino acid content of the light exposed samples was
determined. LCMS analysis was performed following exposure, as explained
above. Exposure of MT to cool white light or UVA light produced an
increase in oxidized amino acid residues, for example, histidine (Table
4-1, Table 4-2 and Table 4-3).
TABLE-US-00025
TABLE 4-1
Photo-Induction Study Design
Cumulative 0.2 0.5 0.8 1.0 2.0
Exposure (xICH) (xICH) (xICH) (xICH)H (xICH)
CW fluorescent 0.24 0.6 0.96 1.2 2.4
exposure (lux*hr) million million million million million
lux*hr lux*hr lux*hr lux*hr lux*hr
Incubation time with 30 75 100 150 300
CW fluorescent light hours hours hours hours hours
(at 8 klux)
UVA exposure 40 100 160 200 400
(W*hr/m.sup.2)
Incubation time with 4 10 16 20 40
UVA (at 10 W/m.sup.2) hours hours hours hours hours
ICH refers to ICH Harmonised Tripartite Guideline: Stability Testing:
Photostability Testing of New Drug Substances and Products Q1B which
specifies photostability studies to be conducted with not less than 1.2
million lux*hours cool white fluorescent light and near ultraviolet
energy of not less than 200 W*hr/m.sup.2.
[0555] Table 4-2 depicts the increase in coloration of the MT sample
exposed to cool-white light and ultra-violet light. For example, b-value
for sample (t=0) was 9.58. On exposing this sample to cool-white light at
2.4 million lux*hr, the b-value increases to 22.14. This increase in
b-value indicates that the exposure of MT to cool-white light at 2.4
million lux*hr increases yellow-brown coloration of the sample as
compared to sample (t=0). Similarly, on exposing MT sample (t=0) to
ultra-violet light at 400 W*h/m.sup.2, the b-value increases to 10.72
from 9.58. This increase in b-value indicates that the exposure of MT
sample to ultra-violet light at 400 W*h/m.sup.2 produces an increased
yellow-brown coloration of the sample as compared to sample (t=0).
TABLE-US-00026
TABLE 4-2
Color of Samples Exposed to Cool White
Light and Ultra-Violet Light
Photo exposure xICH (lux*hr) L* a* b* BY Value
Cool White Light
T = 0 97.37 -1.12 9.58 4.0
0.2x (0.24 million lux*hr) 96.46 -0.72 11.75 3.7
0.5x (0.6 million lux*hr) 95.47 -0.4 11.3 3.7
0.8x (0.96 million lux*hr) 95.33 -0.38 11.96 3.6
1.0x (1.2 million lux*hr) 94.42 -0.2 13.72 3.3
2.0x (2.4 million lux*hr) 92.70 0.41 22.14 2.0
UVA
0.2x (40 W*h/m.sup.2) 97.26 -0.92 12.66 3.5
0.5x (100 W*h/m.sup.2) 100.39 -1.01 11.83 3.7
0.8x (160 W*h/m.sup.2) 79.69 -0.18 10.1 3.6
1.0x (200 W*h/m.sup.2) 97.48 -0.95 11.36 3.7
2.0x (400 W*h/m.sup.2) 97.76 -0.98 10.72 3.8
Sample colors are indicated using the CIELAB color space (L*, a* and b*
variables) and relative to the EP BY color standard; L* = white to black
(L* is lightness); a* = magenta to aqua; b* = yellow to blue, the higher
the b-value the more yellow.
TABLE-US-00027
TABLE 4-3 (I)
2-oxo-His Levels in Peptides from Ultra-Violet Light
Stressed MiniTrap
Peptides Site t0 UV_4 h UV_10 h UV_16 h UV_20 h UV_40 h
DKTHTCPPCPAPEL H209 0.056% 0.067% 0.081% 0.088% 0.077% 0.091%
LG (SEQ ID NO.:
17)
EIGLLTCEATVNGH H86 0.010% 0.020% 0.034% 0.037% 0.033% 0.035%
LYK (SEQ ID NO.:
18)
QTNTIIDVVLSPSH H110 0.024% 0.031% 0.028% 0.028% 0.027% 0.027%
GIELSVGEK (SEQ
ID NO.: 19)
TELNVGIDFNWEYP H145 0.096% 0.147% 0.163% 0.173% 0.147% 0.125%
SSKHQHK (SEQ ID
NO.: 20)
TNYLTHR (SEQ ID H95 0.014% 0.032% 0.044% 0.056% 0.058% 0.078%
NO.: 21)
SDTGRPFVEMYSEI H19 0.007% 0.013% 0.021% 0.025% 0.024% 0.034%
PEIIHMTEGR (SEQ
ID NO.: 22)
VHEKDK (SEQ ID H203 0.040% 0.105% 0.238% 0.255% 0.269% 0.324%
NO.: 23)
TABLE-US-00028
TABLE 4-3 (II)
2-oxo-His Levels in Peptides from Cool White Light
Stressed MiniTrap
Peptides Site t0 CW_30 h CW_75 h CW_100 h CW_150 h CW_300 h
DKTHTCPPCPAPELL H209 0.056% 0.152% 0.220% 0.243% 0.258% 0.399%
G (SEQ ID NO.: 17)
EIGLLTCEATVNGHL H86 0.010% 0.063% 0.110% 0.132% 0.170% 0.308%
YK (SEQ ID NO.: 18)
QTNTIIDVVLSPSHGI H110 0.024% 0.085% 0.120% 0.128% 0.148% 0.180%
ELSVGEK (SEQ ID
NO.: 19)
TELNVGIDFNWEYP H145 0.096% 0.423% 0.585% 0.634% 0.697% 0.748%
SSKHQHK (SEQ ID
NO.: 20)
TNYLTHR (SEQ ID H95 0.014% 0.103% 0.175% 0.198% 0.267% 0.437%
NO.: 21)
SDTGRPFVEMYSEIP H19 0.007% 0.025% 0.043% 0.049% 0.058% 0.115%
EIIHMTEGR (SEQ ID
NO.: 22)
VHEKDK (SEQ ID H203 0.040% 0.426% 0.542% 0.622% 0.702% 1.309%
NO.: 23)
[0556] Exposure of aflibercept MT to cool white light or UVA light tracked
with the appearance of oxidized histidines (2-oxo-his) (Table 4-3).
Referring to Table 4-3, the peptide "SDTGRPFVEMYSEIPEIIHMTEGR (SEQ ID
NO.: 22)" with oxo-histidine was 0.007% in MT sample (t=0), whereas its
abundance increased to 0.324% on exposure to ultra-violet light for 40
hours (Table 4-3(I)) and to 1.309% on exposure to cool-white light for
300 hours (Table 4-3(II)).
[0557] Two species of 2-oxo-histidine were observed, a 13.98 Da species
(as shown in FIG. 2) and a 15.99 Da species (as shown in FIG. 3), with
the 13.98 Da species being predominant in light stressed MiniTrap
samples. The 15.99 Da species is known to be a product of a copper
metal-catalyzed process. Schoneich, J. Pharm. Biomed Anal. 21:1093-1097
(2000). Moreover, the 13.98 Da species is a product of a light-driven
process. Liu et al., Anal. Chem. 86(10: 4940-4948 (2014)).
[0558] Similar to the increased abundance of oxidized histidines in
samples exposed to cool white light and UVA light, exposure of MT to cool
white light or UVA light also induced formation of other PTMs (Table 4-4
and Table 4-5).
TABLE-US-00029
TABLE 4-4 (I)
Other PTMs in Peptides from Ultra-Violet Light
Stressed MiniTrap
Peptides Site t0 UV_4 h UV_10 h UV_16 h UV_20 h UV_40 h
Deamidation
EIGLLTCEATVNGHLYK (SEQ N84 20.8% 21.7% 21.5% 21.5% 22.7% 22.4%
ID NO.: 62)
QTNTIIDVVLSPSHGIELSVGE N99 5.3% 5.4% 5.5% 5.4% 5.5% 5.6%
K (SEQ ID NO.: 63)
Oxidation
SDTGRPFVEMYSEIPEIIHMTE M10 4.5% 8.2% 11.1% 13.3% 13.8% 19.3%
GR (SEQ ID NO.: 64)
SDTGRPFVEMYSEIPEIIHMTE M20 1.1% 2.0% 2.8% 3.4% 3.4% 4.6%
GR (SEQ ID NO.: 65)
TQSGSEMK (SEQ ID NO.: M163 2.0% 2.7% 4.1% 4.6% 7.9% 8.7%
66)
SDQGLYTCAASSGLMTK M192 5.4% 8.1% 10.9% 12.1% 12.5% 18.3%
(SEQ ID NO.: 67)
3-deoxygluconasone
SDTGRPFVEMYSEIPEIIHMTE R5 9.9% 10.0% 9 9% 9.7% 9.8% 9.3%
GR (SEQ ID NO.: 68)
TABLE-US-00030
TABLE 4-4 (II)
Other PTMs in Peptides from Cool White Light Stressed MiniTrap
Peptides Site t0 CW_30 h CW_75 h CW_100 h CW_150 h CW_300 h
Deamidation
EIGLLTCEATVNGHLYK (SEQ N84 20.8% 22.0% 22.9% 20.3% 21.8% 21.3%
ID NO.: 62)
QTNTIIDVVLSPSHGIELSVGEK N99 5.3% 5.6% 5.2% 5.6% 5.5% 5.8%
(SEQ ID NO.: 63)
Oxidation
SDTGRPFVEMYSEIPEIIHMTEG M10 4.5% 11.7% 17.3% 19.9% 25.1% 39.7%
R (SEQ ID NO.: 64)
SDTGRPFVEMYSEIPEIIHMTEG M20 1.1% 3.1% 4.3% 5.1% 6.1% 8.2%
R (SEQ ID NO.: 65)
TQSGSEMK (SEQ ID NO.: 66) M163 2.0% 3.3% 15.7% 11.7% 26.4% 20.5%
SDQGLYTCAASSGLMTK (SEQ M192 5.4% 10.7% 15.3% 18.7% 22.8% 37.6%
ID NO.: 67)
3-deoxygluconasone
SDTGRPFVEMYSEIPEIIHMTEG R5 9.9% 9.9% 9.6% 9.3% 9.3% 9.0%
R (SEQ ID NO.: 68)
TABLE-US-00031
TABLE 4-5 (I)
Oxidation Levels of Tryptophan/Tyrosine/Phenylalanine in Peptides from
Ultra-Violet Light Stressed MiniTrap
Peptides Modification Site t0 UV_4 h UV_10 h UV_16 h UV_20 h UV_40 h
SDQGLYTCAASSGLM +4 W58 0.016% 0.049% 0.089% 0.119% 0.132% 0.221%
TK (SEQ ID NO.: 67) +16 0.047% 0.109% 0.177% 0.225% 0.242% 0.514%
+32 0.200% 0.487% 0.415% 0.481% 0.423% 0.498%
+48 0.000% 0.000% 0.000% 0.001% 0.001% 0.001%
TELNVGIDFNWEYPSS +4 W138 0.435% 0.462% 0.550% 0.557% 0.502% 0.512%
K (SEQ ID NO.: 29) +16 0.083% 0.100% 0.161% 0.206% 0.239% 0.448%
+32 0.009% 0.018% 0.027% 0.039% 0.044% 0.115%
+48 0.284% 0.278% 0.270% 0.302% 0.343% 0.275%
GFIISNATYK (SEQ ID +16 Y64 0.032% 0.041% 0.046% 0.053% 0.054% 0.073%
NO.: 69)
KFPLDTLIPDGK (SEQ +16 F44 0.068% 0.077% 0.087% 0.084% 0.070% 0.096%
ID NO.: 70)
FLSTLTIDGVTR (SEQ +16 F166 0.066% 0.075% 0.085% 0.089% 0.089% 0.124%
ID NO.: 71)
TABLE-US-00032
TABLE 4-5 (II)
Oxidation Levels of Tryptophan/Tyrosine/Phenylalanine in Peptides
from Cool White Light Stressed MiniTrap
Peptides Modification Site t0 CW_30 h CW_75 h CW_100 h CW_150 h CW_300 h
SDQGLYTCAASSG +4 W58 0.016% 0.063% 0.124% 0.161% 0.228% 0.526%
LMTK (SEQ ID NO.: +16 0.047% 0.129% 0.227% 0.283% 0.377% 0.795%
67) +32 0.200% 1.601% 2.706% 3.139% 3.925% 6.974%
+48 0.000% 0.001% 0.002% 0.002% 0.003% 0.005%
TELNVGIDFNWEY +4 W138 0.435% 0.555% 0.481% 0.490% 0.429% 0.522%
PSSK (SEQ ID NO.: +16 0.083% 0.109% 0.364% 0.251% 0.399% 0.753%
29) +32 0.009% 0.019% 0.027% 0.033% 0.048% 0.135%
+48 0.284% 0.284% 0.330% 0.308% 0.347% 0.316%
GFIISNATYK (SEQ +16 Y64 0.032% 0.043% 0.057% 0.063% 0.078% 0.127%
ID NO.: 69)
KFPLDTLIPDGK +16 F44 0.068% 0.087% 0.072% 0.088% 0.079% 0.144%
(SEQ ID NO.: 70)
FLSTLTIDGVTR +16 F166 0.066% 0.091% 0.088% 0.101% 0.112% 0.168%
(SEQ ID NO.: 71)
[0559] Thus, exposure of MT to cool white light or UVA light tracked with
the appearance of oxidized residues (such as histidines/tryptophans
(oxo-Trp)). Four species of oxo-trp were observed: +4 Da, +16 Da, +32 Da
and +48 Da. The +4 Da species is explained by formation of kynurenine
(FIG. 4), whereas the 16 Da, +32 Da and +48 Da are the mono-oxidation,
di-oxidation and tri-oxidation of tryptophan residues. Peptide mapping of
tryptic digests of MT samples monitored at 320 nm as shown in FIG. 20.
The relative presence of oxidized residues comprising peptides can be
compared in FIG. 20. For example, for the peptide, IIW(+4)DSRK, a
significant difference in its presence can be seen for MT sample at t=0,
and MT1 sample exposed to UVA for 40 hour and MT sample exposed to CWL
for 300 hours.
[0560] Exposure of MT to cool white light or UVA light was also evaluated
for the presence of HMW/low molecular weight (LMW) species (Table 4-6).
TABLE-US-00033
TABLE 4-6
HMW/LMW Species Were Generated
After Extended UVA and CWL Stress
Sample: MT1, 80 mg/mL, pH 5.8
Light Dark Light Dark Light Dark
exposed control exposed control exposed control
Samples samples Samples samples Samples samples
% HMW % Native % LMW
Cumulative UVA exposure (x ICH)
t = 0 2.1 NA 96.7 NA 1.2 NA
0.2x ICH (40 2.1 2.1 96.7 96.7 1.2 1.3
W*h/m2)
0.5x ICH (100 11.6 2.2 86.5 96.6 1.9 1.2
W*h/m2)
0.8x ICH (160 14.5 2.2 83.4 96.6 2.1 1.2
W*h/m2)
1.0x ICH (200 15.8 2.2 81.9 96.6 2.3 1.3
W*h/m2)
2.0x ICH (400 22.7 2.3 74.5 96.7 2.8 1.0
W*h/m2)
Cumulative CWL exposure (x ICH)
0.2x ICH (0.24 12.1 2.2 86.6 96.6 1.4 1.2
million lux* h)
0.5x ICH (0.6 20.4 2.3 77.9 96.4 1.6 1.3
million lux*hr)
0.8x ICH 23.2 2.4 75.1 96.2 1.7 1.4
(0.96 million
lux*hr)
1.0x ICH (1.2 30.1 2.6 68.1 96.2 1.9 1.3
million lux*hr)
2.0x ICH (2.4 45.0 2.9 52.6 95.8 2.4 1.4
million lux*hr)
[0561] To track the coloration with respect to HMW/LMW species for each
sample, analytical size-exclusion chromatography with full-spectrum PDA
detection (SEC-PDA) was performed as shown above on all the stressed
samples (CWL and UVA). SEC-PDA analysis of CWL-stressed MT reveals
significant increases in absorbance at .about.350 nm for all size
variants except the LMW species (FIG. 21), whereas SEC-PDA on
UVA-stressed MT reveals no increases in absorbance at .about.350 nm (FIG.
22). Unlike CWL-treated stress samples, UVA-treated stress samples did
not produce any significantly quantifiable yellow-brown color.
[0562] A similar result was obtained after studying absorbance ratios at
320 nm and 280 nm for the samples stressed by UVA and CWL. The A320/A280
ratios, analyzed by either raw intensity or total peak area, tracked with
increasing intensity of yellow color in CWL-stressed samples (FIG. 23),
whereas the A320/A280 ratios did not track with increasing intensity of
yellow color in UVA-stressed samples (FIG. 24). This corroborates the
previous observation that MT1 samples subjected to UVA stress does not
result in the same yellow-brown color observed following CWL stress.
