U.S. patent application number 17/409193 was filed with the patent office on 2021-12-16 for cell culture process for making a glycoprotein.
The applicant listed for this patent is Regeneron Pharmaceuticals, Inc.. Invention is credited to Scott CARVER, John CHEN, Ta-Chun HANG, Amy JOHNSON, Shawn LAWRENCE, Theodore LONEY, Ravindra PANGULE, Bernhard SCHILLING.
Application Number | 20210388408 17/409193 |
Document ID | / |
Family ID | 1000005799066 |
Filed Date | 2021-12-16 |
United States Patent
Application |
20210388408 |
Kind Code |
A1 |
CHEN; John ; et al. |
December 16, 2021 |
CELL CULTURE PROCESS FOR MAKING A GLYCOPROTEIN
Abstract
The instant application provides a method for screening batches
of soy hydrolysate for a desired amount of a component thereof,
such as ornithine or putrescine, and selecting only those batches
of soy hydrolysate that have a desired amount of such component.
The present disclosure also sets forth methods for culturing cells
in media supplemented with selected batches of soy to produce more
consistent, high quality lots of a protein of interest. Further,
the present disclosure provides a plurality of protein preparations
that have each been produced by culturing cells in media
supplemented with separate batches of soy hydrolysate containing a
desired amount of ornithine or putrescine, whereby each batch of
protein produced exhibits improved quality of the protein of
interest or amount of quality protein produced.
Inventors: |
CHEN; John; (Tarrytown,
NY) ; LAWRENCE; Shawn; (Nyack, NY) ; JOHNSON;
Amy; (Briarcliff Manor, NY) ; LONEY; Theodore;
(Averill Park, NY) ; PANGULE; Ravindra;
(Bridgewater, NJ) ; HANG; Ta-Chun; (Morristown,
NJ) ; CARVER; Scott; (Wynantskill, NY) ;
SCHILLING; Bernhard; (Hudson, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Regeneron Pharmaceuticals, Inc. |
Tarrytown |
NY |
US |
|
|
Family ID: |
1000005799066 |
Appl. No.: |
17/409193 |
Filed: |
August 23, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16026539 |
Jul 3, 2018 |
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17409193 |
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62625744 |
Feb 2, 2018 |
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62529471 |
Jul 6, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/7155 20130101;
C12P 21/005 20130101; C07K 14/71 20130101; C07K 2319/30 20130101;
C07K 2319/32 20130101; C07K 14/4705 20130101 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C07K 14/47 20060101 C07K014/47; C07K 14/715 20060101
C07K014/715; C07K 14/71 20060101 C07K014/71 |
Claims
1. A method comprising: a. enzymatically digesting soy extract in a
residue-free reaction vessel to manufacture a soy hydrolysate; b.
measuring the amount of ornithine or putrescine in the soy
hydrolysate; and c. selecting soy hydrolysate with .ltoreq.0.067%
(w/w) ornithine or putrescine for use in a cell culture media.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 16/026,539, filed Jul. 3, 2018, which claims priority to
and the benefit of U.S. Provisional Patent Application Ser. Nos.
62/529,471, filed Jul. 6, 2017, and 62/625,744 filed Feb. 2, 2018,
the contents of each of which are herein incorporated by reference
in their entireties.
INCORPORATION OF THE SEQUENCE LISTING
[0002] The contents of the text filed named
"REGE009D02US_SeqList.txt", which was created on Aug. 23, 2021 and
is 11.3 KB in size, are hereby incorporated by reference in their
entirety.
FIELD
[0003] The invention relates to methods for the culturing of cells
and for the production of recombinant proteins. The invention
specifically relates to methods for the culturing of cells in soy
hydrolysate-containing media to achieve consistent production of
high quality recombinant protein.
BACKGROUND
[0004] Cell culture media containing protein hydrolysates, such as
soy hydrolysate, are commonly used in the production of recombinant
proteins from cultured cells. However, protein hydrolysates may
contain compounds that negatively impact cell growth or recombinant
protein production. Despite these drawbacks, protein hydrolysates
have been widely used as supplements in cell culture.
[0005] Human biological therapeutics (biopharmaceuticals) are
generally produced in mammalian cell culture. However, the quality
and performance of biological therapeutics are highly dependent
upon the manufacturing process. Tebbey, P. and Declerck, P.
Generics and Biosimilars Initiative Journal (2016) 5:2, pp. 70-73
is incorporated herein for manufacturing biological drugs with
consistent glycosylation. Changes to the cell culture process for
the manufacture of glycoproteins may lead to variation in
glycosylation pattern, the presence of acidic species (e.g., sialic
acid) or the amount of glycan on a protein. Id. Such variation
increases heterogeneity of protein isoforms in the resulting
protein production, which can alter stability, efficacy or
immunogenicity of the biological therapeutic and ultimately lead to
the rejection of the lot of proteins.
[0006] Hence, cell culture methods that eliminate lot-to-lot
variability in drug product yield and composition are highly
desirable. The present disclosure identifies certain components in
plant protein hydrolysate (e.g., soy hydrolysate) that can vary
from batch-to-batch and alter the composition and yield of high
quality glycoproteins produced in culture using soy hydrolysate.