Example 5. Upstream Methods for Reducing Coloration
5.1 Chemically Defined Medium Incubation Study
[0563] The effect of various constituents spiked into fresh chemically
defined media (CDM) comprising aflibercept with respect to coloration was
examined.
[0564] One or more 50 mL vent-capped shaker tubes with 10 mL working
volume (fresh CDM1) were incubated for 7 days, taking samples on day 0
and day 7. Aflibercept samples (aflibercept recombinant protein in an
aqueous buffered solution, pH 6.2, comprising 5 mM sodium phosphate, 5 mM
sodium citrate and 100 mM sodium chloride) were spiked into shaker tubes
at a concentration of 6 g/L.
[0565] Components added to reach a cumulative concentration: [0566]
Cysteine: 16.6 mM [0567] Iron: 0.23 mM [0568] Copper: 0.0071 mM [0569]
Zinc: 0.54 mM
[0570] The scaled effect of each constituent added on the b* value (CIE
L*, a*, b* color space) is set forth in FIG. 25A and plot of actual b*
value against predicted b* value is set forth in FIG. 25B. Addition of
cysteine resulted in the largest yellow-brown color increase. Iron and
Zinc also generated color. Folic acid and B vitamin group (including
thiamine, niacinamide, D-pantothenic acid, D-biotin, and pyridoxine)
increased the yellow-brown color. Riboflavin and Vitamin B12 did not
statistically impact color.
5.2 Effect of Decreasing Cysteine and Metals on b* Value
[0571] Bioreactors (e.g., 2L) were inoculated from a seed culture of an
aflibercept producing cell line. The inoculated cultures were grown at a
temperature of 35.5.degree. C., pH of 7.1.+-.0.25, and air sparge set
point of 22 ccm. Glucose, antifoam, and basal feeds were supplemented to
the bioreactors as needed. The effect of lowering the concentration of
cysteine and of metals on color when aflibercept is expressed was
evaluated in CDM1.
Medium at day 0=CDM1, including 1.48 mM of cysteine [0572] Nutrient
Feeds: [0573] Day 2=Chemically defined feed (CDF)+1.3-2.1 mM cysteine
[0574] Day 4=CDF+1.6-1.7 mM cysteine [0575] Day 6=CDF+1.6-1.7 mM cysteine
[0576] Day 8=CDF+1.6-1.7 mM cysteine
[0577] The bioreactor conditions were as follows: [0578] Cysteine was
added at a cumulative concentration of about 6-7 millimoles per L of
culture, 8-9 millimoles per L of culture, or 10-11 millimoles per L of
culture. [0579] Metals in CDM1 (0.5.times., 1.times., or 1.5.times.CDM1
levels) at 1.times. levels are listed below (where the concentrations are
prior to inoculum addition): [0580] Fe=68-83 micromoles per liter of
culture [0581] Zn=6-7 micromoles per liter of culture [0582] Cu=0.1-0.2
micromoles per liter of culture [0583] Ni=0.5-1 micromoles per liter of
culture Decreasing cumulative cysteine levels to 6-7 millimoles/L reduced
yellow-brown color with no significant impact to titer. Decreasing metal
concentrations to 0.5.times. in the medium reduced color with significant
increase in titer. There was a minimal impact to titer, VCC (viable cell
concentration), viability, ammonia or osmolality (See FIG. 26A-E). The
predicted scale effect of metal content and cysteine on b* value and
titer is set forth in FIG. 27.
5.3 Evaluation of the Effect of Antioxidants on b* Value
[0584] The effect of the antioxidants, taurine, hypotaurine, thioctic
acid, glutathione, glycine and vitamin C, on color, spiked into spent CDM
comprising aflibercept, was evaluated. One or more 50 mL vent-capped
shaker tubes with 10 mL working volume (CDM1) were incubated for 7 days,
taking samples on day 0 and day 7.
[0585] The conditions for component additions to spent CDM1 were as
follows: [0586] Aflibercept sample (aflibercept recombinant protein in
an aqueous buffered solution, pH 6.2, comprising 5 mM sodium phosphate, 5
mM sodium citrate and 100 mM sodium chloride) spiked into shaker tubes at
6 g/L concentration [0587] Antioxidants added to spent CDM1 at the
following concentrations: [0588] Taurine=10 mM of culture [0589]
Hypotaurine=10 mM of culture [0590] Glycine=10 mM of culture [0591]
Thioctic Acid=0.0024 mM of culture [0592] Glutathione, reduced=2 mM of
culture [0593] Hydrocortisone=0.0014 mM of culture [0594] Vitamin C
(ascorbic acid)=0.028 mM of culture
[0595] Multiple antioxidants decreased color formation in spent medium: a
combination of hypotaurine, taurine and glycine; thioctic acid; and
vitamin C. Glutathione increased b* value.
TABLE-US-00034
TABLE 5-1
Summary of Antioxidant Effect on Color Formation of MiniTrap
Condition b* value
Spent Medium Day 0 0.37
Spent Medium Day 7 Control 1.47
Spent Medium Day 7 + Antioxidants* 1.02
*Antioxidants that significantly decreased b* value:
Hypotaurine/Taurine/Glycine, Thioctic Acid, Vitamin C.
[0596] A summary of the predicted effect of various anti-oxidants on b*
value (CIE L*, a*, b* color space) is set forth in FIG. 28 (A-C).
[0597] The effect of the further addition to the antioxidants on color,
spiked into spent CDM comprising aflibercept, was evaluated. One or more
50 mL vent-capped shaker tubes with 10 mL working volume (CDM1) were
incubated for 7 days, taking samples on day 0 and day 7.
[0598] The conditions for component additions to spent CDM1 were as
follows: [0599] Aflibercept sample (aflibercept recombinant protein in
an aqueous buffered solution, pH 6.2, comprising 5 mM sodium phosphate, 5
mM sodium citrate and 100 mM sodium chloride) spiked into shaker tubes at
6 g/L concentration [0600] Two DOE experiments were run: [0601] (i)
Antioxidants added to spent CDM1 at the following concentrations: [0602]
Taurine=10 mM of culture [0603] Hypotaurine=10 mM of culture [0604]
Glycine=10 mM of culture [0605] Thioctic Acid=0.0024 mM of culture [0606]
Vitamin C (ascorbic acid)=0.028 mM of culture [0607] (ii) Antioxidants
added to reach the following cumulative concentrations: [0608] ATA=2.5-5
.mu.M [0609] Deferoxamine mesylate (DFO)=5-10 .mu.M [0610] Catalase=101.5
mg/L [0611] S-carboxymethyl-L-Cysteine=10 mM
[0612] Hypotaurine was found to decrease the color formation in spent
medium (FIG. 28D). DFO also significantly decreased the color formation
in spent medium (FIG. 28D). The other anti-oxidants did not have a
statistical impact on the color formation.
TABLE-US-00035
TABLE 5-2
Summary of Antioxidant Effect on Color Formation of MiniTrap
Condition b* value
Spent Medium Day 0 0.44
Spent Medium Day 7 Control 1.73
Spent Medium Day 7 + Hypotaurine 1.32
Spent Medium Day 7 + DFO 0.92
Shake--Flask Antioxidant Study:
[0613] Taurine, hypotaurine, glycine, thioctic acid and vitamin C were
individually evaluated and in combination for their ability to decrease
the color formation in cell culture (Table 5-3).
[0614] 250 mL shake flasks were inoculated from a seed culture of an
aflibercept producing cell line. The inoculated cells were grown at
35.5.degree. C. in incubators with 5% CO.sub.2 control. Glucose and basal
feeds were supplemented to the shake flasks as needed. The process
described above was used wherein metals were present at 0.5.times.
concentration in CDM1 and cysteine was added at a cumulative
concentration of 6-7 mM.
TABLE-US-00036
TABLE 5-3
Level 1 Level 1 Level 1
Antioxidant 0x 0.5x 1x
Taurine 0 3.75 mM 7.5 mM
Hypotaurine 0 3.75 mM 7.5 mM
Glycine 2.0 mM 5.75 mM 9.5 mM
Thioctic acid 1.0 .mu.M 1.9 .mu.M 2.8 .mu.M
Vitamin C 0 11.0 .mu.M 21.0 .mu.M
[0615] FIG. 28E shows the predicted effect of the anti-oxidants in Table
5-3 on b* value (CIE L*, a*, b* color space) and final titler. Taurine,
hypotaurine, glycine significantly reduced b* value without negatively
impacting titer.
Example 6. Glycosylation and Viability Studies for Aflibercept Production
Using CDM
[0616] Bioreactors (e.g., 2L) were inoculated from a seed culture of an
aflibercept producing cell line. The inoculated cultures were grown at a
temperature of 35.5.degree. C., pH of 7.1.+-.0.25, and air sparge set
point of 22 ccm. Glucose, antifoam, and basal feeds were supplemented to
the bioreactors as needed. Production of aflibercept protein was carried
out using CDM1 (proprietary). Production of a host cell line expressing
aflibercept fusion protein was carried out using CDM 1 (proprietary), CDM
2 (commercially obtained), and CDM 3 (commercially obtained). A set of
experiments was carried out using CDM 1, 2, and 3 with no additional
media components. Another set of experiments was performed using CDMs 1-3
to which manganese (manganese chloride tetrahydrate, Sigma, 3.2 mg/L),
galactose (Sigma, 8 g/L), and uridine (Sigma, 6 g/L) were added to the
feeds to modify the galactosylation profile. Lastly, a set of experiments
was performed using CDMs 1-3 to which manganese (manganese chloride
tetrahydrate, Sigma, 3.2 mg/L), galactose (Sigma, 8 g/L), and uridine
(Sigma, 6 g/L) were added to the feeds to modify the galactosylation
profile and dexamethasone (Sigma, 12 mg/L) was added to the feeds to
modify the sialyation profile of the composition. A clarified harvest
using each of the CDM was prepared by centrifugation followed by 0.45
.mu.m filtration.
[0617] Samples were processed by Protein A prior to N-glycan analysis.
Titer Measurements
[0618] Throughout these examples, unless stated otherwise, aflibercept
titers were measured daily using an Agilent (Santa Clara, Calif.) 1200
Series HPLC, or equivalent, operating with a low pH, and step elution
gradient with detection at 280 nm. Concentrations were assigned with
respect to a reference standard calibration curve.
Viable Cell Density (VCD) and Cell Viability Values
[0619] Throughout these examples, unless stated otherwise, viable cell
density (VCD) and cell viability values were measured through trypan blue
exclusion via Nova BioProfile Flex automated cell counters (Nova
Biomedical, Waltham, Mass.). Glucose, lactate, offline pH, dissolved
oxygen (DO), pCO2 measurements, and osmolality were measured with the
Nova BioProfile Flex (Nova Biomedical, Waltham, Mass.).
N-Glycan Oligosaccharide Profiling
[0620] Approximately 15 .mu.g of Protein A processed samples from CDM 1-3
harvests were prepared for N-glycan analysis in accordance with the
Waters GlycoWorks protocol using the GlycoWorks Rapid Deglycosylation and
GlycoWorks RapiFluor-MS Label kits (Waters part numbers 186008939 and
186008091, respectively). N-glycans were removed from the aflibercept
protein by treating the samples with PNGase-F at 50.5.degree. C. for 5
minutes, followed by a cool down at 25.degree. C. for 5 minutes. The
released glycans were labeled with RapiFluor-MS fluorescent dye through
reaction at room temperature for 5 minutes. The protein was precipitated
by adding acetonitrile to the reaction mixture and pelletized to the
bottom of the well through centrifugation at 2,204.times.g for 10
minutes. The supernatant comprising the labeled glycans was collected and
analyzed on an UPLC using hydrophilic interaction liquid chromatography
(Waters BEH Amide column) with post-column fluorescence detection. After
binding to the column, the labeled glycans were separated and eluted
using a binary mobile phase gradient comprised of acetonitrile and
aqueous 50 mM ammonium formate (pH 4.4). The labeled glycans were
detected using a fluorescence detector with an excitation wavelength of
265 nm and an emission wavelength of 425 nm. Using the relative area
percentages of the N-glycan peaks in the resultant chromatograms, the
N-glycan distribution is reported as the total percentage of N-glycans
(1) containing a core fucose residue (Total Fucosylation, Table 6-1), (2)
containing at least one sialic acid residue (Total Sialylation, Table
6-2), (3) identified as Mannose-5 (Mannose-5, Table 6-3), (4) containing
at least one galactose residue (Total Galactosylation, Table 6-4), and
(5) of known identity (Total Identified Peaks, Table 6-5).
Results
[0621] The viable cell count (VCC), viability, and harvest titer results
are shown in FIGS. 29-31 for CDMs 1-3 with and without additional
components.
[0622] Amongst the nine cultures, the CDM1 culture comprising uridine,
manganese, and galactose showed the highest titer at 12 days (5.5 g/L).
CDM1 without additional components also showed a high titer at 12 days
(about 4.25 g/L) compared to the other seven cultures (FIG. 29).
[0623] Cell viability results were similar across the various conditions
up to process day 6. After process day 7, the CDM2 and CDM3 cultures with
or without additional media components showed more than about 90%
viability (FIG. 30).
[0624] CDM1 culture with uridine, manganese and galactose showed the
highest VCC around day 6 (FIG. 31).
[0625] The impact of cultures and supplements had a significant impact on
the overall N-glycan distribution (Tables 6-1 to 6-5). The glycan levels
were compared using Protein A processed aflibercept (two samples were
evaluated) made using soy hydrolysate. The total identified peaks are
listed in Table 6-5.
TABLE-US-00037
TABLE 6-1
Total Fucosylation (%)
Condition Day 6 Day 10 Day 12
CDM1 48.75 -- 46.26
CDM1 + UMG 49.21 -- 44.38
CDM1 + UMG + Dex 48.88 -- 46.23
CDM2 -- 45.68 45.14
CDM2 + UMG -- 46.36 45.27
CDM2 + UMG + Dex -- 46.92 --
CDM3 49.24 -- 45.59
CDM3 + UMG 48.71 -- 42.61
CDM3 + UMG + Dex 49.36 -- 44.56
Soy hydrolysate 1 51.37
Soy hydrolysate 2 52.43
U is uridine,
M is manganese,
G is galactose,
Dex is dexamethasone
TABLE-US-00038
TABLE 6-2
Total Sialylation (%)
Condition Day 6 Day 10 Day 12
CDM1 44.06 -- 39.14
CDM1 + UMG 43.72 -- 35.8
CDM1 + UMG + Dex 43.2 -- 36.72
CDM2 -- 37.62 36.67
CDM2 + UMG -- 37.57 36.29
CDM2 + UMG + Dex -- 38.06 --
CDM3 44 -- 31.21
CDM3 + UMG 42.48 -- 30.84
CDM3 + UMG + Dex 43.82 -- 32.74
Soy hydrolysate 1 58.24
Soy hydrolysate 2 59.23
U is uridine,
M is manganese,
G is galactose,
Dex is dexamethasone
TABLE-US-00039
TABLE 6-3
Mannose-5 (%)
Condition Day 6 Day 10 Day 12
CDM1 6.76 -- 10.1
CDM1 + UMG 6.9 -- 13.17
CDM1 + UMG + Dex 6.23 -- 8.86
CDM2 -- 9.71 11.96
CDM2 + UMG -- 9.44 10.93
CDM2 + UMG + Dex -- 8.21 --
CDM3 2.31 -- 12.63
CDM3 + UMG 2.71 -- 13.38
CDM3 + UMG + Dex 2.05 -- 11.98
Soy hydrolysate 1 5.19
Soy hydrolysate 2 5.24
U is uridine,
M is manganese,
G is galactose,
Dex is dexamethasone
TABLE-US-00040
TABLE 6-4
Total Galactosylation (%)
Condition Day 6 Day 10 Day 12
CDM1 68.44 -- 62.9
CDM1 + UMG 69.25 -- 59.02
CDM1 + UMG + Dex 69.05 -- 63.26
CDM2 -- 65.33 63.68
CDM2 + UMG -- 68.13 66
CDM2 + UMG + Dex -- 69.35 --
CDM3 74.57 -- 62.28
CDM3 + UMG 74.82 -- 62.2
CDM3 + UMG + Dex 76.48 -- 65.18
Soy hydrolysate 1 79.64
Soy hydrolysate 2 80.55
U is uridine,
M is manganese,
G is galactose,
Dex is dexamethasone
TABLE-US-00041
TABLE 6-5
Total Identified Peaks (%)
Condition Day 6 Day 10 Day 12
CDM1 87.28 -- 84.67
CDM1 + UMG 88.43 -- 83.82
CDM1 + UMG + Dex 87.36 -- 83.44
CDM2 -- 86.23 86.67
CDM2 + UMG -- 87.81 86.87
CDM2 + UMG + Dex -- 87.53 --
CDM3 86.38 -- 86.31
CDM3 + UMG 87.07 -- 86.13
CDM3 + UMG + Dex 87.18 -- 87.43
Soy hydrolysate 1 93.93
Soy hydrolysate 2 94.74
U is uridine,
M is manganese,
G is galactose,
Dex is dexamethasone
[0626] The total fucosylation, total sialylation, total galactosylation
and mannose-5 observed on day 12 of the cultures of the various CDMs was
42.61% to 46.26%, 30.84% to 39.14%, 59.02 to 66% and 8.86% to 13.38%,
respectively. These values for glycosylation differ from the
glycosylation values obtained using soy hydrolysate.
[0627] Lastly, color measurements were carried out for the clarified
harvests obtained from cells expressing aflibercept in CDM1, CDM2, and
CDM3 supplemented with uridine, manganese, and galactose. The operating
parameters for the bioreactor study steps will be known to one of
ordinary skill in the art.
Example 7. Affinity Production of Anti-VEGF Proteins
7.1 Expression of VEGF MiniTrap
[0628] The coding regions of recombinant VEGF MiniTrap (e.g., MT5, SEQ ID
NO.: 46) was operably linked to a signal sequence, cloned into a
mammalian expression vector and transfected into Chinese hamster ovary
(CHO-K1) cells; the stably transfected pools were isolated after
selection with 400 .mu.g/mL hygromycin for 12 days. The stable CHO cell
pools, grown in chemically defined protein-free medium, were used to
produce proteins for testing. The recombinant polypeptides were secreted
from the cells into the growth medium.
[0629] Sequences of constituent domains of the VEGF MiniTrap [0630]
Human Flt1 (accession # NP 001153392.1) [0631] Human Flk1 (accession # NP
002244.1) [0632] Human Fc (IGHG1, accession # P01857-1)
[0633] The recombinant VEGF MiniTrap having (MT5) was obtained from this
process and was further processed.
7.2 Preparation of Affinity Chromatography Columns
[0634] Five distinct proteins capable of binding to the VEGF MiniTrap
(MT5) were evaluated. The proteins used include a VEGF.sub.165 (SEQ ID
NO.: 72), mAb1 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 73
is a heavy chain and SEQ ID NO.: 74 is a light chain); mAb2 (a mouse
anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 75 is a heavy chain and SEQ
ID NO.: 76 is a light chain); mAb3 (a mouse anti-VEGF-R1 mAb mouse IgG1
where SEQ ID NO.: 77 is a heavy chain and SEQ ID NO.: 78 is a light
chain) and mAb4 (a mouse anti-VEGFR1 mAb mouse IgG1 where SEQ ID NO.: 79
is a heavy chain and SEQ ID NO.: 80 is a light chain).
[0635] The column was activated by washing the columns with 6 column
volumes (CV) of 1 mM ice-cold hydrochloric acid at a flow rate not
exceeding 1 mL/min. Ten mg of each of the proteins were loaded onto three
HiTrap NETS-Activated HP affinity columns (1 mL, GE Healthcare, Cat
#17-0716-01) and the columns were closed to allow coupling to take place
for 30 minutes at room temperature. The columns were washed with 18
column volumes of 0.5 M sodium acetate, 0.5 M NaCl, pH 4.0 and the open
sites were blocked with 18 column volumes of 0.5 M Tris-HCl, 0.5 M NaCl
pH 8.3 (the wash was carried out in the following order: 6 column volumes
of 0.5 M Tris-HCl, 0.5 M NaCl, pH 8.3; 6 column volumes of 0.5 M sodium
acetate (sodium acetate: JT Baker, Cat #3470-01) 0.5 M NaCl, pH 4.0; 6
column volumes of 0.5 M Tris, pH 8.3; incubate the column for 30 minutes
at room temperature; 6 column volumes of 0.5 M sodium acetate buffer, 0.5
M NaCl pH 4.0; 6 column volumes of 0.5 M Tris-HCl, 0.5 NaCl, pH 8.3 and 6
column volumes of 0.5 M sodium acetate buffer, 0.5 M NaCl pH 4.0). The
columns were stored in DPBS, pH 7.5. The five columns evaluated are
designated as column 1 (comprising VEGF.sub.165), column 2 (comprising
mAb1), column 3 (comprising mAb2), column 4 (comprising mAb3) and column
5 (comprising mAb4).
7.3 Production of MiniTrap Using Affinity Chromatography
[0636] Sample Preparation. Two different production processes for the
MiniTrap were performed. In one case, material comprising a MiniTrap
sample was produced using each of the affinity columns where the parent
material (MiniTrap) was diluted in 1.times.DPBS buffer to 20 mg/mL and
was applied to the column and included at RT for 30 minutes. Using the
affinity column, the MiniTrap was isolated from 7000 ppm of HCP.
[0637] Alternatively, harvested culture supernatant was used which
comprised 0.4 mg/mL of protein in the supernatant and loaded onto the
different affinity columns (1-5) separately. No further dilution was
performed. The affinity columns were then washed with 9 CV of
1.times.DPBS buffer followed by eluting the proteins with IgG elution
buffer, pH 2.8 (Thermo, Cat #21009).
[0638] MiniTrap material obtained as described above was then filtered
through a 0.45 .mu.m filter or centrifuged before loading onto the
columns prepared as described in Section 7.2 above. Twenty-five mL of
loading solution comprising approximately 0.4 mg/mL protein was loaded
onto each of the columns and incubated for 20 minutes. Each column was
washed with 9 CV of DPBS (Invitrogen, Cat #14190-144) before elution for
equilibration. The amount of MT5 in the wash fractions is shown in Table
7-1. The washes were followed by elution using 6 CV of pH 2.8 (Commercial
Elution Buffer, (Thermo, Cat #21009)) and 100 mM glycine buffer pH 2.5
and fractions were quickly neutralized with the addition of 1 M Tris, pH
7.5 (Invitrogen, Cat #15567-027). The amount of MiniTrap in the eluted
fractions is also shown in Table 7-1.
[0639] The MiniTrap (MT5) was successfully produced from all five affinity
columns. The yield from the column with VEGF.sub.165 was higher than
compared to mAb1 and mAb2 columns. The mAb3 and mAb4 comprising humanized
anti-VEGFR1 mAb also showed successful production of MT5 with similar
yield to mAb1 and mAb2. In Table 7-1, the expected yield was calculated
based on 100% conjugation efficiency and 1:1 molar ratio of
affinity-captured protein to MT5.
[0640] Multiple runs were carried out using columns 1 and 2 following the
method discussed in Section 7.3 (Table 7-2 for column 1 and Table 7-3 for
column 2).
[0641] The columns were stored at 4.degree. C. for about 5 weeks. A
similar amount of MT5 was eluted from each production demonstrating good
column stability.
7.5 Stability Study of the Produced VEGF MiniTrap
[0642] SDS-PAGE analysis of the eluted fractions from the three columns
(column 1, column 2, and column 3) was performed. The samples were
prepared in non-reducing and reducing SDS-PAGE sample buffer and run on a
4-12% gradient NuPage bis-Tris gel using 1.times.MES (Cat. No. NP0322,
Invitrogen, Carlsbad, Calif.).
[0643] The wells were loaded with (1) molecular weight standard, (2)
loading solution, (3) column wash from column 1, (4) eluted fraction from
column 2, (5) eluted fraction from column 1, (6) eluted fraction from
column 3, (7) MT5 stored at pH 2.8 for 1 min, (8) MT5 stored at pH 2.8
for 30 min, and (9) molecular weight standard (FIG. 33 and FIG. 34). The
analysis demonstrated that fractions obtained from the eluted fractions
from all the three affinity columns (columns 1-3) showed similar size
profiles and the use of the affinity columns did not destabilize the
MiniTrap.
7.6 Host Cell Protein Level Calculations
[0644] A standard curve of concentration of host cell proteins was
obtained using CHO HCP ELISA Kit, 3G (F550) (Cygus Technologies) (FIG. 32
and Table 7-4). The amount of HCPs in the loading solutions and the
eluted fractions was calculated using the standard curve as depicted in
FIG. 32 and curve formula listed in Table 7-4.
TABLE-US-00045
TABLE 7-4
Low EC.sub.50 High
Curve Formula Asymptote Slope (ng/mL) Asymptote R.sup.2
Y = (A - D)/(1 + 0.2 1.9 32.9 2.3 1
(X/C){circumflex over ( )}B) + D
[0645] The total HCPs were calculated using the standard curve and the
chart with the total amount of host cell proteins is shown in FIG. 35A.
FIG. 35B also shows total amount of host cell proteins in the load
compared to the washes and eluted fractions from columns 1, 2, 4 and 5.
Multiple runs were carried out using the columns and the ( #) in FIG. 35B
represent the run from which the fraction was evaluated.
[0646] The use of affinity capture using proteins capable of binding to
MiniTrap showed an efficient reduction of HCPs from about 7000 ppm to
about 25-50 ppm. As observed for the yield, the column with VEGF.sub.165
showed higher purity of MiniTrap from HCPs than shown by mAb1 and mAb2
columns.
7.7 SEC Profiles of VEGF MiniTrap Before and After Affinity Production
[0647] SEC profiles of the eluted fractions from three columns (columns
1-3) were compared to the SEC profile of MiniTrap in the loading
solution. As seen in FIG. 36 and Table 7-5, the SEC profiles of the MT5
before or after affinity production were highly similar.
TABLE-US-00046
TABLE 7-5
Peak No. Retention % Peak Retention % Peak Retention % Peak Retention %
Peak
as in Time Area Time Area Time Area Time Area
FIG. 36 Loading solution Column 1 Eluate Column 2 Eluate Column 3 Eluate
1 6.8 1.8 6.8 1.2 6.9 1.1 7.0 1.2
2 7.8 94.6 7.9 97.2 7.9 97.3 7.9 98.3
3 9.4 3.6 10.2 1.7 11.4 1.6 11.3 0.5
7.8 Kinetics of VEGF MiniTrap Pre and Post Column Samples Binding to
mAb1, mAb2 and VEGF.sub.165
[0648] Kinetic studies were performed using a Biacore T200 instrument.
[0649] Equilibrium dissociation constants (K.sub.D values) for
VEGF.sub.165 binding to MiniTrap in the eluates from columns 1 and 2 and
loading solution were determined using a real-time surface plasmon
resonance biosensor using a Biacore T200 instrument. All binding studies
were performed in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v
Surfactant Tween-20, pH 7.4 (HBS-ET) running buffer at 25.degree. C. The
Biacore sensor surface was first derivitized by amine coupling with a mAb
1 to capture MT5. A cartoon representation of the binding study is shown
in FIG. 37.
[0650] Briefly, the eluates from the columns and loading solution were
diluted into an HBS-EP (Biacore) buffer and injected across the
immobilized protein matrices at a capture level of .about.70 RUs. The
VEGF.sub.165 was then injected at a flow rate of 50 .mu.L/min. Equivalent
concentration of analyte was simultaneously injected over an untreated
reference surface to serve as blank sensorgrams for subtraction of bulk
refractive index background. The sensor chip surface was regenerated
between cycles with two 5-min injections of 10 mM Glycine, at 25
.mu.L/min. The resultant experimental binding sensorgrams were then
evaluated using the BIA evaluation 4.0.1 software to determine kinetic
rate parameters. Datasets for each sample were fit to the 1:1 Langmuir
model. For these studies, binding and dissociation data were analyzed
under the Global Fit Analysis protocol while selecting fit locally for
maximum analyte binding capacity (RU) or Rmax attribute. In this case,
the software calculated a single dissociation constant (kd), association
constant (ka), and affinity constant (Kd). The equilibrium dissociation
constant is K.sub.D=kd/ka. The kinetic on-rate, the kinetic off rate, and
the overall affinities were determined by using different VEGF.sub.165
concentrations in the range of 0.03-2 nM (Table 7-6). The dissociative
half-lives (t1/2) were calculated from the kinetic rate constants as:
t.sub.1/2=ln(2)/60*Kd. Binding kinetic parameters for MT5 to VEGF.sub.165
obtained from before and after the affinity chromatography production at
25.degree. C. are shown in Table 7-6.
[0651] The affinity (KD), on rate (ka, M-1s-1) and off rate (kd) for MT5
produced by affinity chromatography compared with loading solution to
assess the effect(s) of affinity chromatography step showed no change in
the kinetics of MT5 from different samples. The SPR sensorgrams of the
VEGF MiniTrap constructs are shown in FIG. 38.
[0652] Chromatographic production of harvest as obtained by step 7.1 was
carried out using column 1 (hVEGF.sub.165) and column 2 (mAb1) as shown
in 7.3. The columns were used for multiple chromatographic cycles. The
yields in the columns did not vary significantly due to additional runs,
suggesting that the columns retained binding capacity (Table 7-7).
[0653] HCP calculations in the loading solutions, wash fractions and
eluted fractions for columns 1 and 2 were obtained using the method
described in 7.4 (FIG. 39). The total HCPs calculated showed that
repeated use of the columns did not reduce the ability of the columns to
bind to MiniTrap.
7.10 Optimizing the Affinity Chromatographic Columns
[0654] The chromatographic production of harvest material as obtained in
Section 7.1 was performed using column 1 (VEGF.sub.165) and column 2
(mAb1). For the optimization studies, 14 mg or 45 mg instead of 10 mg of
the VEGF165 or the anti-VEGF R1 mAb were loaded onto two HiTrap
NETS-Activated HP affinity columns (1 mL, GE Healthcare) and the columns
were closed to allow coupling to take place for 30 minutes at room
temperature. The column preparation and production of the harvest
including the MiniTrap was carried out as discussed in 7.2 and 7.3 above.
The amount of MT5 in the wash and eluted fractions is shown in Table 7-8.
The comparison of affinity column with 14 mg or 45 mg (VEGF.sub.165 or
anti-VEGF R1 mAb (mAb1)) conjugation amount instead of 10 mg shows an
increased yield of MiniTrap from both columns. Thus, the column yield
using the outlined method can be improved by optimizing the protein to
column ratio or by increasing the conjugation efficiency by changing the
pH, incubation time, incubation temperature, etc.
[0655] A cell culture sample from MT5 expression was produced using column
1 as discussed in Section 7.3 above. The eluate obtained was subjected to
cation exchange chromatography (CEX) column (HiTrap Capto S, 1 mL). The
operating conditions of the column are shown in Table 7-9.
TABLE-US-00050
TABLE 7-9
Steps Affinity Cation Exchange (CEX)
Column Affinity Column, 1 mL HiTrap Capto S, 1 mL
Load MT5 CM2926 20 mM Acetate,
pH 5.0 (Load/Wash1)
Wash 1X DPBS pH 7.2 10 mM Phosphate, pH 7.0
Elution Pierce .TM. IgG 50 mM Tris, 62.5 mM
Elution Buffer (NH.sub.4).sub.2SO.sub.4, pH 8.5
Regeneration/ 10 mM Glycine 50 mM Tris, 1 M
Strip pH 2.5 (NH.sub.4).sub.2SO.sub.4, pH 8.5
[0656] The total HCP in the original/starting cell culture sample, the
affinity chromatography column 1 eluate and CEX eluate was about 230,000
ng/mL, about 9,000 ng/mL and about 850 ng/mL, respectively. The HCP
amounts were quantitated determined using the Cygnus CHO HCP ELISA Kit,
3G, as mentioned above.
7.12 Use of Affinity Chromatography to Produce Other Anti-VEGF Proteins
[0657] Column 1 was evaluated to study its ability to produce other
anti-VEGF proteins. Aflibercept and a scFv fragment with VEGF binding
potential were used for this study. The production processes were carried
out as discussed in Section 7.3. Table 7-10 demonstrates that column 1
was successful in binding and eluting other anti-VEGF proteins.
Example 8. Mass Spectrometry-Based Characterization of VEGF MiniTrap
Constructs
[0658] Materials. VEGF MiniTrap (MT1) was produced from aflibercept as
described in Example 1. VEGF MiniTrap 5 (MT5) was produced as described
in Example 7. VEGF MiniTrap (MT6) was produced by the following method:
the coding regions of recombinant VEGF MiniTrap (MT5) were operably
linked to a signal sequence and cloned into a mammalian expression
vector, transfected into Chinese hamster ovary (CHO-K1) cells and stably
transfected pools were isolated after selection with 400 .mu.g/mL
hygromycin for 12 days. The stable CHO cell pools, grown in CDM were used
to produce proteins for analysis.
8.1 Deglycosylation of Glycoproteins.
[0659] Samples from clarified harvest of MT1, MT2 and MT3 were diluted or
reconstituted to a concentration of 0.52 mg/mL into a 28.8 .mu.L solution
of 1% (w/v) RG surfactant (RapiGest SF, Waters, Milford, Mass.) and 50 mM
HEPES (pH 7.9). These solutions were heated to approximately 95.degree.
C. over 2 min, allowed to cool to 50.degree. C., and mixed with 1.2 .mu.L
of PNGase F solution (GlycoWorks Rapid PNGase F, Waters, Milford, Mass.).
Deglycosylation was completed by incubating the samples at 50.degree. C.
for 5 min.