The present disclosure addresses the need for improved cell culture
methods by, among other things, screening batches of plant protein
hydrolysate and selecting those batches that include a desirable
concentration of a component of plant protein hydrolysate for use
in the production of biopharmaceuticals.
SUMMARY
[0007] The present disclosure is predicated in part on the
discovery that the concentration of ornithine or putrescine in a
batch of soy hydrolysate affects the quality and composition of
proteins produced in cell culture using soy hydrolysate. The
present disclosure also provides that cells cultured in media
including soy hydrolysate comprising certain concentrations of
ornithine or putrescine produce greater amounts of high quality
proteins exhibiting more consistent glycosylation patterns, amounts
of glycan and sialic acid profiles from lot-to-lot.
[0008] In one aspect, the invention relates to a method of
culturing a population of cells expressing a recombinant
heterologous glycoprotein in cell culture media comprising soy
hydrolysate to produce the recombinant heterologous glycoprotein,
and wherein the soy hydrolysate comprises .ltoreq.0.067% (w/w)
ornithine or putrescine.
[0009] In some embodiments, the method includes the steps of
culturing a population of cells expressing a recombinant
heterologous glycoprotein in cell culture media comprising soy
hydrolysate containing .ltoreq.less than 0.67 milligram (mg) of
ornithine per gram (g) of soy (w/w), or about 0.003%-0.067% (w/w)
ornithine. In one embodiment, the culture media contains .ltoreq.5
mg/L ornithine, or about 0.6-3 mg/L ornithine. In some embodiments,
the population of cells is obtained by clonal expansion of a cell
expressing a recombinant heterologous glycoprotein.
[0010] In one aspect, the invention relates to a method for
producing a glycoprotein. In one embodiment, the method includes
the steps of culturing a population of cells expressing a
recombinant heterologous glycoprotein in culture media containing
soy hydrolysate containing .ltoreq.less than 0.67 milligram (mg) of
putrescine per gram (g) of soy (w/w), or about 0.003%-0.067% (w/w)
putrescine. In one embodiment, the culture media contains .ltoreq.5
mg/L putrescine, or about 0.6-3 mg/L putrescine. In some
embodiments, the population of cells is obtained by clonal
expansion of a cell expressing a recombinant heterologous
glycoprotein.
[0011] In one embodiment, the glycoprotein is a trap molecule, such
as rilonacept (IL1-trap, disclosed, e.g., in U.S. Pat. No.
6,927,004), aflibercept (VEGF-trap, disclosed, e.g., in U.S. Pat.
No. 7,087,411), conbercept (VEGF-trap, disclosed, e.g., in U.S.
Pat. Nos. 7,750,138 and 8,216,575), and etanercept (TNF-trap,
disclosed, e.g., in U.S. Pat. No. 5,610,279.) In one embodiment,
.gtoreq.10% (w/w) of the total amount of all N-glycan species of
the glycoprotein is an A1 N-glycan.
[0012] In one aspect, the invention relates to a method of
producing a glycoprotein. In another aspect, the invention relates
to a method of using a soy hydrolysate in the producing of a
glycoprotein. In another aspect, the invention relates to a method
of selecting a soy hydrolysate for use in producing a glycoprotein
by evaluating the quality of the produced glycoprotein. In one
embodiment, the method comprises culturing a cell expressing a
glycosylated protein in a cell culture media to produce the
glycoprotein, purifying the glycosylated protein, subjecting the
purified glycosylated protein to oligosaccharide fingerprint
analysis, determining the relative amount of an A1 N-glycan
compared to total amount of N-glycan species of the glycoprotein;
and selecting a soy hydrolysate that provides for at least 10%
(w/w) A1 N-glycan compared to total amount of N-glycan species of
the glycoprotein.
[0013] In one embodiment, the method includes the steps of
preparing a cell culture media containing a soy hydrolysate,
culturing a cell that expresses the glycoprotein in the cell
culture media, purifying the glycosylated protein, subjecting the
purified glycosylated protein to oligosaccharide fingerprint
analysis, determining the relative amount of an A1 N-glycan
compared to total amount of N-glycan species of the glycoprotein,
and then selecting the soy hydrolysate that provides for the
production of a glycoprotein with at least 10% (w/w) A1 N-glycan
compared to total amount of N-glycan species of the
glycoprotein.
[0014] In one embodiment, the selected soy hydrolysate contains
.ltoreq.0.67 mg ornithine per g soy (w/w), or about 0.003%-0.067%
(w/w) ornithine. In one embodiment, the culture media contains
.ltoreq.5 mg/L ornithine, or about 0.6-3 mg/L ornithine.
[0015] In one embodiment, the selected soy hydrolysate contains
.ltoreq.0.67 mg putrescine per g soy (w/w), or about 0.003%-0.067%
(w/w) putrescine. In one embodiment, the culture media contains
.ltoreq.5 mg/L putrescine, or about 0.6-3 mg/L putrescine.