8.2 HILIC-Fluorescence-ESI-MS (MS/MS) Analysis.
[0660] MT1 was analyzed via HILIC separation combined with fluorescence
and mass spectrometric detection. MT2 and MT3 were analyzed using only
HILIC. Chromatography was performed using a Waters 2D Acquity UPLC
equipped with photodiode array and fluorescence (FLR) detectors and
interfaced with a Waters Synapt G2-S mass spectrometer (MS conditions). A
hydrophilic interaction chromatography (HILIC) mode of separation was
used with a Waters UPLC Glycan BEH Amide column, 150.times.2.1 mm, 1.7
.mu.m. The column temperature was set to 60.degree. C. and the
autosampler temperature was set to 5.degree. C. The injection volume was
50 .mu.L. The photodiode array scan range was 190-700 nm. The FLR was set
to excitation 265 nm, emission 425 nm for RapiFluor-labeled glycans and
excitation 274 nm, and emission 303 nm for tyrosine present in the
glycopeptides. The initial flow rate was 0.4 mL/min with mobile phase A
comprising of 100 mM ammonium formate (pH 4.4) and mobile phase B being
acetonitrile.
8.3 MS Conditions
[0661] Liquid chromatography/mass spectrometry (LC/MS) experiments were
conducted using a Waters Synapt G2-S mass spectrometer. The scan range
was mass-to-charge ratio 100-2400 for positive and negative ion mode
analyses. Scan time was 1s, and glu-fibrinopeptide B was constantly
infused (2 .mu.L/min) as a calibrant ("lock mass"). The capillary voltage
was set to 2.5 kV, with a source temperature of 120.degree. C. and
desolvation temperature of 500.degree. C. The nitrogen nebulizer gas flow
was set to 7001/h.
8.4 Native SEC-MS
[0662] ACQUITY UPLC I class system (Waters, Milford, Mass.) was coupled to
Q Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo
Scientific, Bremen, Germany) for all online SEC-MS analyses. ACQUITY UPLC
Protein BEH SEC Column (200 .ANG., 1.7 .mu.m, 4.6.times.300 mm) was set
at 30.degree. C. and used for protein separation. The mobile phase was
100 mM ammonium acetate at pH 6.8. Each separation was 30 minutes with a
flow rate of 0.3 mL/min, and the injection amount was set to 40 .mu.g.
The following MS parameters were used for online SEC-nano-ESI-MS data
acquisition. Each acquisition was 25 minutes beginning immediately after
sample injection. The deglycosylated samples were ionized in positive
mode with 3 kV spray voltage, 200.degree. C. capillary temperature, and
70 S-lens RF level. In-source CID was set at 75 eV. Full MS scans were
acquired at 15 K resolving power with mass range between m/z 2000-8000. A
maximum injection time of 100 ms, automatic gain control target value of
3e6, and 10 microscans were used for full MS scans.
8.5 Peptide Mapping
[0663] Sample preparation for peptide mapping. Reduction was achieved by
the addition of 500 mmol/L dithiothreitol (DTT) to a final concentration
of 5 mmol/L followed by incubation at 4.degree. C. for 60 min. Alkylation
was performed by adding 500 mmol/L iodoacetamide (IAM) to a final
concentration of 10 mmol/L and incubating at 4.degree. C. for 60 min in
the dark. The denaturing buffer was exchanged for digestion buffer (1
mol/L urea in 0.1 mol/L Tris, pH 7.8) using Zeba.TM. Spin 7 K MWCO
size-exclusion desalting columns (P/N 89882) (Thermo Scientific, Waltham,
Mass.) according to the manufacturer's instructions. Recombinant porcine
trypsin (purchased from Sigma, Cat #03708985001) was added at a 1:18
(enzyme: sample) mass ratio (based on VEGF MiniTrap protein concentration
as measured by UV-Vis spectrophotometry after buffer exchange), the
concentration of VEGF MiniTrap proteins was adjusted to 0.5 .mu.g/.mu.L
and digestion allowed to proceed during a 4 h incubation at room
temperature. When the digestion was complete, 0.1% formic acid in LC-MS
grade water was added at a 1:1 volume ratio. Digests were stored at
-80.degree. C. until analysis.
[0664] LC-MS/MS analysis of tryptic digests. One or more 2.5 .mu.g (10
.mu.L) of peptide digests were loaded via autosampler onto a C18 column
enclosed in a thermostatted column oven set to 40.degree. C. Samples were
held at 7.degree. C. while queued for injection. The chromatographic
method was initiated with 98% Mobile Phase A (0.1% volume fraction of
formic acid in water) and 2% Mobile Phase B (0.1% volume fraction of
formic acid in acetonitrile) with the flow rate set at a constant 0.200
mL/min. After a 10 min wash, peptides were eluted over a 110 min gradient
in which Mobile Phase B content rose at a rate of 0.39% per min to reach
a final composition comprising 45% Mobile Phase B. Prior to the next
sample injection, the column was washed for 15 min with 97% Mobile Phase
B, then equilibrated at 98% Mobile Phase A for 25 min. The eluate was
diverted to waste for the first 1.5 minutes and final 5 minutes of the
run. Peptides eluting from the chromatography column were analyzed by UV
absorption at 214 nm followed by mass spectrometry on the LTQ Orbitrap
Elite or Discovery XL. Replicate peptide mapping data were collected for
PS 8670 and RM 8671 samples to include three tandem MS (MS/MS) analyses
and one MS-only analysis each. The MS/MS analyses were performed for
peptide identification in data-dependent mode in which one cycle of
experiments consisted of one full MS scan of 300 m/z to 2000 m/z followed
by five sequential MS/MS events performed on the first through fifth most
intense ions detected at a minimum threshold count of 500 in the MS scan
initiating that cycle. The sequential mass spectrometry (MS'') AGC target
was set to 1E4 with microscans=3. The ion trap was used in centroid mode
at normal scan rate to analyze MS/MS fragments. Full MS scans were
collected in profile mode using the high resolution FTMS analyzer
(R=30,000) with a full scan AGC target of 1E6 and microscans=1. Ions were
selected for MS/MS using an isolation width of 2 Da, then fragmented by
collision induced dissociation (CID) with helium gas using a normalized
CID energy of 35, an activation Q of 0.25 and an activation time of 10
msec. A default charge state was set at z=2. Data dependent masses were
placed on the exclusion list for 45s if the precursor ion triggered an
event twice within 30s; the exclusion mass width was set at .+-.1 Da.
Charge state rejection was enabled for unassigned charge states. A
rejection mass list included common contaminants at 122.08 m/z, 185.94
m/z, 355.00 m/z, 371.00 m/z, 391.00 m/z, 413.30 m/z, 803.10 m/z, 1222.10
m/z, 1322.10 m/z, 1422.10 m/z, 1522.10 m/z, 1622.10 m/z, 1722.10 m/z,
1822.10 m/z, and 1922.10 m/z. MS-only analyses were performed for the
generation of the TIC non-reduced peptide map and reduced maps.
8.6 Results
[0665] Structure of VEGF MiniTrap constructs. Structure of VEGF MiniTraps
MT1, MT5 and MT6 are depicted in FIG. 40, FIG. 41, FIG. 43 and FIG. 44.
[0666] Initial mass analysis using SEC-MS confirmed the identities of all
three molecules at intact protein level after deglycosylation (FIG. 42).
The Total Ion Chromatogram (TIC) of the native SEC-MS analysis
demonstrates detection of an intact VEGF MiniTrap molecules at around
12-13 minutes. The expansion of the low molecular weight (LMW) region of
the TIC showed presence of LMW impurities in all the three protein
samples.
[0667] The deconvoluted mass spectra for the VEGF MiniTraps further
confirmed their identity and provided data for elucidation of the major
PTMs present in the samples comprising MT1 and MT5 (FIG. 43), which are
dimers and MT6 (FIG. 44) which is a single chain protein.
[0668] Analysis of MT1 sample. The LMW species identified from the TIC of
the SEC-MS analysis of the samples comprising MT1 was extracted to
examine three distinct LMW impurities--LMW1, LMW2, and LMW3 (FIG. 45A and
FIG. 45B). LMW1 species comprised a truncated species of aflibercept.
LMW2 species comprised the Fc impurity present in the sample form the
cleavage of aflibercept which was performed to produce MiniTrap. LMW3
species comprised a monomer possibly cleaved from the MT1 (dimer)
molecule.
[0669] MT1 sample did not show presence of FabRICATOR enzyme, which had
been used to cleave aflibercept to form a MiniTrap protein. The enzyme,
if present, is detected at about 11.5 and 12.5 minutes. No such peak was
detected during the SEC-MS analysis of the MT1 sample (FIG. 46).
[0670] Analysis of MT5 sample. The LMW species identified from the TIC of
the SEC-MS analysis of the samples comprising MT5 was extracted to
examine the presence of two distinct LMW impurities--LMW1 and LMW2 (FIG.
47).
[0671] Analysis of MT6 sample. The LMW species identified from the TIC of
the SEC-MS analysis of the samples comprising MT1 was extracted to
examine the presence of three distinct LMW impurities--LMW1, LMW2, and
LMW3 (FIG. 48). LMW2 species comprised a fragment of the MT6 wherein the
cleavage produced the fragment of VEGF MiniTrap with the G4S linker. LMW5
species comprised a fragment of the MT6 wherein the cleavage occurred
right before or after the G4S linker.
[0672] The glycans in the MT6 sample were identified by their mass and
elution order in the HILIC chromatography method using the glucose unit
value pioneered by Waters and the National Institute for Bioprocessing
Research and Training (Dublin, Ireland) (FIG. 49A and FIG. 49B).
[0673] Free thiol Quantification. Cysteine residues of the VEGF MiniTrap
constructs may be involved in the formation of intra- and inter-molecular
disulfide bond(s) or they may exist as free thiols. The presence of a
sulfide bonds in peptides and proteins has been shown to impose
conformational rigidity on a protein. Thiols can be detected by a variety
of reagents and separation techniques. The analysis of the three VEGF
MiniTrap constructs for a very low level of free thiols is shown in Table
8-1.
TABLE-US-00052
TABLE 8-1
Peptide (site of
Location free cysteine) MT1 MT5 MT6
VEGF R1 ELVIPCR <0.1% <0.1% <0.1%
(SEQ ID NO.: 81)
VEGF R2 LVLNCTAR 0.3% 0.3% 0.3%
(SEQ ID NO.: 82)
Fc Hinge THTCPPCPAPELLG 0.0% 0.0% N/A
(SEQ ID NO.: 83)
[0674] Trisulfide Quantification. Similar to free thiols in Cys residues
of the VEGF MiniTrap constructs, trisulfide bonds can influence the
structure of the protein. The analysis of the three VEGF MiniTrap
constructs under conditions with very low level of free thiols is shown
in Table 8-2.
TABLE-US-00053
TABLE 8-2
Location Peptide MT1 MT5 MT6
VEGF R1 ELVIPCR- 0.1% <0.1% 0.1%
EIGLLTCEATVNGHLYK
(SEQ ID NO.: 84)
VEGF R2 LVLNCTAR- <0.1% <0.1% <0.1%
SDQGLYTCAASSGLMTK(K)
(SEQ ID NO.: 85)
Fc Hinge THTCPPCPAPELLG- 1.5% 3.7% N/A
THTCPPCPAPELL(G)
(SEQ ID NO.: 86)
[0675] Intra-chain disulfide in the Hinge region. Mispaired disulfide
bonds in the hinge region can have implications on the structure,
function and stability of the VEGF MiniTrap constructs. The analysis of
the three VEGF MiniTrap constructs for a very low or no intra-chain
disulfide binds in the hinge region of the VEGF MiniTrap constructs
[THTC*PPC*PAPELLG, C* shows where intra-chain sulfide bond can be formed]
is shown in Table 8-3.
[0676] Cross and parallel disulfide linkage isomer quantification. For MT1
and MT5, which are dimers connected by parallel disulfide bonds in the
hinge regions, there is a possibility of isomers wherein the disulfide
bonds in the hinge region can be crossed (FIG. 50).
[0677] The quantification of types of disulfide bond, parallel versus
cross, showed that MT2 recombinantly expressed protein had a slightly
higher level of cross disulfide bridge in the Fc hinge region compared to
the MT1--which is a FabRICATOR digested molecule (Table 8-4).
TABLE-US-00056
TABLE 8-5
PTM Site Modified Peptide MT1 MT5 MT6
Deamidation Asn84 EIGLLTCEATVNGHLYK (SEQ Succinimide 3.1% 3.2% 3.2%
(Asn319) ID NO.: 87) Asp/iso Asp 21.9% 18.9% 20.9%
Asn99 QTNTIIDVVLSPSHGIELSVGEK Succinimide 4.6% 4.6% 4.0%
(Asn334) (SEQ ID NO.: 88) Asp/iso Asp 0.7% 0.5% 0.6%
Oxidation Met10 SDTGRPFVEMYSEIPEIIHMTEGR (SEQ ID NO.: 1.8% 2.1% 2.1%
89)
Met20 SDTGRPFVEMYSEIPEIIHMTEGR (SEQ ID NO.: 2.9% 3.0% 2.7%
90)
Met245 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSS -- -- 1.4%
DTGRPFVEMYSEIPEIIHMTEGR (SEQ ID NO.:
91)
Met255 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSS -- -- 2.7%
DTGRPFVEMYSEIPEIIHMTEGR (SEQ ID NO.:
92)
Met163 TQSGSEMK (SEQ ID NO.: 93) 4.3% 4.3% 3.8%
(Met398)
Met192 SDQGLYTCAASSGLMTK (SEQ ID NO.: 94) 5.0% 5.0% 4.2%
(Met427)
C-term G1y211 THTCPPCPAPELLG (SEQ ID NO.: 95) 0.1% 2.0% --
Glycine loss
[0679] Evaluation of PTMs in all the three VEGF MiniTrap constructs showed
comparable levels of PTMs (Table 8-5). The deamidation observed at Asn84
to form succinimide was in the range of about 3.1-3.2% and to form
aspartic acid/iso aspartic acid was 18.9-21.9%. Oxidation of several
methionine residues (e.g., Met10, Met 20m Met163 and Met192) was observed
in the range of about 0.7-6.8% for all the three VEGF MiniTrap
constructs. MT6, which, in contrast to MT1 and MT5, comprises a linker,
showed additional oxidation of methionine residues on the linker (e.g.,
Met245 and Met255). About 0.1% and 2.0% of the C-terminal glycine
(Gly211) in MT1 and MT5 showed a glycine loss. This was not observed for
MT6, which lacks a C-terminal glycine.
[0680] Advanced glycation end-product modifications related to lysine and
arginine glycation. Glycation of the VEGF MiniTrap constructs can alter
their structure and function, leading to impaired anti-VEGF activity.
[0681] Evaluation of modifications in all three VEGF MiniTrap constructs
showed comparable levels (Table 8-6).
[0682] Modified sites. The modified sites on the VEGF MiniTrap constructs,
as elucidated by the intact mass analysis as per Section 8.4, were
confirmed and quantified using reduced peptide mapping as illustrated in
Section 8.5 (Table 8-7). The site T90N91 for peptide sequence TNYLTHR,
the ** represents that asparagine was converted to aspartic acid after
truncation, whereas for site N99T100 the peptide sequence
QTNTIIDVVLSPSHGIELSVGEK, the * represents a high level of no-specific
cleavage by trypsin. These two truncation sites were found to form LMW
species impurities during evaluation of MT1 and MT5. The truncation at
M245Y246 was found only on MT3 which had the unique linker and was
responsible for the LMW2 species impurity during the MT3 preparation.
TABLE-US-00058
TABLE 8-7
Site Peptide Sequence MT1 MT5 MT6
N99T100 QTNTIIDVVLSPSHGIELSV 12.6% 13.2% 13.6%
GEK* (SEQ ID NO.: 96)
T90N91 TNYLTHR** 0.5% 0.1% 0.3%
(SEQ ID NO.: 97)
M245Y246 GGGGSGGGGSGGGGSGGGGSG -- -- 1.8%
GGGSGGGGSSDTGRPFVEMYS
EIPEIIHMTEGR (SEQ ID
NO.: 98)
M10Y11 SDTGRPFVEMYSEIPEIIHMT 0.2% 1.5% 1.7%
EGR (SEQ ID NO.: 99)
[0683] Glycosites occupancy quantification. N-glycosylation is a common
PTM. Characterizing the site-specific N-glycosylation including N-glycan
macroheterogeneity (glycosylation site occupancy) and microheterogeneity
(site-specific glycan structure) is important for the understanding of
glycoprotein biosynthesis and function. The extent of glycosylation can
change depending on how the protein is expressed. The levels of
glycosylation at N36 were similar for all the three VEGF MiniTraps (Table
8-8 and FIG. 51). Similarly, the levels of glycosylation at N68 were also
similar for all the three VEGF MiniTraps (Table 8-8 and FIG. 52). The
levels of glycosylation at N123 were also similar for all the three VEGF
MiniTraps (Table 8-8 and FIG. 53), but mannose-5 was found to be elevated
in the MT1 preparation. For the VEGF MiniTrap constructs, glycosylation
at Asn196 was lower for MT5 and MT6, compared to MT1 (Table 8-8 and FIG.
54). Additionally, the mannose-5 was also elevated for the MT1
preparation than MT4 and MT6 preparations.
TABLE-US-00059
TABLE 8-8
Site Peptide MT1 MT5 MT6
N36 (R)VTSPNITVTLK 98.3% 98.1% 99.4%
(SEQ ID NO.: 100)
N68 (K)GFIISNATYK 51.9% 55.4% 64.9%
(SEQ ID NO.: 101)
N123 (K)LVLNCTAR 99.9% 99.4% 98.4%
(SEQ ID NO.: 102)
N196 (K)NSTFVR 98.6% 44.5% 55.1%
(SEQ ID NO.: 103)
[0684] Analysis of N-glycans. The glycosylation at N36 is shown in Table
8-9. G2F, G2FS, G2FS2 were the major N-glycans found in all the three
VEGF MiniTraps. For glycosylation at N68 shown in Table 8-10, G2F and
G2FS were the major N-glycans found in all the three VEGF MiniTraps. For
glycosylation at N123 is shown in Table 8-11, G2F and G2S were the major
N-glycans found in all the three VEGF MiniTraps and Mannose-5 was
detected at high levels in MT1 compared to MT5 and MT6. For glycosylation
at N196 shown in Table 8-12, G2, G2S, G2S2 were the major N-glycans found
in all the three VEGF MiniTraps and Mannose-5 was detected at high levels
in MT1 compared to MT5 and MT6.
[0685] O-glycans at the linker for MT3. The GS linker for MT3 was
evaluated to study 0-glycans on MT3. O-xylosylation was found to on
serine residues located on the GS linker of MT3
(GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGR, underlined
serine residues were glycosylated). The composition of the 0-glycans is
shown in Table 8-13.
TABLE-US-00064
TABLE 8-13
Composition Mass Annotation Number Level
Xylosylation +132.0 Tri <0.1%
di 1.5%
mono 15%
Xylose + Galactose +294.1 mono 0.9%
Xylose + Galactose + +585.2 mono 0.7%
Sialic Acid
[0686] HILIC-FLR-MS analysis. HILIC-FLR-MS analysis was performed for all
the VEGF MiniTrap proteins as described in Section 8.2. The analysis
showed that the N-linked glycans for MT5 and MT6 were similar but were
different than the ones obtained for MT1 (FIG. 55 shows the full scale
and stacked chromatograms, FIG. 56 shows full scale and overlaid
chromatograms and FIG. 57 shows the full scale, stacked and normalized
chromatograms).
[0687] Finally, the percent glycosylation and detailed glycan
identification and quantification for all three VEGF MiniTrap proteins is
listed in Table 8-14 and FIG. 58A-C, respectively. As observed in all the
glycan analysis, the glycosylation profile and mannose levels for MT5 and
MT6 are similar, but different from MT1.
Example 9. Production and Color Quantification Using Upstream Medium and
Feed Process Optimization
(A) Un-optimized CDM (Control Bioreactor)
[0688] The manufacture of MiniTrap described in Example 5 was employed.
[0689] The operating parameters for the study steps are as known to one of
ordinary skill in the art.
[0690] Medium at day 0=CDM1 and included the following nutrients,
antioxidants and metals: [0691] Cysteine was added at a cumulative
concentration of 8-9 mM [0692] Metals in Starting Medium are listed below
at 1.times. concentration (where the concentrations are prior to inoculum
addition): [0693] Fe=68-83 micromoles per liter of culture [0694] Zn=6-7
micromoles per liter of culture [0695] Cu=0.1-0.2 micromoles per liter of
culture [0696] Ni=0.5-1 micromoles per liter of culture
[0697] On harvesting MT1, the production procedure as shown in FIG. 59 was
followed. The operating parameters for the chromatography are known to
one of ordinary skill in the art. The operating parameters for the
affinity capture (step 3 of FIG. 59), affinity Flowthrough (step 5 of
FIG. 59), AEX (step 8 of FIG. 59), and HIC (step 9 of FIG. 59) are
outlined in Table 9-1. The proteolytic cleavage of aflibercept following
affinity capture and filtration step was carried out using the procedure
as outlined in Example 1.2.
TABLE-US-00066
TABLE 9-1
Affinity Affinity
Capture flowthrough AEX HIC
Steps MabSelect MabSelect POROS Capto
Resin SuRe SuRe 50 HQ Phenyl HS
Load 30 g/L 30 g/L 40 g/L 100 g/L
resin resin pH resin pH resin pH
6.80-7.20 8.30-8.50, 4.40-4.60
1.90-2.10 7.50-10.50
mS/cm mS/cm
Equilibration 20 mM 26 mM 50 mM 40 mM
Sodium Tris, Tris pH Tris,
Phosphate 16 mM 8.30-8.50, 30 mM
pH Sodium 1.90-2.10 Sodium
7.10-7.30, Phosphate, mS/cm Citrate,
2.60-3.20 18 mM 74 mM
mS/cm Acetate pH Acetate pH
6.90-7.10, 4.40-4.60,
2.00-4.00 7.50-10.50
mS/cm mS/cm
Wash 1 10 mM 26 mM 50 mM 40 mM
Sodium Tris, Tris pH Tris,
Phosphate, 16 mM 8.30-8.50, 30 mM
500 mM Sodium 1.90-2.10 Sodium
NaCl pH Phosphate, mS/cm Citrate,
7.10-7.30, 18 mM 74 mM
40-50 Acetate pH Acetate pH
mS/cm 6.90-7.10, 4.40-4.60,
2.00-4.00 7.50-10.50
mS/cm mS/cm
Wash 2 20 mM N/A N/A N/A
Sodium
Phosphate
pH
7.10-7.30,
2.60-3.20
mS/cm
Elution 40 mM 40 mM N/A N/A
Acetic Acid Acetic Acid
pH pH
2.80-3.20, 2.80-3.20,
0.28-0.36 0.28-0.36
mS/cm mS/cm
Regeneration/ 500 mM 500 mM 2 M Proprietary
Strip 1 Acetic Acid, Acetic Acid, Sodium buffer
pH pH Chloride
2.25-2.65, 2.25-2.65, (NaCl)
0.90-1.25 0.90-1.25
mS/cm mS/cm
Regeneration/ N/A N/A 1 N N/A
Strip 2 Sodium
Hydroxide
(NaOH)
[0698] Table 9-2 shows the color quantification of the pools obtained on
performing various chromatographic steps. The color quantification was
carried using samples from the pool having a protein concentration of 5
g/L.
[0699] Affinity Capture Pool refers to the eluate collected on performing
the affinity capture step (step 3 of FIG. 59). Enzymatic Pool refers to
the flowthrough collected on performing the enzymatic cleavage step (step
4 of FIG. 59). Affinity flowthrough Pool refers to the flowthrough
collected on performing the affinity flowthrough step (step 5 of FIG. 59)
and Affinity flowthrough Eluate refers to the eluate collected on
performing the affinity flowthrough step (step 5 of FIG. 59). AEX Pool
and AEX Strip refer to the flowthrough and stripped fractions obtained on
performing anion exchange chromatography step (step 8 of FIG. 59). HIC
Pool refers to the flowthrough collected on performing the hydrophobic
interaction chromatography step (step 9 of FIG. 59).
[0700] Each step as seen in Table 9-2 shows a reduction in coloration (as
observed from the reduction in b* values of the pools). For example, on
performing affinity flowthrough chromatography, the flowthrough fraction
has a b* value of 2.16 (reduced from a b* value of 2.52 for the
flowthrough collected from the affinity capture step). The flowthrough
and wash following the AEX separation further reduced the coloration, as
observed by reduction in the b* value from 2.16 to 0.74. As expected,
stripping the AEX column led to a sample with a yellow-brown color which
was significantly more intense than the coloration from the flowthrough
and wash following the AEX separation as seen from the b* values (8.10
versus 0.74). Lastly, a HIC step afforded a further reduction in color
(the b* value can be normalized for 5 g/L protein concentration from the
b* value obtained for HIC pool at 28.5 g/L protein concentration).
TABLE-US-00067
TABLE 9-2
Color Quantification of Samples at Various Production Steps
Sample Conc. (g/L) L* a* b*
Affinity Capture Pool 5.0 .+-. 0.1 98.75 -0.12 2.52
Enzymatic Cleavage Pool 5.0 .+-. 0.1 99.03 -0.07 1.61
Affinity flowthrough Pool 5.0 .+-. 0.1 98.95 -0.08 2.16
Affinity flowthrough Eluate 5.0 .+-. 0.1 98.92 -0.01 0.83
AEX Pool 5.0 .+-. 0.1 99.72 -0.03 0.74
AEX 2 M NaCl Strip 5.0 .+-. 0.1 96.25 -0.42 8.10
HIC Pool 28.5 98.78 -0.28 3.11
[0701] The effect of lowering the concentration of cysteine, lowering the
concentration of metals, and increasing anti-oxidants on coloration was
evaluated using the following protocols:
[0702] Medium at day 0=CDM1 [0703] Cysteine was added at a cumulative
concentration of 5-6 mM [0704] Antioxidants were added to CDM1 to reach
the following cumulative concentrations (where the concentrations are
prior to inoculum addition): [0705] Taurine=10 mM of culture [0706]
Glycine=10 mM of culture [0707] Thioctic Acid=0.0024 mM of culture [0708]
Vitamin C (ascorbic acid)=0.028 mM of culture [0709] Metals in Starting
Medium are listed below for the 1.times. level. [0710] Fe=68-83
micromoles per liter of culture [0711] Zn=6-7 micromoles per liter of
culture [0712] Cu=0.1-0.2 micromoles per liter of culture [0713] Ni=0.5-1
micromoles per liter of culture. [0714] The reduction of all the metals
included using 0.25.times. the concentrations noted above for the medium.
[0715] Upon harvesting of the MT1 sample, the production procedure as
shown in FIG. 59 was followed. The operating parameters for the
chromatography are known to one of ordinary skill in the art. The
operating parameters for the affinity capture, affinity flowthrough, and
HIC are outlined in Table 9-1. The proteolytic cleavage of aflibercept
following affinity capture and filtration step was carried out using the
procedure as outlined in Example 1.2.
[0716] Table 9-3 shows the color quantification of the pools obtained on
performing the various chromatographic steps. The color quantification
was carried using samples from the pool having a protein concentration of
5 g/L. The steps as seen in Table 9-3 afforded a similar production as
seen for steps in Table 9-2.
TABLE-US-00068
TABLE 9-3
Color Quantification of Samples at Various
Production Steps of MiniTrap
Sample Conc. (g/L) L* a* b*
Affinity Capture Pool 5.0 .+-. 0.1 99.18 -0.09 1.77
Enzymatic Cleavage Pool 5.0 .+-. 0.1 99.44 -0.06 1.17
Affinity flowthrough Pool 5.0 .+-. 0.1 99.32 -0.10 1.58
Affinity flowthrough Eluate 5.0 .+-. 0.1 99.74 -0.05 0.60
AEX Pool 5.0 .+-. 0.1 99.63 -0.07 0.50
AEX 2 M NaCl Strip 5.0 .+-. 0.1 97.63 -0.49 6.10
HIC Pool 27.6 99.07 -0.29 2.32
[0717] Comparing Table 9-2 and Table 9-3, it is evident that the "Low
Cysteine, Low Metals, and Increased Antioxidants Bioreactor Condition"
had lower color in affinity capture pool (b* value of 1.77) compared to
the "Control Bioreactor Condition" (b* value 2.52).
[0718] An MT sample with a concentration of 160 g/L, where the MT is
formed using the steps listed in Table 9-2 and Table 9-3, is predicted to
have a b* value of 13.45 for the "Low Cysteine, Low Metals, and Increased
Antioxidants Bioreactor Condition" and a b* value of 17.45 for the
"Control Bioreactor Condition." A 23% reduction in color is achieved
through optimization of the upstream media and feeds. Similarly, an MT
sample with a concentration of 110 g/L, where the MT is formed using the
steps listed in Table 9-2 and Table 9-3, is predicted to have a b* value
of 9.25 for the "Low Cysteine, Low Metals, and Increased Antioxidants
Bioreactor Condition" and a b* value of 12 for the "Control Bioreactor
Condition."
[0719] To understand how each production unit operation contributes to
color reduction, the b* value for each production process intermediate as
a percentage of the color of affinity capture pool was calculated (Table
9-4).
TABLE-US-00069
TABLE 9-4
b* as
% of
Affinity
Conc. Capture
Sample (g/L) b* .DELTA.b* Pool
Control Affinity 5.0 .+-. 0.1 2.52 N/A 100.0
Bioreactor Capture Pool
Enzymatic 5.0 .+-. 0.1 1.61 -0.91 63.8
Cleavage Pool
Affinity 5.0 .+-. 0.1 2.16 0.55 85.7
flowthrough
Pool
AEX Pool 5.0 .+-. 0.1 0.74 -1.42 29.4
HIC Pool 5.0 .+-. 0.1 0.55 -0.19 21.8
Low Cysteine, Affinity 5.0 .+-. 0.1 1.77 N/A 100.0
Low Metals, Capture Pool
and Increased Enzymatic 5.0 .+-. 0.1 1.17 -0.60 66.1
Antioxidants Cleavage
Bioreactor Pool
Affinity 5.0 .+-. 0.1 1.58 0.41 89.2
flowthrough
Pool
AEX Pool 5.0 .+-. 0.1 0.50 -1.08 28.2
HIC Pool 5.0 .+-. 0.1 0.42 -0.08 23.7
[0720] The AEX unit operation provides the most color reduction (1.08 to
1.42 change in b*) while the HIC unit operation provides some additional
color reduction (0.08 to 0.19 change in b*). The unit operations
evaluated overall remove 76.3%-78.2% of the color present in affinity
capture pool.
[0721] The color of various production process intermediates for "Control
Bioreactor Condition" and "Low Cysteine, Low Metals, and Increased
Antioxidants Bioreactor Condition" were also studied for the percentage
of 2-oxo-histidines and percentage of oxo-tryptophans in the
oligopeptides that were generated by protease digestion, as measured by
mass spectrometry as shown in Table 9-5 and Table 9-6, respectively. The
peptide mapping was performed as discussed in Example 3.
[0722] Referring to Table 9-5, on comparing the histidine oxidation levels
in the pools in different production steps, it is evident that relative
abundance of the percentage of histidine oxidation levels for MT formed
reduces in the pool as the production process progresses. For example,
for H209 in the "Control Bioreactor Condition", the percent histidine
oxidation level was 0.062 for the enzymatic cleavage pool and this was
reduced to 0.029 for AEX flowthrough and further reduced to 0.020 for the
HIC pool. Similarly, for H209 in the "Low Cysteine, Low Metals, and
Increased Antioxidants Bioreactor Condition", the percent histidine
oxidation level was 0.039 for the enzymatic cleavage pool and this was
reduced to 0.023 for AEX flowthrough and further reduced to 0.016 for the
HIC pool. Thus, the production strategy led to a reduction in percentage
of histidine oxidation levels in MT. As the coloration reduced, presence
of some of the oxidized residues in the sample also reduced. Similar to
histidine oxidation, tryptophan oxidation levels were also tracked for
the pools in different production steps for both the "Control Bioreactor
Condition" and "Low Cysteine, Low Metals, and Increased Antioxidants
Bioreactor Condition" (Table 9-6).