[0016] In one aspect, the invention relates to a method of
selecting a soy hydrolysate for use in producing a glycoprotein by
measuring the amount of ornithine or putrescine in the soy
hydrolysate. In one embodiment, the method includes the steps of
measuring the amount of ornithine in a soy hydrolysate, selecting a
soy hydrolysate with .ltoreq.0.67 mg ornithine per g soy, or about
0.003%-0.067% (w/w) ornithine, and combining the selected soy
hydrolysate with an additional ingredient to form a cell culture
media with .ltoreq.5 mg/L ornithine, or about 0.6-3 mg/L ornithine.
In one embodiment, the method includes the steps of measuring the
amount of putrescine in a potentially useful soy hydrolysate,
selecting a soy hydrolysate with .ltoreq.0.67 mg putrescine per g
soy, or about 0.003%-0.067% (w/w) putrescine, and combining the
selected soy hydrolysate with an additional ingredient to form a
cell culture media with .ltoreq.5 mg/L putrescine, or about 0.6-3
mg/L putrescine.
[0017] In one aspect, the invention relates to a glycoprotein
comprising an A1 N-glycan and at least one other N-glycan species
in which the relative amount of the A1 N-glycan is at least 10%
(w/w) of the total amount of N-glycans of the glycoprotein is
provided. In one embodiment, the relative amount of the A1 N-glycan
is about 10%-17% (w/w).
[0018] In one embodiment, the glycoprotein also has an A2 N-glycan,
an A2F N-glycan, an A1F N-glycan, an NGA2F N-glycan, an NA2G1F
N-glycan, an NA2 N-glycan, and an NA2F N-glycan.
[0019] In one embodiment, the glycoprotein contains 8-65 moles of
sialic acid per mole of glycoprotein. In one embodiment in which
the glycoprotein is rilonacept, any one of asparagine residues N37,
N98, N418, and N511 of SEQ ID NO: 1 contains an A1 N-glycan. In one
embodiment in which the glycoprotein is aflibercept, any one of
asparagine residues N123 and N196 of SEQ ID NO: 2 contains an A1
N-glycan.
[0020] In one embodiment, the relative amount of the A1 N-glycan of
the glycoprotein is determined by comparing the area under the peak
of the A1 N-glycan to the total areas under the peak for all
N-glycans obtained from an oligosaccharide fingerprint of the
glycoprotein obtained by capillary electrophoresis.
[0021] In one aspect, a method of manufacturing soy hydrolysate
having a reduced amount of ornithine or putrescine is provided. In
one embodiment, the method comprises the steps of enzymatically
digesting soy extract in a residue-free reaction vessel, measuring
the amount of ornithine in the soy hydrolysate, and selecting those
lots of soy hydrolysate containing .ltoreq.0.067% (w/w) ornithine
or putrescine for use in a cell culture media. In one embodiment,
the method comprises the steps of enzymatically digesting soy
extract in a residue-free reaction vessel, measuring the amount of
putrescine in the soy hydrolysate, and selecting soy hydrolysate
with .ltoreq.0.067% (w/w) ornithine or putrescine for use in a cell
culture media.
[0022] The term "about" can be understood as within 10%, 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the
stated value. Unless otherwise clear from the context, all
numerical values provided herein are modified by the term
"about."
[0023] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present disclosure, suitable methods and materials are
described below. All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference
in their entirety. The references cited herein are not admitted to
be prior art to the claimed disclosure. In the case of conflict,
the present Specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and are not intended to be limiting. Other features and
advantages of the disclosure will be apparent from the following
detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] 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.
[0025] Any of the above aspects and embodiments can be combined
with any other aspect or embodiment as disclosed here in the
Summary and/or Detailed Description sections.
[0026] Various objects and advantages and a more complete
understanding of the present invention are apparent and more
readily appreciated by reference to the following Detailed
Description and to the appended claims when taken in conjunction
with the accompanying Drawing wherein:
[0027] FIG. 1 depicts a chromatographic elution profile of
ninhydrin-derived amino acids. The X-axis depicts time of elution
from a chromatography column (retention time), and the Y-axis
depicts light absorbance at 570 nm. Panel A depicts batch that does
not meet the criteria for producing an acceptable N-glycan mixture
by FDA standards. Panel B depicts an acceptable amino acid analysis
of soy protein hydrolysate. The peak representing ornithine is
circled in both chromatograms.
[0028] FIG. 2 depicts a capillary electrophoretogram of
oligosaccharides released from a glycoprotein by
peptide:N-glycosidase F (PNGase F) digestion. The X-axis depicts
time of elution from a capillary, and the Y-axis depicts light
absorbance or fluorescence intensity. The peaks are numbered 1-21.
Peak 1 represents the N-glycan A2; peak 4 represents the N-glycan
A2F; peak 11 represents the N-glycan A1; peak 14 represents the
N-glycan A1F; peak 16 represents the N-glycan NGA2F; peak 19
represents the N-glycan NA2G1F; peak 20 represents the N-glycan
NA2; and peak 21 represents the N-glycan NA2F.