1031339PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 1Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala
Ala Val1 5 10 15Thr Leu
Phe Val Leu Ser Val Asp Arg Gly Val Ile Ala Asp Ser Phe 20
25 30Ser Ala Asn Gln Glu Ile Arg Tyr Ser
Glu Val Thr Pro Tyr His Val 35 40
45Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln 50
55 60Gly Glu Asp Val Phe His Ala Pro Tyr
Val Ala Asn Gln Gly Trp Tyr65 70 75
80Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys
Gly Ala 85 90 95Ala Thr
Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp 100
105 110Gln Ile Lys Arg Tyr Leu Glu Glu His
Pro Glu Lys Gln Lys Ile Asn 115 120
125Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys
130 135 140Asn His Gln Leu Asp Ser Lys
Leu Phe Glu Tyr Phe Lys Glu Lys Ala145 150
155 160Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe
Pro Asp His Val 165 170
175Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly
180 185 190Pro Thr Pro Val Lys Glu
Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe 195 200
205Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr
Ser Arg 210 215 220His Asp Phe Lys Glu
Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys225 230
235 240Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly
Leu Ser His Thr Tyr Ala 245 250
255Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp
260 265 270Ser Asn Gly Asn Leu
Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn 275
280 285Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val
Asn Ser Ala Gly 290 295 300Lys Val Ala
Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala305
310 315 320Gln Val Leu Gly Leu Phe Thr
Leu Ser Thr Gly Gln Asp Ser Trp Asn 325
330 335Gln Thr Asn2339PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 2Met Arg Lys Arg Cys
Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val1 5
10 15Thr Leu Phe Val Leu Ser Val Asp Arg Gly Val
Ile Ala Asp Ser Phe 20 25
30Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val
35 40 45Thr Ser Val Trp Thr Lys Gly Val
Thr Pro Pro Ala Asn Phe Thr Gln 50 55
60Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr65
70 75 80Asp Ile Thr Lys Thr
Phe Asp Gly Lys Asp Asp Leu Leu Cys Gly Ala 85
90 95Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe
Asp Gln Asn Lys Asp 100 105
110Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn
115 120 125Phe Asn Gly Glu Gln Met Phe
Asp Val Lys Glu Ala Ile Asp Thr Lys 130 135
140Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys
Ala145 150 155 160Phe Pro
Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val
165 170 175Ile Asp Met Phe Ile Asn Gly
Tyr Arg Leu Ser Leu Thr Asn His Gly 180 185
190Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly
Ile Phe 195 200 205Asp Ala Val Phe
Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg 210
215 220His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser
Asp Leu Ile Lys225 230 235
240Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala
245 250 255Asn Val Arg Ile Asn
His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp 260
265 270Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp
Ser Asp Ser Asn 275 280 285Ala Ser
Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly 290
295 300Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu
Asp Asn Ile Gly Ala305 310 315
320Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn
325 330 335Gln Thr
Asn3339PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 3Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala
Ala Val1 5 10 15Thr Leu
Phe Val Leu Ser Val Asp Arg Gly Val Ile Ala Asp Ser Phe 20
25 30Ser Ala Asn Gln Glu Ile Arg Tyr Ser
Glu Val Thr Pro Tyr His Val 35 40
45Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln 50
55 60Gly Glu Asp Val Phe His Ala Pro Tyr
Val Ala Asn Gln Gly Trp Tyr65 70 75
80Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys
Gly Ala 85 90 95Ala Thr
Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp 100
105 110Gln Ile Lys Arg Tyr Leu Glu Glu His
Pro Glu Lys Gln Lys Ile Asn 115 120
125Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys
130 135 140Asn His Gln Leu Asp Ser Lys
Leu Phe Glu Tyr Phe Lys Glu Lys Ala145 150
155 160Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe
Pro Asp His Val 165 170
175Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly
180 185 190Pro Thr Pro Val Lys Glu
Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe 195 200
205Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr
Ser Arg 210 215 220His Asp Phe Lys Glu
Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys225 230
235 240Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly
Leu Ser His Thr Tyr Ala 245 250
255Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp
260 265 270Ser Asn Gly Asn Leu
Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn 275
280 285Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val
Asn Ser Ala Gly 290 295 300Lys Val Ala
Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala305
310 315 320Gln Val Leu Gly Leu Phe Thr
Leu Ser Thr Gly Gln Asp Ser Trp Asn 325
330 335Gln Thr Asn4339PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 4Met Arg Lys Arg Cys
Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val1 5
10 15Thr Leu Phe Val Leu Ser Val Asp Arg Gly Val
Ile Ala Asp Ser Phe 20 25
30Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val
35 40 45Thr Ser Val Trp Thr Lys Gly Val
Thr Pro Pro Ala Asn Phe Thr Gln 50 55
60Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr65
70 75 80Asp Ile Thr Lys Thr
Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala 85
90 95Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe
Asp Gln Asn Lys Asp 100 105
110Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn
115 120 125Phe Asn Gly Glu Gln Met Phe
Asp Val Lys Glu Ala Ile Asp Thr Lys 130 135
140Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys
Ala145 150 155 160Phe Pro
Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val
165 170 175Ile Asp Met Phe Ile Leu Gly
Tyr Arg Leu Ser Leu Thr Asn His Gly 180 185
190Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly
Ile Phe 195 200 205Asp Ala Val Phe
Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg 210
215 220His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser
Asp Leu Ile Lys225 230 235
240Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala
245 250 255Asn Val Arg Ile Asn
His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp 260
265 270Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp
Ser Asp Ser Asn 275 280 285Ala Ser
Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly 290
295 300Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu
Asp Asn Ile Gly Ala305 310 315
320Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn
325 330 335Gln Thr
Asn5339PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 5Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala
Ala Val1 5 10 15Thr Leu
Phe Val Leu Ser Val Asp Arg Gly Val Ile Ala Asp Ser Phe 20
25 30Ser Ala Asn Gln Glu Ile Arg Tyr Ser
Glu Val Thr Pro Tyr His Val 35 40
45Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln 50
55 60Gly Glu Asp Val Phe His Ala Pro Tyr
Val Ala Asn Gln Gly Trp Tyr65 70 75
80Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys
Gly Ala 85 90 95Ala Thr
Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp 100
105 110Gln Ile Lys Arg Tyr Leu Glu Glu His
Pro Glu Lys Gln Lys Ile Asn 115 120
125Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys
130 135 140Asn His Gln Leu Asp Ser Lys
Leu Phe Glu Tyr Phe Lys Glu Lys Ala145 150
155 160Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe
Pro Asp His Val 165 170
175Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly
180 185 190Pro Thr Pro Val Lys Glu
Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe 195 200
205Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr
Ser Arg 210 215 220His Asp Phe Lys Glu
Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys225 230
235 240Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly
Leu Ser His Thr Tyr Ala 245 250
255Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp
260 265 270Ser Asp Gly Asn Leu
Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn 275
280 285Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val
Asn Ser Ala Gly 290 295 300Lys Val Ala
Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala305
310 315 320Gln Val Leu Gly Leu Phe Thr
Leu Ser Thr Gly Gln Asp Ser Trp Asn 325
330 335Gln Thr Asn6339PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 6Met Arg Lys Arg Cys
Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val1 5
10 15Thr Leu Phe Val Leu Ser Val Asp Arg Gly Val
Ile Ala Asp Ser Phe 20 25
30Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val
35 40 45Thr Ser Val Trp Thr Lys Gly Val
Thr Pro Pro Ala Asn Phe Thr Gln 50 55
60Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr65
70 75 80Asp Ile Thr Lys Thr
Phe Asp Gly Lys Asp Asp Leu Leu Cys Gly Ala 85
90 95Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe
Asp Gln Asn Lys Asp 100 105
110Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn
115 120 125Phe Arg Gly Glu Gln Met Phe
Asp Val Lys Glu Ala Ile Asp Thr Lys 130 135
140Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys
Ala145 150 155 160Phe Pro
Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val
165 170 175Ile Asp Met Phe Ile Asn Gly
Tyr Arg Leu Ser Leu Thr Asn His Gly 180 185
190Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly
Ile Phe 195 200 205Asp Ala Val Phe
Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg 210
215 220His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser
Asp Leu Ile Lys225 230 235
240Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala
245 250 255Asn Val Arg Ile Asn
His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp 260
265 270Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp
Ser Asp Ser Asn 275 280 285Ala Ser
Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly 290
295 300Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu
Asp Asn Ile Gly Ala305 310 315
320Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn
325 330 335Gln Thr
Asn7339PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 7Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala
Ala Val1 5 10 15Thr Leu
Phe Val Leu Ser Val Asp Arg Gly Val Ile Ala Asp Ser Phe 20
25 30Ser Ala Asn Gln Glu Ile Arg Tyr Ser
Glu Val Thr Pro Tyr His Val 35 40
45Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln 50
55 60Gly Glu Asp Val Phe His Ala Pro Tyr
Val Ala Asn Gln Gly Trp Tyr65 70 75
80Asp Ile Thr Lys Thr Phe Asp Gly Lys Asp Asp Leu Leu Cys
Gly Ala 85 90 95Ala Thr
Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp 100
105 110Gln Ile Lys Arg Tyr Leu Glu Glu His
Pro Glu Lys Gln Lys Ile Asn 115 120
125Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys
130 135 140Asn His Gln Leu Asp Ser Lys
Leu Phe Glu Tyr Phe Lys Glu Lys Ala145 150
155 160Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe
Pro Asp His Val 165 170
175Ile Asp Met Phe Ile Leu Gly Tyr Arg Leu Ser Leu Thr Asn His Gly
180 185 190Pro Thr Pro Val Lys Glu
Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe 195 200
205Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr
Ser Arg 210 215 220His Asp Phe Lys Glu
Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys225 230
235 240Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly
Leu Ser His Thr Tyr Ala 245 250
255Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp
260 265 270Ser Asn Gly Asn Leu
Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn 275
280 285Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val
Asn Ser Ala Gly 290 295 300Lys Val Ala
Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala305
310 315 320Gln Val Leu Gly Leu Phe Thr
Leu Ser Thr Gly Gln Asp Ser Trp Asn 325
330 335Gln Thr Asn8339PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 8Met Arg Lys Arg Cys
Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val1 5
10 15Thr Leu Phe Val Leu Ser Val Asp Arg Gly Val
Ile Ala Asp Ser Phe 20 25
30Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val
35 40 45Thr Ser Val Trp Thr Lys Gly Val
Thr Pro Pro Ala Asn Phe Thr Gln 50 55
60Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr65
70 75 80Asp Ile Thr Lys Thr
Phe Asp Gly Lys Asp Asp Leu Leu Cys Gly Ala 85
90 95Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe
Asp Gln Asn Lys Asp 100 105
110Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn
115 120 125Phe Asn Gly Glu Gln Met Phe
Asp Val Lys Glu Ala Ile Asp Thr Lys 130 135
140Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys
Ala145 150 155 160Phe Pro
Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val
165 170 175Ile Asp Met Phe Ile Asn Gly
Tyr Arg Leu Ser Leu Thr Asn His Gly 180 185
190Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly
Ile Phe 195 200 205Asp Ala Val Phe
Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg 210
215 220His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser
Asp Leu Ile Lys225 230 235
240Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala
245 250 255Asn Val Arg Ile Asn
His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp 260
265 270Ser Asp Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp
Ser Asp Ser Asn 275 280 285Ala Ser
Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly 290
295 300Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu
Asp Asn Ile Gly Ala305 310 315
320Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn
325 330 335Gln Thr
Asn9339PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 9Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala
Ala Val1 5 10 15Thr Leu
Phe Val Leu Ser Val Asp Arg Gly Val Ile Ala Asp Ser Phe 20
25 30Ser Ala Asn Gln Glu Ile Arg Tyr Ser
Glu Val Thr Pro Tyr His Val 35 40
45Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln 50
55 60Gly Glu Asp Val Phe His Ala Pro Tyr
Val Ala Asn Gln Gly Trp Tyr65 70 75
80Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys
Gly Ala 85 90 95Ala Thr
Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp 100
105 110Gln Ile Lys Arg Tyr Leu Glu Glu His
Pro Glu Lys Gln Lys Ile Asn 115 120
125Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys
130 135 140Asn His Gln Leu Asp Ser Lys
Leu Phe Glu Tyr Phe Lys Glu Lys Ala145 150
155 160Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe
Pro Asp His Val 165 170
175Ile Asp Met Phe Ile Leu Gly Tyr Arg Leu Ser Leu Thr Asn His Gly
180 185 190Pro Thr Pro Val Lys Glu
Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe 195 200
205Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr
Ser Arg 210 215 220His Asp Phe Lys Glu
Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys225 230
235 240Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly
Leu Ser His Thr Tyr Ala 245 250
255Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp
260 265 270Ser Asn Gly Asn Leu
Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn 275
280 285Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val
Asn Ser Ala Gly 290 295 300Lys Val Ala
Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala305
310 315 320Gln Val Leu Gly Leu Phe Thr
Leu Ser Thr Gly Gln Asp Ser Trp Asn 325
330 335Gln Thr Asn10339PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 10Met Arg Lys Arg Cys
Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val1 5
10 15Thr Leu Phe Val Leu Ser Val Asp Arg Gly Val
Ile Ala Asp Ser Phe 20 25
30Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val
35 40 45Thr Ser Val Trp Thr Lys Gly Val
Thr Pro Pro Ala Asn Phe Thr Gln 50 55
60Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr65
70 75 80Asp Ile Thr Lys Thr
Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala 85
90 95Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe
Asp Gln Asn Lys Asp 100 105
110Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn
115 120 125Phe Arg Gly Glu Gln Met Phe
Asp Val Lys Glu Ala Ile Asp Thr Lys 130 135
140Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys
Ala145 150 155 160Phe Pro
Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val
165 170 175Ile Asp Met Phe Ile Asn Gly
Tyr Arg Leu Ser Leu Thr Asn His Gly 180 185
190Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly
Ile Phe 195 200 205Asp Ala Val Phe
Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg 210
215 220His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser
Asp Leu Ile Lys225 230 235
240Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala
245 250 255Asn Val Arg Ile Asn
His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp 260
265 270Ser Asp Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp
Ser Asp Ser Asn 275 280 285Ala Ser
Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly 290
295 300Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu
Asp Asn Ile Gly Ala305 310 315
320Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn
325 330 335Gln Thr
Asn11339PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 11Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala
Val Leu Ala Ala Val1 5 10
15Thr Leu Phe Val Leu Ser Val Asp Arg Gly Val Ile Ala Asp Ser Phe
20 25 30Ser Ala Asn Gln Glu Ile Arg
Tyr Ser Glu Val Thr Pro Tyr His Val 35 40
45Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr
Gln 50 55 60Gly Glu Asp Val Phe His
Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr65 70
75 80Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp
Leu Leu Cys Gly Ala 85 90
95Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp
100 105 110Gln Ile Lys Arg Tyr Leu
Glu Glu His Pro Glu Lys Gln Lys Ile Asn 115 120
125Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp
Thr Lys 130 135 140Asn His Gln Leu Asp
Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala145 150
155 160Phe Pro Tyr Leu Ser Thr Lys His Leu Gly
Val Phe Pro Asp His Val 165 170
175Ile Asp Met Phe Ile Leu Gly Tyr Arg Leu Ser Leu Thr Asn His Gly
180 185 190Pro Thr Pro Val Lys
Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe 195
200 205Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu
Leu Thr Ser Arg 210 215 220His Asp Phe
Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys225
230 235 240Lys Glu Leu Thr Glu Gly Lys
Ala Leu Gly Leu Ser His Thr Tyr Ala 245
250 255Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly
Ala Asp Phe Asp 260 265 270Ser
Asp Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn 275
280 285Ala Ser Ile Gly Met Lys Lys Tyr Phe
Val Gly Val Asn Ser Ala Gly 290 295
300Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala305
310 315 320Gln Val Leu Gly
Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn 325
330 335Gln Thr Asn12339PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
12Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val1
5 10 15Thr Leu Phe Val Leu Ser
Val Asp Arg Gly Val Ile Ala Asp Ser Phe 20 25
30Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro
Tyr His Val 35 40 45Thr Ser Val
Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln 50
55 60Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn
Gln Gly Trp Tyr65 70 75
80Asp Ile Thr Lys Thr Phe Asp Gly Lys Asp Asp Leu Leu Cys Gly Ala
85 90 95Ala Thr Ala Gly Asn Met
Leu His Trp Trp Phe Asp Gln Asn Lys Asp 100
105 110Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys
Gln Lys Ile Asn 115 120 125Phe Arg
Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys 130
135 140Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr
Phe Lys Glu Lys Ala145 150 155
160Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val
165 170 175Ile Asp Met Phe
Ile Leu Gly Tyr Arg Leu Ser Leu Thr Asn His Gly 180
185 190Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro
Arg Gly Gly Ile Phe 195 200 205Asp
Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg 210
215 220His Asp Phe Lys Glu Lys Asn Leu Lys Glu
Ile Ser Asp Leu Ile Lys225 230 235
240Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr
Ala 245 250 255Asn Val Arg
Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp 260
265 270Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val
Thr Asp Ser Asp Ser Asn 275 280
285Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly 290
295 300Lys Val Ala Ile Ser Ala Lys Glu
Ile Lys Glu Asp Asn Ile Gly Ala305 310
315 320Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln
Asp Ser Trp Asn 325 330
335Gln Thr Asn13339PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 13Met Arg Lys Arg Cys Tyr Ser Thr Ser
Ala Ala Val Leu Ala Ala Val1 5 10
15Thr Leu Phe Val Leu Ser Val Asp Arg Gly Val Ile Ala Asp Ser
Phe 20 25 30Ser Ala Asn Gln
Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val 35
40 45Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala
Asn Phe Thr Gln 50 55 60Gly Glu Asp
Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr65 70
75 80Asp Ile Thr Lys Thr Phe Asp Gly
Lys Asp Asp Leu Leu Cys Gly Ala 85 90
95Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn
Lys Asp 100 105 110Gln Ile Lys
Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn 115
120 125Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu
Ala Ile Asp Thr Lys 130 135 140Asn His
Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala145
150 155 160Phe Pro Tyr Leu Ser Thr Lys
His Leu Gly Val Phe Pro Asp His Val 165
170 175Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu
Thr Asn His Gly 180 185 190Pro
Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe 195
200 205Asp Ala Val Phe Thr Arg Gly Asp Gln
Ser Lys Leu Leu Thr Ser Arg 210 215
220His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys225
230 235 240Lys Glu Leu Thr
Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala 245
250 255Asn Val Arg Ile Asn His Val Ile Asn Leu
Trp Gly Ala Asp Phe Asp 260 265
270Ser Asp Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn
275 280 285Ala Ser Ile Gly Met Lys Lys
Tyr Phe Val Gly Val Asn Ser Ala Gly 290 295
300Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly
Ala305 310 315 320Gln Val
Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn
325 330 335Gln Thr Asn14339PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
14Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val1
5 10 15Thr Leu Phe Val Leu Ser
Val Asp Arg Gly Val Ile Ala Asp Ser Phe 20 25
30Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro
Tyr His Val 35 40 45Thr Ser Val
Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln 50
55 60Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn
Gln Gly Trp Tyr65 70 75
80Asp Ile Thr Lys Thr Phe Asp Gly Lys Asp Asp Leu Leu Cys Gly Ala
85 90 95Ala Thr Ala Gly Asn Met
Leu His Trp Trp Phe Asp Gln Asn Lys Asp 100
105 110Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys
Gln Lys Ile Asn 115 120 125Phe Asn
Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys 130
135 140Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr
Phe Lys Glu Lys Ala145 150 155
160Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val
165 170 175Ile Asp Met Phe
Ile Leu Gly Tyr Arg Leu Ser Leu Thr Asn His Gly 180
185 190Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro
Arg Gly Gly Ile Phe 195 200 205Asp
Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg 210
215 220His Asp Phe Lys Glu Lys Asn Leu Lys Glu
Ile Ser Asp Leu Ile Lys225 230 235
240Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr
Ala 245 250 255Asn Val Arg
Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp 260
265 270Ser Asp Gly Asn Leu Lys Ala Ile Tyr Val
Thr Asp Ser Asp Ser Asn 275 280
285Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly 290
295 300Lys Val Ala Ile Ser Ala Lys Glu
Ile Lys Glu Asp Asn Ile Gly Ala305 310
315 320Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln
Asp Ser Trp Asn 325 330
335Gln Thr Asn15339PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 15Met Arg Lys Arg Cys Tyr Ser Thr Ser
Ala Ala Val Leu Ala Ala Val1 5 10
15Thr Leu Phe Val Leu Ser Val Asp Arg Gly Val Ile Ala Asp Ser
Phe 20 25 30Ser Ala Asn Gln
Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val 35
40 45Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala
Asn Phe Thr Gln 50 55 60Gly Glu Asp
Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr65 70
75 80Asp Ile Thr Lys Thr Phe Asn Gly
Lys Asp Asp Leu Leu Cys Gly Ala 85 90
95Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn
Lys Asp 100 105 110Gln Ile Lys
Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn 115
120 125Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu
Ala Ile Asp Thr Lys 130 135 140Asn His
Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala145
150 155 160Phe Pro Tyr Leu Ser Thr Lys
His Leu Gly Val Phe Pro Asp His Val 165
170 175Ile Asp Met Phe Ile Leu Gly Tyr Arg Leu Ser Leu
Thr Asn His Gly 180 185 190Pro
Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe 195
200 205Asp Ala Val Phe Thr Arg Gly Asp Gln
Ser Lys Leu Leu Thr Ser Arg 210 215
220His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys225
230 235 240Lys Glu Leu Thr
Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala 245
250 255Asn Val Arg Ile Asn His Val Ile Asn Leu
Trp Gly Ala Asp Phe Asp 260 265
270Ser Asp Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn
275 280 285Ala Ser Ile Gly Met Lys Lys
Tyr Phe Val Gly Val Asn Ser Ala Gly 290 295
300Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly
Ala305 310 315 320Gln Val
Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn
325 330 335Gln Thr Asn16339PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
16Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val1
5 10 15Thr Leu Phe Val Leu Ser
Val Asp Arg Gly Val Ile Ala Asp Ser Phe 20 25
30Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro
Tyr His Val 35 40 45Thr Ser Val
Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln 50
55 60Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn
Gln Gly Trp Tyr65 70 75
80Asp Ile Thr Lys Thr Phe Asp Gly Lys Asp Asp Leu Leu Cys Gly Ala
85 90 95Ala Thr Ala Gly Asn Met
Leu His Trp Trp Phe Asp Gln Asn Lys Asp 100
105 110Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys
Gln Lys Ile Asn 115 120 125Phe Arg
Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys 130
135 140Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr
Phe Lys Glu Lys Ala145 150 155
160Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val
165 170 175Ile Asp Met Phe
Ile Leu Gly Tyr Arg Leu Ser Leu Thr Asn His Gly 180
185 190Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro
Arg Gly Gly Ile Phe 195 200 205Asp
Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg 210
215 220His Asp Phe Lys Glu Lys Asn Leu Lys Glu
Ile Ser Asp Leu Ile Lys225 230 235
240Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr
Ala 245 250 255Asn Val Arg
Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp 260
265 270Ser Asp Gly Asn Leu Lys Ala Ile Tyr Val
Thr Asp Ser Asp Ser Asn 275 280
285Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly 290
295 300Lys Val Ala Ile Ser Ala Lys Glu
Ile Lys Glu Asp Asn Ile Gly Ala305 310
315 320Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln
Asp Ser Trp Asn 325 330
335Gln Thr Asn1716PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 17Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly1 5 10
151817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn
Gly His Leu Tyr1 5 10
15Lys1923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro
Ser His Gly Ile1 5 10
15Glu Leu Ser Val Gly Glu Lys 202021PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Thr
Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser1
5 10 15Lys His Gln His Lys
20217PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 21Thr Asn Tyr Leu Thr His Arg1
52224PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 22Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro
Glu1 5 10 15Ile Ile His
Met Thr Glu Gly Arg 20236PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 23Val His Glu Lys Asp Lys1
52424PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 24Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser
Glu Ile Pro Glu1 5 10
15Ile Ile His Met Thr Glu Gly Arg 202524PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Ser
Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu1
5 10 15Ile Ile His Met Thr Glu Gly
Arg 20268PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 26Thr Gln Ser Gly Ser Glu Met Lys1
52717PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser
Gly Leu Met Thr1 5 10
15Lys286PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Ile Ile Trp Asp Ser Arg1
52917PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 29Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser
Ser1 5 10
15Lys3010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys1
5 103112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 31Lys Phe Pro Leu Asp Thr Leu
Ile Pro Asp Gly Lys1 5
103212PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 32Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg1
5 1033227PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMOD_RES(8)..(8)Leu or
ProMOD_RES(173)..(173)Ala or Thr 33Asp Lys Thr His Thr Cys Pro Xaa Cys
Pro Ala Pro Glu Leu Leu Gly1 5 10
15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70
75 80Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90
95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile 100 105 110Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115
120 125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145
150 155 160Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Xaa Thr Pro Pro 165
170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195
200 205His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 210 215
220Pro Gly Lys22534103PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 34Ser Asp Thr Gly Arg Pro Phe Val Glu
Met Tyr Ser Glu Ile Pro Glu1 5 10
15Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg
Val 20 25 30Thr Ser Pro Asn
Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35
40 45Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser
Arg Lys Gly Phe 50 55 60Ile Ile Ser
Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu65 70
75 80Ala Thr Val Asn Gly His Leu Tyr
Lys Thr Asn Tyr Leu Thr His Arg 85 90
95Gln Thr Asn Thr Ile Ile Asp
1003593PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 35Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile
His Met Thr1 5 10 15Glu
Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile 20
25 30Thr Val Thr Leu Lys Lys Phe Pro
Leu Asp Thr Leu Ile Pro Asp Gly 35 40
45Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala
50 55 60Thr Tyr Lys Glu Ile Gly Leu Leu
Thr Cys Glu Ala Thr Val Asn Gly65 70 75
80His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr
85 9036102PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 36Val Val Leu Ser Pro
Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys1 5
10 15Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu
Asn Val Gly Ile Asp 20 25
30Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val
35 40 45Asn Arg Asp Leu Lys Thr Gln Ser
Gly Ser Glu Met Lys Lys Phe Leu 50 55
60Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr65
70 75 80Thr Cys Ala Ala Ser
Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe 85
90 95Val Arg Val His Glu Lys
1003797PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 37Pro Phe Val Ala Phe Gly Ser Gly Met Glu Ser Leu Val
Glu Ala Thr1 5 10 15Val
Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu Gly Tyr Pro Pro 20
25 30Pro Glu Ile Lys Trp Tyr Lys Asn
Gly Ile Pro Leu Glu Ser Asn His 35 40
45Thr Ile Lys Ala Gly His Val Leu Thr Ile Met Glu Val Ser Glu Arg
50 55 60Asp Thr Gly Asn Tyr Thr Val Ile
Leu Thr Asn Pro Ile Ser Lys Glu65 70 75
80Lys Gln Ser His Val Val Ser Leu Val Val Tyr Val Pro
Pro Gly Pro 85 90
95Gly3892PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 38Phe Val Ala Phe Gly Ser Gly Met Glu Ser Leu
Val Glu Ala Thr Val1 5 10
15Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu Gly Tyr Pro Pro Pro
20 25 30Glu Ile Lys Trp Tyr Lys Asn
Gly Ile Pro Leu Glu Ser Asn His Thr 35 40
45Ile Lys Ala Gly His Val Leu Thr Ile Met Glu Val Ser Glu Arg
Asp 50 55 60Thr Gly Asn Tyr Thr Val
Ile Leu Thr Asn Pro Ile Lys Ser Glu Lys65 70
75 80Gln Ser His Val Val Ser Leu Val Val Tyr Val
Pro 85 90399PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 39Asp
Lys Thr His Thr Cys Pro Pro Cys1 54012PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 40Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Pro Cys1 5
104115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 41Asp Lys Thr His Thr Cys Pro Pro Cys Pro Pro Cys
Pro Pro Cys1 5 10
154216PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 42Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly1 5 10
154316PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 43Asp Lys Thr His Thr Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu
Gly1 5 10
15446PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 44Asp Lys Thr His Thr Cys1 54512PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 45Asp
Lys Thr His Thr Cys Pro Leu Cys Pro Ala Pro1 5
1046221PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 46Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr
Ser Glu Ile Pro Glu1 5 10
15Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
20 25 30Thr Ser Pro Asn Ile Thr Val
Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40
45Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly
Phe 50 55 60Ile Ile Ser Asn Ala Thr
Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu65 70
75 80Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn
Tyr Leu Thr His Arg 85 90
95Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile
100 105 110Glu Leu Ser Val Gly Glu
Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120
125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser
Ser Lys 130 135 140His Gln His Lys Lys
Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150
155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu
Thr Ile Asp Gly Val Thr 165 170
175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met
180 185 190Thr Lys Lys Asn Ser
Thr Phe Val Arg Val His Glu Lys Asp Lys Thr 195
200 205His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly 210 215 22047315PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
47Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His1
5 10 15Met Thr Glu Gly Arg Glu
Leu Val Ile Pro Cys Arg Val Thr Ser Pro 20 25
30Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
Leu Ile Pro 35 40 45Asp Gly Lys
Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser 50
55 60Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys
Glu Ala Thr Val65 70 75
80Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn
85 90 95Thr Ile Ile Asp Val Val
Leu Ser Pro Ser His Gly Ile Glu Leu Ser 100
105 110Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg
Thr Glu Leu Asn 115 120 125Val Gly
Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His 130
135 140Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln
Ser Gly Ser Glu Met145 150 155
160Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp
165 170 175Gln Gly Leu Tyr
Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys 180
185 190Asn Ser Thr Phe Val Arg Val His Glu Asn Leu
Ser Val Ala Phe Gly 195 200 205Ser
Gly Met Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg 210
215 220Ile Pro Ala Lys Tyr Leu Gly Tyr Pro Pro
Pro Glu Ile Lys Trp Tyr225 230 235
240Lys Asn Gly Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly
His 245 250 255Val Leu Thr
Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr 260
265 270Val Ile Leu Thr Asn Pro Ile Ser Lys Glu
Lys Gln Ser His Val Val 275 280
285Ser Leu Val Val Tyr Val Pro Pro Gly Pro Gly Asp Lys Thr His Thr 290
295 300Cys Pro Leu Cys Pro Ala Pro Glu
Leu Leu Gly305 310 31548214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
48Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu1
5 10 15Ile Ile His Met Thr Glu
Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25
30Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro
Leu Asp Thr 35 40 45Leu Ile Pro
Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50
55 60Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu
Leu Thr Cys Glu65 70 75
80Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
85 90 95Gln Thr Asn Thr Ile Ile
Asp Val Val Leu Ser Pro Ser His Gly Ile 100
105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys
Thr Ala Arg Thr 115 120 125Glu Leu
Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130
135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu
Lys Thr Gln Ser Gly145 150 155
160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr
165 170 175Arg Ser Asp Gln
Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180
185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His
Glu Lys Asp Lys Thr 195 200 205His
Thr Cys Pro Pro Cys 21049217PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 49Ser Asp Thr Gly Arg Pro
Phe Val Glu Met Tyr Ser Glu Ile Pro Glu1 5
10 15Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile
Pro Cys Arg Val 20 25 30Thr
Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35
40 45Leu Ile Pro Asp Gly Lys Arg Ile Ile
Trp Asp Ser Arg Lys Gly Phe 50 55
60Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu65
70 75 80Ala Thr Val Asn Gly
His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85
90 95Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser
Pro Ser His Gly Ile 100 105
110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr
115 120 125Glu Leu Asn Val Gly Ile Asp
Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130 135
140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser
Gly145 150 155 160Ser Glu
Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr
165 170 175Arg Ser Asp Gln Gly Leu Tyr
Thr Cys Ala Ala Ser Ser Gly Leu Met 180 185
190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp
Lys Thr 195 200 205His Thr Cys Pro
Pro Cys Pro Pro Cys 210 21550220PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
50Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu1
5 10 15Ile Ile His Met Thr Glu
Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25
30Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro
Leu Asp Thr 35 40 45Leu Ile Pro
Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50
55 60Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu
Leu Thr Cys Glu65 70 75
80Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
85 90 95Gln Thr Asn Thr Ile Ile
Asp Val Val Leu Ser Pro Ser His Gly Ile 100
105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys
Thr Ala Arg Thr 115 120 125Glu Leu
Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130
135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu
Lys Thr Gln Ser Gly145 150 155
160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr
165 170 175Arg Ser Asp Gln
Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180
185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His
Glu Lys Asp Lys Thr 195 200 205His
Thr Cys Pro Pro Cys Pro Pro Cys Pro Pro Cys 210 215
22051440PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 51Ser Asp Thr Gly Arg Pro Phe Val Glu
Met Tyr Ser Glu Ile Pro Glu1 5 10
15Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg
Val 20 25 30Thr Ser Pro Asn
Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35
40 45Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser
Arg Lys Gly Phe 50 55 60Ile Ile Ser
Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu65 70
75 80Ala Thr Val Asn Gly His Leu Tyr
Lys Thr Asn Tyr Leu Thr His Arg 85 90
95Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His
Gly Ile 100 105 110Glu Leu Ser
Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115
120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu
Tyr Pro Ser Ser Lys 130 135 140His Gln
His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145
150 155 160Ser Glu Met Lys Lys Phe Leu
Ser Thr Leu Thr Ile Asp Gly Val Thr 165
170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser
Ser Gly Leu Met 180 185 190Thr
Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly 195
200 205Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly 210 215
220Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg225
230 235 240Pro Phe Val Glu
Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr 245
250 255Glu Gly Arg Glu Leu Val Ile Pro Cys Arg
Val Thr Ser Pro Asn Ile 260 265
270Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly
275 280 285Lys Arg Ile Ile Trp Asp Ser
Arg Lys Gly Phe Ile Ile Ser Asn Ala 290 295
300Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn
Gly305 310 315 320His Leu
Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile
325 330 335Ile Asp Val Val Leu Ser Pro
Ser His Gly Ile Glu Leu Ser Val Gly 340 345
350Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn
Val Gly 355 360 365Ile Asp Phe Asn
Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys 370
375 380Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser
Glu Met Lys Lys385 390 395
400Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly
405 410 415Leu Tyr Thr Cys Ala
Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser 420
425 430Thr Phe Val Arg Val His Glu Lys 435
44052425PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 52Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr
Ser Glu Ile Pro Glu1 5 10
15Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
20 25 30Thr Ser Pro Asn Ile Thr Val
Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40
45Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly
Phe 50 55 60Ile Ile Ser Asn Ala Thr
Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu65 70
75 80Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn
Tyr Leu Thr His Arg 85 90
95Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile
100 105 110Glu Leu Ser Val Gly Glu
Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120
125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser
Ser Lys 130 135 140His Gln His Lys Lys
Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150
155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu
Thr Ile Asp Gly Val Thr 165 170
175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met
180 185 190Thr Lys Lys Asn Ser
Thr Phe Val Arg Val His Glu Lys Gly Gly Gly 195
200 205Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Ser Asp Thr Gly 210 215 220Arg Pro Phe
Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met225
230 235 240Thr Glu Gly Arg Glu Leu Val
Ile Pro Cys Arg Val Thr Ser Pro Asn 245
250 255Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
Leu Ile Pro Asp 260 265 270Gly
Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn 275
280 285Ala Thr Tyr Lys Glu Ile Gly Leu Leu
Thr Cys Glu Ala Thr Val Asn 290 295
300Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr305
310 315 320Ile Ile Asp Val
Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val 325
330 335Gly Glu Lys Leu Val Leu Asn Cys Thr Ala
Arg Thr Glu Leu Asn Val 340 345
350Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys
355 360 365Lys Leu Val Asn Arg Asp Leu
Lys Thr Gln Ser Gly Ser Glu Met Lys 370 375
380Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp
Gln385 390 395 400Gly Leu
Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn
405 410 415Ser Thr Phe Val Arg Val His
Glu Lys 420 42553455PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
53Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu1
5 10 15Ile Ile His Met Thr Glu
Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25
30Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro
Leu Asp Thr 35 40 45Leu Ile Pro
Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50
55 60Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu
Leu Thr Cys Glu65 70 75
80Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
85 90 95Gln Thr Asn Thr Ile Ile
Asp Val Val Leu Ser Pro Ser His Gly Ile 100
105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys
Thr Ala Arg Thr 115 120 125Glu Leu
Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130
135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu
Lys Thr Gln Ser Gly145 150 155
160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr
165 170 175Arg Ser Asp Gln
Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180
185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His
Glu Lys Gly Gly Gly 195 200 205Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 210
215 220Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser225 230 235
240Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg
Pro 245 250 255Phe Val Glu
Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu 260
265 270Gly Arg Glu Leu Val Ile Pro Cys Arg Val
Thr Ser Pro Asn Ile Thr 275 280
285Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys 290
295 300Arg Ile Ile Trp Asp Ser Arg Lys
Gly Phe Ile Ile Ser Asn Ala Thr305 310
315 320Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr
Val Asn Gly His 325 330
335Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile
340 345 350Asp Val Val Leu Ser Pro
Ser His Gly Ile Glu Leu Ser Val Gly Glu 355 360
365Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val
Gly Ile 370 375 380Asp Phe Asn Trp Glu
Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu385 390
395 400Val Asn Arg Asp Leu Lys Thr Gln Ser Gly
Ser Glu Met Lys Lys Phe 405 410
415Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu
420 425 430Tyr Thr Cys Ala Ala
Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr 435
440 445Phe Val Arg Val His Glu Lys 450
45554470PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 54Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr
Ser Glu Ile Pro Glu1 5 10
15Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
20 25 30Thr Ser Pro Asn Ile Thr Val
Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40
45Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly
Phe 50 55 60Ile Ile Ser Asn Ala Thr
Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu65 70
75 80Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn
Tyr Leu Thr His Arg 85 90
95Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile
100 105 110Glu Leu Ser Val Gly Glu
Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120
125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser
Ser Lys 130 135 140His Gln His Lys Lys
Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150
155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu
Thr Ile Asp Gly Val Thr 165 170
175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met
180 185 190Thr Lys Lys Asn Ser
Thr Phe Val Arg Val His Glu Lys Gly Gly Gly 195
200 205Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly 210 215 220Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser225
230 235 240Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly 245
250 255Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr
Gly Arg Pro Phe 260 265 270Val
Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly 275
280 285Arg Glu Leu Val Ile Pro Cys Arg Val
Thr Ser Pro Asn Ile Thr Val 290 295
300Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg305
310 315 320Ile Ile Trp Asp
Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr 325
330 335Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala
Thr Val Asn Gly His Leu 340 345
350Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp
355 360 365Val Val Leu Ser Pro Ser His
Gly Ile Glu Leu Ser Val Gly Glu Lys 370 375
380Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile
Asp385 390 395 400Phe Asn
Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val
405 410 415Asn Arg Asp Leu Lys Thr Gln
Ser Gly Ser Glu Met Lys Lys Phe Leu 420 425
430Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly
Leu Tyr 435 440 445Thr Cys Ala Ala
Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe 450
455 460Val Arg Val His Glu Lys465
47055434PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 55Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr
Ser Glu Ile Pro Glu1 5 10
15Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
20 25 30Thr Ser Pro Asn Ile Thr Val
Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40
45Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly
Phe 50 55 60Ile Ile Ser Asn Ala Thr
Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu65 70
75 80Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn
Tyr Leu Thr His Arg 85 90
95Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile
100 105 110Glu Leu Ser Val Gly Glu
Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120
125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser
Ser Lys 130 135 140His Gln His Lys Lys
Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150
155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu
Thr Ile Asp Gly Val Thr 165 170
175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met
180 185 190Thr Lys Lys Asn Ser
Thr Phe Val Arg Val His Glu Lys Asp Lys Thr 195
200 205His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser 210 215 220Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg225
230 235 240Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro 245
250 255Glu Val Lys Phe Met Trp Tyr Val Asp Gly Ser Ser
Val Glu Val His 260 265 270Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 275
280 285Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys 290 295
300Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu305
310 315 320Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 325
330 335Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu 340 345
350Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
355 360 365Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val 370 375
380Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp385 390 395 400Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
405 410 415Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 420 425
430Gly Lys566PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 56Ile Ile Trp Asp Ser Arg1
55717PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 57Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu
Tyr1 5 10
15Lys5823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 58Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro
Ser His Gly Ile1 5 10
15Glu Leu Ser Val Gly Glu Lys 205921PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 59Thr
Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser1
5 10 15Lys His Gln His Lys
206016PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 60Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly1 5 10
15617PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 61Thr Asn Tyr Leu Thr His Arg1
56217PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 62Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu
Tyr1 5 10
15Lys6323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 63Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro
Ser His Gly Ile1 5 10
15Glu Leu Ser Val Gly Glu Lys 206424PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 64Ser
Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu1
5 10 15Ile Ile His Met Thr Glu Gly
Arg 206524PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 65Ser Asp Thr Gly Arg Pro Phe Val Glu Met
Tyr Ser Glu Ile Pro Glu1 5 10
15Ile Ile His Met Thr Glu Gly Arg 20668PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 66Thr
Gln Ser Gly Ser Glu Met Lys1 56717PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 67Ser
Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr1
5 10 15Lys6824PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 68Ser
Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu1
5 10 15Ile Ile His Met Thr Glu Gly
Arg 206910PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 69Gly Phe Ile Ile Ser Asn Ala Thr Tyr
Lys1 5 107012PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 70Lys
Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys1 5
107112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 71Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg1
5 1072165PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
72Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys1
5 10 15Phe Met Asp Val Tyr Gln
Arg Ser Tyr Cys His Pro Ile Glu Thr Leu 20 25
30Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr
Ile Phe Lys 35 40 45Pro Ser Cys
Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu 50
55 60Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile
Thr Met Gln Ile65 70 75
80Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe
85 90 95Leu Gln His Asn Lys Cys
Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg 100
105 110Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg
Lys His Leu Phe 115 120 125Val Gln
Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser 130
135 140Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu
Arg Thr Cys Arg Cys145 150 155
160Asp Lys Pro Arg Arg 16573451PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
73Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly1
5 10 15Ser Leu Lys Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser 20 25
30Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu
Glu Trp Val 35 40 45Ala Thr Ile
Thr Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Leu Tyr65 70 75
80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys
85 90 95Ala Arg Leu Gly Asn Tyr
Gly Gly Tyr Tyr Ala Met Asp Tyr Trp Gly 100
105 110Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser 115 120 125Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130
135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180
185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His 195 200 205Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210
215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260
265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290
295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310
315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 325 330
335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390
395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435
440 445Pro Gly Lys 45074213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
74Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly1
5 10 15Gly Lys Val Thr Ile Thr
Cys Thr Thr Ser Gln Asp Ser Asn Asn Tyr 20 25
30Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg
Leu Leu Ile 35 40 45His Tyr Ala
Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser
Asn Leu Glu Pro65 70 75
80Glu Asp Ile Ala Thr Tyr Phe Cys Leu Gln Tyr Asp Tyr Leu Trp Thr
85 90 95Phe Gly Gly Gly Thr Lys
Leu Glu Leu Lys Arg Thr Val Ala Ala Pro 100
105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly Thr 115 120 125Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130
135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu145 150 155
160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180
185 190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser Phe 195 200 205Asn
Arg Gly Glu Cys 21075451PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 75Glu Val Gln Leu Val Glu
Ser Gly Gly Asp Leu Val Lys Pro Gly Gly1 5
10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asp Ser 20 25 30Gly
Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35
40 45Ala Thr Ile Ser Ser Gly Gly Gly Tyr
Thr Tyr Tyr Ser Asp Ser Val 50 55
60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Thr Leu Phe65
70 75 80Leu Gln Met Ser Ser
Leu Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys 85
90 95Ala Arg Leu Gly Asn Tyr Gly Gly Tyr Tyr Gly
Met Asp Tyr Trp Gly 100 105
110Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135
140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185
190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210
215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260
265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290
295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340
345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 355 360 365Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370
375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420
425 430His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 435 440
445Pro Gly Lys 45076213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 76Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Leu Gly1 5 10
15Gly Lys Val Thr Ile Thr Cys Lys Thr Ser Gln Asp Ser Asn Lys
Tyr 20 25 30Ile Ala Trp Tyr
Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile 35
40 45His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser
Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro65 70
75 80Glu Asp Ile Ala Thr Tyr Tyr Cys
Leu Gln Tyr Asp Asn Leu Trp Thr 85 90
95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
Ala Pro 100 105 110Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115
120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys 130 135 140Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145
150 155 160Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165
170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala 180 185 190Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195
200 205Asn Arg Gly Glu Cys
21077445PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 77Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu
Val Lys Pro Gly Gly1 5 10
15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser
20 25 30Gly Met Ser Trp Val Arg Gln
Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40
45Ala Thr Ile Thr Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70
75 80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr
Ala Met Phe Tyr Cys 85 90
95Ala Arg Leu Gly Asn Tyr Gly Gly Tyr Tyr Ala Met Asp Tyr Trp Gly
100 105 110Gln Gly Thr Ser Val Thr
Val Ser Ser Ala Lys Thr Thr Pro Pro Ser 115 120
125Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser
Met Val 130 135 140Thr Leu Gly Cys Leu
Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val145 150
155 160Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly
Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro
180 185 190Ser Ser Thr Trp Pro
Ser Glu Thr Val Thr Cys Asn Val Ala His Pro 195
200 205Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro
Arg Asp Cys Gly 210 215 220Cys Lys Pro
Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile225
230 235 240Phe Pro Pro Lys Pro Lys Asp
Val Leu Thr Ile Thr Leu Thr Pro Lys 245
250 255Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp
Pro Glu Val Gln 260 265 270Phe
Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln 275
280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr
Phe Arg Ser Val Ser Glu Leu 290 295
300Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg305
310 315 320Val Asn Ser Ala
Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325
330 335Thr Lys Gly Arg Pro Lys Ala Pro Gln Val
Tyr Thr Ile Pro Pro Pro 340 345
350Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr
355 360 365Asp Phe Phe Pro Glu Asp Ile
Thr Val Glu Trp Gln Trp Asn Gly Gln 370 375
380Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp
Gly385 390 395 400Ser Tyr
Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu
405 410 415Ala Gly Asn Thr Phe Thr Cys
Ser Val Leu His Glu Gly Leu His Asn 420 425
430His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
435 440 44578213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
78Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly1
5 10 15Gly Lys Val Thr Ile Thr
Cys Thr Thr Ser Gln Asp Ser Asn Asn Tyr 20 25
30Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg
Leu Leu Ile 35 40 45His Tyr Ala
Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser
Asn Leu Glu Pro65 70 75
80Glu Asp Ile Ala Thr Tyr Phe Cys Leu Gln Tyr Asp Tyr Leu Trp Thr
85 90 95Phe Gly Gly Gly Thr Lys
Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro 100
105 110Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu
Thr Ser Gly Gly 115 120 125Ala Ser
Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 130
135 140Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
Asn Gly Val Leu Asn145 150 155
160Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser
165 170 175Thr Leu Thr Leu
Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr 180
185 190Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
Ile Val Lys Ser Phe 195 200 205Asn
Arg Gly Glu Cys 21079445PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 79Glu Val Gln Leu Val Glu
Ser Gly Gly Asp Leu Val Lys Pro Gly Gly1 5
10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asp Ser 20 25 30Gly
Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35
40 45Ala Thr Ile Ser Ser Gly Gly Gly Tyr
Thr Tyr Tyr Ser Asp Ser Val 50 55
60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Thr Leu Phe65
70 75 80Leu Gln Met Ser Ser
Leu Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys 85
90 95Ala Arg Leu Gly Asn Tyr Gly Gly Tyr Tyr Gly
Met Asp Tyr Trp Gly 100 105
110Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser
115 120 125Val Tyr Pro Leu Ala Pro Gly
Ser Ala Ala Gln Thr Asn Ser Met Val 130 135
140Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr
Val145 150 155 160Thr Trp
Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Asp Leu Tyr
Thr Leu Ser Ser Ser Val Thr Val Pro 180 185
190Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala
His Pro 195 200 205Ala Ser Ser Thr
Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly 210
215 220Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser
Ser Val Phe Ile225 230 235
240Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys
245 250 255Val Thr Cys Val Val
Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln 260
265 270Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr
Ala Gln Thr Gln 275 280 285Pro Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu 290
295 300Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys
Glu Phe Lys Cys Arg305 310 315
320Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335Thr Lys Gly Arg
Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro 340
345 350Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu
Thr Cys Met Ile Thr 355 360 365Asp
Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln 370
375 380Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro
Ile Met Asp Thr Asp Gly385 390 395
400Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp
Glu 405 410 415Ala Gly Asn
Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn 420
425 430His His Thr Glu Lys Ser Leu Ser His Ser
Pro Gly Lys 435 440
44580200PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 80Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Leu Gly1 5 10
15Gly Lys Val Thr Ile Thr Cys Lys Thr Ser Gln Asp Ser Asn Lys Tyr
20 25 30Ile Ala Trp Tyr Gln His Lys
Pro Gly Lys Gly Pro Arg Leu Leu Ile 35 40
45His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Arg Asp
Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro65 70
75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr
Asp Asn Leu Trp Thr 85 90
95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro
100 105 110Thr Val Ser Ile Phe Pro
Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120
125Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp
Ile Asn 130 135 140Val Lys Trp Lys Ile
Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn145 150
155 160Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Met Ser Ser 165 170
175Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr
180 185 190Cys Glu Ala Thr His
Lys Thr Ser 195 200817PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 81Glu
Leu Val Ile Pro Cys Arg1 5828PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 82Leu
Val Leu Asn Cys Thr Ala Arg1 58314PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 83Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5
108424PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 84Glu Leu Val Ile Pro Cys Arg Glu Ile Gly
Leu Leu Thr Cys Glu Ala1 5 10
15Thr Val Asn Gly His Leu Tyr Lys 208526PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMISC_FEATURE(26)..(26)May or may not be present 85Leu Val Leu Asn
Cys Thr Ala Arg Ser Asp Gln Gly Leu Tyr Thr Cys1 5
10 15Ala Ala Ser Ser Gly Leu Met Thr Lys Lys
20 258628PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMISC_FEATURE(28)..(28)May or
may not be present 86Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Thr His1 5 10 15Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 20
258717PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 87Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His
Leu Tyr1 5 10
15Lys8823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 88Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro
Ser His Gly Ile1 5 10
15Glu Leu Ser Val Gly Glu Lys 208924PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 89Ser
Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu1
5 10 15Ile Ile His Met Thr Glu Gly
Arg 209024PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 90Ser Asp Thr Gly Arg Pro Phe Val Glu Met
Tyr Ser Glu Ile Pro Glu1 5 10
15Ile Ile His Met Thr Glu Gly Arg 209154PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
91Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1
5 10 15Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp 20 25
30Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro
Glu Ile Ile 35 40 45His Met Thr
Glu Gly Arg 509254PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 92Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly1 5 10
15Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser
Asp 20 25 30Thr Gly Arg Pro
Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile 35
40 45His Met Thr Glu Gly Arg 50938PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 93Thr
Gln Ser Gly Ser Glu Met Lys1 59417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 94Ser
Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr1
5 10 15Lys9514PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 95Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5
109623PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 96Gln Thr Asn Thr Ile Ile Asp Val Val Leu
Ser Pro Ser His Gly Ile1 5 10
15Glu Leu Ser Val Gly Glu Lys 20977PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideSee
specification as filed for detailed description of substitutions and
preferred embodiments 97Thr Asn Tyr Leu Thr His Arg1
59854PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 98Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly1 5 10 15Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp 20
25 30Thr Gly Arg Pro Phe Val Glu Met Tyr
Ser Glu Ile Pro Glu Ile Ile 35 40
45His Met Thr Glu Gly Arg 509924PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 99Ser Asp Thr Gly Arg Pro Phe
Val Glu Met Tyr Ser Glu Ile Pro Glu1 5 10
15Ile Ile His Met Thr Glu Gly Arg
2010012PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptideMISC_FEATURE(1)..(1)May or may not be present 100Arg Val Thr
Ser Pro Asn Ile Thr Val Thr Leu Lys1 5
1010111PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptideMISC_FEATURE(1)..(1)May or may not be present 101Lys Gly Phe
Ile Ile Ser Asn Ala Thr Tyr Lys1 5
101029PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptideMISC_FEATURE(1)..(1)May or may not be present 102Lys Leu Val
Leu Asn Cys Thr Ala Arg1 51037PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMISC_FEATURE(1)..(1)May or may not be present 103Lys Asn Ser Thr
Phe Val Arg1 5
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