[0029] FIG. 3 depicts a dot blot of the relative amount of A1
N-glycan as a function of ornithine and citrulline concentration in
soy protein hydrolysate. The X-axis depicts the concentration of
citrulline or ornithine in mg/L. The Y-axis depicts the relative
area of peak 11, which represents A1 N-glycan.
[0030] FIG. 4 is a correlation plot depicting the negative
correlation of ornithine concentration in soy hydrolysate (lower
right quadrant) to the relative amount of peak 11 in aflibercept
(A1 N-glycan, upper left quadrant).
[0031] FIG. 5 is a correlation plot depicting (i) the negative
correlation of ornithine concentration in soy hydrolysate (lower
left quadrant) to the final titer of rilonacept (upper right
quadrant); and (ii) the positive correlation of ornithine
concentration in soy hydrolysate (lower left quadrant) to the
accumulation of lactate in media (lower left quadrant).
[0032] FIG. 6A is a pair of graphs depicting the amount of
polyamine synthesized from a CHO cell culture, depicted as either
IVCD.times.10.sup.6 cell-day/ml or as titer (as grams per ml) as a
function of batch day under various conditions including, control,
high and low ornithine concentrations, putrescine, MFC and IPC.
[0033] FIG. 6B is a table providing the experimental conditions of
each study group depicted in FIG. 6A.
DETAILED DESCRIPTION
[0034] It is to be understood that the scope of the present
disclosure is not limited to the particular methods and
experimental conditions described, as such methods and conditions
may vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting.
[0035] Unless defined otherwise, all technical and scientific terms
used in this application have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. Although any methods and materials similar or
equivalent to those described in this application can be used in
the practice or testing of the present invention, certain specific
methods and materials are now described. Units, prefixes, and
symbols may be denoted in their standard, industry accepted form.
Numeric ranges recited herein are open-bracketed, meaning that they
include the numbers defining the range. Unless otherwise noted, the
terms "a" or "an" are to be construed as meaning "at least one
of".
[0036] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. The methods and techniques described herein are
generally performed according to conventional methods known in the
art and as described in various general and more specific
references that are cited and discussed throughout the present
specification unless otherwise indicated. See, e.g., Sambrook et
al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and
Ausubel et al., Current Protocols in Molecular Biology, Greene
Publishing Associates (1992), Harlow and Lane Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1990), and Julio E. Celis, Cell Biology: A Laboratory
Handbook, 2nd ed., Academic Press, New York, N.Y. (1998), and
Dieffenbach and Dveksler, PCR Primer: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1995).
All publications mentioned throughout this disclosure are
incorporated herein by reference in their entirety.
Definitions
[0037] Unless defined otherwise, all technical and scientific terms
used in this application have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs.
[0038] The phrase "relative amount" means the amount of a molecular
species over the total amount of all molecular species of a general
type. For example, the relative amount of an A1 glycan (i.e.,
(GlcNAc)2(Man)3(GlcNAc)2(Gal)2(SA)1) is calculated as amount of
A1/sum of the amount of all N glycans. The relative amount can be
expresses as absolute mass-to-mass amounts (i.e. gram per gram) or
a percentage i.e., % (w/w).
[0039] "Ornithine" is a non-protein coding amino acid involved in
the urea cycle, polyamine synthesis and arginine metabolism.
Ornithine is also known to influence glycoform content of
recombinant proteins. See PCT/US2014/069378. Ornithine is acted on
by several enzymes. For example, ornithine decarboxylase catalyzes
the conversion of ornithine to putrescine in the polyamine
biosynthetic pathway. See Pegg A, J. of Biol. Chem. (2006) 281:21
pp. 14532. Additionally, ornithine conversion to citrulline is
catalyzed by ornithine transcarbamylase as part of the urea cycle.
Ornithine metabolism occurs in both cytosol and mitochondria of
cells in culture. The presence of putrescine or presence of
ornithine has been deemed critical for growth and productivity of
cells cultured in chemically defined media, however the impact on
critical quality attributes of a protein produced by such cells has
not been described.
[0040] "Putrescine" is a non-protein coding amino acid, a
polyamine, involved in the urea cycle. Putrescine (also known as
1,4-Diaminobutane, having a chemical formula of
C.sub.4H.sub.12N.sub.2) is produced by the decarboxylation of
ornithine and serves as a precursor to gamma-aminobutyrate
(.gamma.-aminobutyrate).
[0041] As used herein "peptide", "polypeptide" and "protein" are
used interchangeably throughout and refer to a molecule comprising
two or more amino acid residues joined to each other by a peptide
bond. Peptides, polypeptides and proteins may also include
modifications such as glycosylation, lipid attachment, sulfation,
gamma-carboxylation of glutamic acid residues, alkylation,
hydroxylation and ADP-ribosylation. Peptides, polypeptides, and
proteins can be of scientific or commercial interest, including
protein-based drugs (biotherapeutics). Peptides, polypeptides, and
proteins include, among other things, antibodies and chimeric or
fusion proteins. Peptides, polypeptides, and proteins can be
produced by recombinant animal cell lines such as mammalian cell
lines using cell culture methods.
[0042] The term "polynucleotide sequence" or "peptide sequence", as
used herein refers to nucleic acid polymers encoding proteins of
interest, such as chimeric proteins (like trap molecules),
antibodies or antibody portions (e.g., VH, VL, CDR3) that are
produced as a biopharmaceutical drug substance. The polynucleotide
sequence may be manufactured by genetic engineering techniques
(e.g., a sequence encoding a chimeric protein, or a codon-optimized
sequence, an intron-less sequence) and introduced into a cell,
where it may reside as an episome or be integrated into the genome
of the cell. The polynucleotide sequence may be a naturally
occurring sequence that is introduced into an ectopic site within
the host cell genome. The peptide sequence may be heterologous,
such as a naturally occurring sequence from another organism, a
recombinant sequence, a genetically modified sequence, or inter
alia a sequence expressed under the control of a
different-than-wild type promoter, for example a nucleotide
sequence encoding a human ortholog, whereby the host (production)
cell is a CHO cell.
[0043] The phrase "antigen-binding protein" includes a protein that
has at least one CDR and is capable of selectively recognizing an
antigen, i.e., is capable of binding an antigen with a KD that is
at least in the micromolar range. Therapeutic antigen-binding
proteins (e.g., therapeutic antibodies) frequently require a KD
that is in the nanomolar or the picomolar range. Typically, an
antigen-binding protein includes two or more CDRs, e.g., 2, 3, 4,
5, or 6 CDRs. Examples of antigen binding proteins include
antibodies, antigen-binding fragments of antibodies such as
polypeptides containing the variable regions of heavy chains and
light chains of an antibody (e.g., Fab fragment, F(ab')2 fragment),
and proteins containing the variable regions of heavy chains and
light chains of an antibody and containing additional amino acids
from the constant regions of heavy and/or light chains (such as one
or more constant domains, i.e., one or more of CL, CH1, hinge, CH2,
and CH3 domains).
[0044] "Antibody" refers to an immunoglobulin molecule consisting
of four polypeptide chains, two heavy (H) chains and two light (L)
chains inter-connected by disulfide bonds. Each heavy chain has a
heavy chain variable region (HCVR or VH) and a heavy chain constant
region. The heavy chain constant region contains three domains,
CH1, CH2 and CH3. Each light chain has a light chain variable
region (VL) and a light chain constant region. The light chain
constant region consists of one domain (CL). The VH and VL regions
can be further subdivided into regions of hypervariability, termed
complementarity determining regions (CDR), 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 the amino-terminus to the carboxy-terminus in the following
order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The term
"antibody" includes both glycosylated and non-glycosylated
immunoglobulins of any isotype or subclass. The term "antibody"
includes antibody molecules prepared, expressed, created or
isolated by recombinant means, such as antibodies isolated from a
host cell transfected with a nucleotide sequence in order to
express the antibody. The term "antibody" also includes a
bispecific antibody, which includes a heterotetrameric
immunoglobulin that can bind to more than one epitope. Bispecific
antibodies are generally described in U.S. Patent Application
Publication No. 2010/0331527, which is incorporated by reference
herein.
[0045] The term "antigen-binding portion" of an antibody (or
antibody fragment) or a protein of interest refers to one or more
fragments of an antibody or a protein of interest that retain the
ability to specifically bind to an antigen. Non-limiting examples
of protein binding fragments encompassed within the term
"antigen-binding portion" of an antibody include (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and
CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al.,
Nature (1989) 241:544-546), which consists of a VH domain, (vi) an
isolated CDR, and (vii) an scFv, which consists of the two domains
of the Fv fragment, VL and VH, joined by a synthetic linker to form
a single protein chain in which the VL and VH regions pair to form
monovalent molecules. Other forms of single chain antibodies, such
as diabodies are also encompassed under the term "antibody". See,
e.g., Holliger et al., PNAS USA (1993) 90:6444-6448; Poljak et al.,
Structure (1994) 2:1121-1123.
[0046] Still further, an antibody or antigen-binding portion
thereof may be part of a larger immunoadhesion molecule, formed by
covalent or noncovalent association of the antibody or antibody
portion with one or more other proteins or peptides. Non-limiting
examples of such immunoadhesion molecules include use of the
streptavidin core region to make a tetrameric scFv molecule
(Kipriyanov et al., Human Antibodies and Hybridomas (1995)
6:93-101) and use of a cysteine residue, a marker peptide and a
C-terminal polyhistidine tag to make bivalent and biotinylated scFv
molecules (Kipriyanov et al. Mol. Immunol. (1994) 31:1047-1058).
Antibody portions, such as Fab and F(ab')2 fragments, can be
prepared from whole antibodies using conventional techniques, such
as via papain or pepsin digestion of whole antibodies. Moreover,
antibodies, antibody portions and immunoadhesion molecules can be
obtained using standard recombinant DNA techniques commonly known
in the art (see Sambrook et al., 1989).
[0047] The term "human antibody" is intended to include antibodies
having variable and constant regions derived from human germline
immunoglobulin sequences. Human antibodies of the present
disclosure may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo), for example in the CDRs and in particular CDR3. The term
"recombinant human antibody", as used herein, is intended to
include all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as antibodies expressed
using a recombinant expression vector transfected into a host cell,
antibodies isolated from a recombinant, combinatorial human
antibody library, antibodies isolated from an animal (e.g., a
mouse) that is transgenic for human immunoglobulin genes (see,
e.g., Taylor et al. Nucl. Acids Res. (1992) 20:6287-6295) or
antibodies prepared, expressed, created or isolated by any other
means that involves splicing of human immunoglobulin gene sequences
to other DNA sequences. Such recombinant human antibodies have
variable and constant regions derived from human germline
immunoglobulin sequences. In certain embodiments, however, such
recombinant human antibodies are subjected to in vitro mutagenesis
(or, when an animal transgenic for human Ig sequences is used, in
vivo somatic mutagenesis), and thus the amino acid sequences of the
VH and VL regions of the recombinant antibodies are sequences that,
while derived from and related to human germline VH and VL
sequences, may not naturally exist within the human antibody
germline repertoire in vivo.
[0048] "Fc fusion proteins" comprise part or all of two or more
proteins, one of which is an Fc portion of an immunoglobulin
molecule, which are not otherwise found together in nature.
Preparation of fusion proteins comprising certain heterologous
polypeptides fused to various portions of antibody-derived
polypeptides (including the Fc domain) has been described, e.g., by
Ashkenazi et al., PNAS USA (1991) 88:10535; Byrn et al., Nature
(1990) 344:677; and Hollenbaugh et al., Current Protocols in
Immunology (1992) Suppl. 4, pp. 10.19.1-10.19.11. "Receptor Fe
fusion proteins" comprise one or more extracellular domain(s) of a
receptor coupled to an Fc moiety, which in some embodiments
comprises a hinge region followed by a CH2 and CH3 domain of an
immunoglobulin. In some embodiments, the Fc-fusion protein contains
two or more distinct receptor chains that bind to a one or more
ligand(s).
[0049] In certain embodiments, an "Fc-fusion protein" is a "trap"
molecule, which is a decoy receptor molecule that includes two
distinct receptor components that mimic the binding domains of a
corresponding endogenous receptor and the Fc portion of an
antibody. Non-limiting examples of trap molecules include an IL-1
trap (e.g., rilonacept, which contains the IL-1RAcP ligand binding
region fused to the IL-1R1 extracellular region which in turn is
fused to the Fc of hIgG1) (e.g., SEQ ID NO:1) (see U.S. Pat. No.
6,927,004), or a VEGF trap (e.g., aflibercept, which contains the
Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of
the VEGF receptor Flk1 which in turn is fused to Fc of hIgG1. See,
e.g., U.S. Pat. Nos. 7,087,411, 7,279,159; see also U.S. Pat. No.
5,610,279 for etanercept (TNF trap).
[0050] "Glycosylation" includes the formation of glycoproteins
where oligosaccharides are attached either to the side chain of an
asparagine (Asn) residue (i.e., N-linked), or a serine (Ser) or
threonine (Thr) residue (i.e., O-linked) of a protein.
"Glycoproteins" include any protein that contains an O-linked
glycan or an N-linked glycan. Glycans can be homo- or
heteropolymers of monosaccharide residues, which can be linear or
branched. N-linked glycosylation is known to initiate primarily in
the endoplasmic reticulum, whereas O-linked glycosylation is shown
to initiate in either the ER or Golgi apparatus. The term
"N-glycan" is used interchangeably with "N-linked oligosaccharide."
The term "O-glycan" is used interchangeably with "O-linked
oligosaccharide."
[0051] An "N-glycan protein" includes proteins that contain or can
accept N-linked oligosaccharides. N-glycans can be composed of
N-acetyl galactosamine (GalNAc), mannose (Man), fucose (Fuc),
galactose (Gal), neuraminic acid (NANA), and other monosaccharides,
however N-glycans usually have a common core pentasaccharide
structure including: three mannose and two N-acetylglucosamine
(GlcNAc) sugars. Proteins with the consecutive amino acid sequence,
Asn-X-Ser or Asn-X-Thr, where X is any amino acid except proline,
can provide an attachment site for N-glycans.
[0052] N-glycans include those N-linked oligosaccharides listed in
Table 1. The shorthand designations of the listed oligosaccharides
are used herein as simplified names to describe the
oligosaccharide. Thus for example, an A1 N-glycan contains an
arginine linked to a oligosaccharide consisting of
(SA)(Gal)2(GlcNAc)2(Man)3(GlcNAc)3.
TABLE-US-00001 TABLE 1 N-Linked Oligosaccharides Expected Shorthand
Mass Graphic Name of Molecule* Designation (g/mol) Depiction**
(SA)(Gal)2(GlcNAc)2(Man)3(GlcNAc)3 A1 2051.7 ##STR00001##
(SA)(Gal)2(GlcNAc)2(Man)3(GlcNAc)3(Fuc) A1F 2197.7 ##STR00002##
(SA)2(Gal)2(GlcNAc)2(Man)3(GlcNAc)3 A2 2343.2 ##STR00003##
(SA)2(Gal)2(GlcNAc)2(Man)3(GlcNAc)3(Fuc) A2F 2488.8 ##STR00004##
(Man)5(GlcNAc)2 Man5 1354.4 ##STR00005##
(Gal)2(GlcNAc)2(Man)3(GlcNAc)3 NA2 1760.6 ##STR00006##
(Gal)2(GlcNAc)2(Man)3(GlcNAc)3(Fuc) NA2F 1906.6 ##STR00007##
(Gal)(GlcNAc)2(Man)3(GlcNAc)3 NA2G1 1598.5 ##STR00008##
(Gal)(GlcNAc)2(Man)3(GlcNAc)3(Fuc) NA2G1F 1744.1 ##STR00009##
(GlcNAc)2(Man)3(GlcNAc)2 NGA2 1436.5 ##STR00010##
(GlcNAc)2(Man)3(GlcNAc)2(Fuc) NGA2F 1582.5 ##STR00011##
*Abbreviations for monosaccharides are sialic acid (SA), galactose
(Gal), mannose (Man), GlcNAc (N-acetylglucosamine), and fucose
(Fuc). **Glycan key: triangle = fucose; square = N-acetyl
glucosamine; circle = mannose; star = sialic acid.
Screening
[0053] "Hydrolysates" are complex materials derived from the
hydrolysis of plant material, animal material, whey, yeast, and the
like. The term "hydrolysate" is used interchangeably with "protein
hydrolysate". "Plant hydrolysates" (plant protein hydrolysates) are
hydrolysed plant material such as rice flour, wheat flour, corn
flour, soy flour, and the like. Protein hydrolysates can be
manufactured by three general methods: acid hydrolysis, alkaline
hydrolysis, and enzymatic hydrolysis. For biological applications,
including biotherapeutic manufacturing, protein hydrolysates are
mostly made by enzymatic hydrolysis. For example, a soy hydrolysate
made by pepsin digestion may be called "soy peptone", or a yeast
hydrolysate made by trpsin digestion may be called "yeast
tryptone." Franek et al., Biotechnol. Prog. 16(5): 688-92 (2000) is
incorporated herein for plant protein hydrolysates and methods of
manufacturing them.
[0054] In some embodiments, the subject hydrolysate is a plant
hydrolysate. In a specific embodiment, the subject protein
hydrolysate is a soy hydrolysate. "Soy hydrolysate" is an
enzymatically digested soy product derived from soybean grit, that
is largely, chemically undefined. Generally, soy hydrolysate is
composed of a conglomerate of amino acids, proteins, carbohydrates,
minerals and vitamins. Soy hydrolysate is a plant-derived protein
hydrolysate that is commercially available, for example, in high
concentration solution (e.g., HyClone.TM. HyQ Soy Hydrolysate
solution) or powder (e.g., Sigma Aldrich.RTM. S1674 (Amisoy.TM.),
soy protein hydrolysate) form. A "batch" or "lot" of soy
hydrolysate, as used herein refers to a manufactured amount of soy
hydrolysate resulting from hydrolysis of soybean grit. For example,
each hydrolysis process can result in a unique "batch" or "lot" of
soy hydrolysate with varying concentrations of components, such as
vitamins, amino acids, peptides and sugars. Soy hydrolysate is
commonly used with animal protein-free cell culture medium for the
growth of mammalian cell lines during the production of commercial
biotherapeutics, such as antibodies. More specifically, soy
hydrolysate is added to a cell culture media prior to or during
inoculation with cells. The cells are then cultured in the
hydrolysate-containing medium, until they are harvested. Due to the
undefined nature of soy hydrolysate, batches of soy hydrolysate
will vary from batch-to-batch (or lot-to-lot), which can lead to
inconsistencies in commercial manufacturing of biotherapeutics.
[0055] The present disclosure has identified that concentrations of
certain components in a batch of soy hydrolysate affect the quality
and composition of proteins produced in cell culture using soy
hydrolysate. The present disclosure provides methods for screening
batches of soy hydrolysate in order to select certain batches of
soy hydrolysate that contain a desirable amount of a component such
as, for example, ornithine, putrescine, citrulline, arginine or a
combination thereof.
[0056] In certain embodiments, the screening method includes
measuring the amount of ornithine or putrescine in at least a
portion (i.e., a sample) of a batch of soy hydrolysate. In a
specific embodiment, a soy hydrolysate sample is weighed and a
portion thereof is dissolved to a desired concentration. In some
embodiments, the soy hydrolysate solution is then diluted in a
solvent to a second desired concentration (e.g., 1 g/L to 25 g/L)
and the composition of the resulting soy hydrolysate solutions can
then be determined.
[0057] In some embodiments, the measuring step employs a suitable
method for determining the molecular composition of a soy
hydrolysate sample including, for example, colorimetric detection
performed following post-column ninhydrin reaction, or
chromatography f eluted ninhydrin-positive compounds, such as HPLC
or UPLC, and the units used to express the measured amount of each
component (e.g., ornithine or putrescine) can be any suitable units
(e.g., micromoles/L, mg/L or g/L). In some embodiments, measuring
the amount of ornithine or putrescine includes measuring the
concentration of ornithine in a sample or measuring the total
amount of ornithine in a soy hydrolysate sample. However, an amount
of ornithine or putrescine is measured and whatever units are used
to express the measured amount, the concentration of ornithine or
putrescine in the selected batch of soy hydrolysate is less than or
equal to 0.67 mg ornithine or putrescine per g soy.
[0058] In one embodiment, a sample of a batch of soy hydrolysate is
obtained and the ornithine or putrescine contents of the sample are
measured by chromatography of the amino acids on an ion exchange
column with post column ninhydrin detection. More specifically, in
a specific embodiment screening methods include acid hydrolysis of
a soy hydrolysate sample and reconstitution in a sample buffer. The
hydrolyzed sample is then subject to high performance cation
exchange separation on, for example, a column of a sulphonated
polystyrene resin (Dowex 50) followed by a post-column
derivatization that allows sensitive detection of individual amino
acids within a sample. See, e.g., Moore and Stein. J. Biol. Chem.
(1954) Vol. 211 pp. 907-913; Nemkov, et al., Amino Acids 2015
November; 47(11): 2345-2357; Wahl and Holzgrabe, "Amino acid
analysis for pharmacopoeial purposes," Talanta 154:150-163, 1 Jul.
2016. Following post-column color development by Ninhydrin reagent,
absorbance is measured in the ninhydrin purple range, e.g., 570 nm.
Data acquisition is accomplished using chromatography software
(e.g., EZChrom Elite for Hitachi version 3.1.5b chromatography
software) to provide a quantitative chromatogram showing
micromoles/L per amino acid, mg/L per amino acid, or g/L per amino
acid.
[0059] One of ordinary skill in the art will appreciate that other
methods for the identification and measurement of amino acids in a
sample composition can be used in accordance with the methods of
the presence disclosure, such as pre-column derivatization
chromatography or reverse phase liquid chromatography methods using
liquid chromatography and mass spectroscopy.
[0060] In certain embodiments, liquid chromatography-mass
spectrometry is used to screen a soy hydrolysate sample. For
example, a sample of a batch of soy hydrolysate may be obtained as
set forth herein and subjected to a chromatography run, or series
of chromatography runs on a high-performance liquid chromatography
(HPLC) system, such as Agilent 1100 or Agilent 1200SL. Mass
spectrometry analysis can be carried out to provide high-resolution
quantitative data describing the composition of the soy hydrolysate
sample being measured.
[0061] In some embodiments, the present disclosure provides a
method that includes screening batches of soy hydrolysate for a
desired amount of a component, such as ornithine, putrescine and/or
citrulline, and selecting those batches of soy hydrolysate that
have a desired amount of such component. For example, a sample
including a portion of a batch of soy hydrolysate powder can be
screened as described above, and compared to an amino acid standard
profile generated under the same conditions as the sample run. As
shown in FIGS. 1A-1B, the resulting chromatogram(s) will provide
the concentration of each amino acid component present in the soy
hydrolysate sample (e.g., micromoles/L per amino acid, mg/L per
amino acid, or g/L per amino acid). Analyzing the chromatogram,
facilitates identification of batches of soy hydrolysate (i.e.,
samples) that contain a desired concentration of a component, such
as ornithine, putrescine and/or citrulline. Each batch of soy
hydrolysate that includes a desired amount of a specific component
or components is then selected for further use, e.g., in cell
culture, as describe herein. FIG. 1, panel A depicts a rejected
batch following amino acid identification. FIG. 1, panel B depicts
an example of an acceptable soy hydrolysate batch run under
identical conditions. The amino acid peak corresponding to
ornithine is circled in both figures. The concentration of
ornithine or putrescine can be determined generating a standard
curve and interpolating the sample ornithine or putrescine
concentration. Alternatively, the relative amount of ornithine or
putrescine can be determined by determining the area under the
curve for the ornithine or putrescine peak and dividing it by the
total of the areas under the peak for all amino acids, or comparing
the peak area to a standard.
[0062] In certain embodiments, the desired concentration of the
component of soy hydrolysate (e.g., ornithine or putrescine) to be
selected is 5 mg/L or less. In one embodiment, the desired
concentration of ornithine or putrescine in a batch of soy
hydrolysate to be selected ranges from 0.5 mg/L to 5.0 mg/L or from
0.5 mg/L to 2.0 mg/L. In other embodiments, the concentration of
ornithine or putrescine in a selected batch of soy hydrolysate
ranges from 0.5 mg/L to 4.5 mg/L, 0.5 mg/L to 4.0 mg/L, 0.5 mg/L to
3.5 mg/L, 0.5 mg/L to 3.0 mg/L, 0.5 mg/L to 2.5 mg/L, 0.5 mg/L to
2.0 mg/L, 0.5 mg/L to 1.5 mg/L or 0.5 mg/L to 1.0 mg/L. In some
embodiments, the concentration of ornithine or putrescine in a
selected batch of soy hydrolysate ranges from 1.0 mg/L to 5.0 mg/L,
1.5 mg/L to 5.0 mg/L, 2.0 mg/L to 5.0 mg/L, 2.5 mg/L to 5.0 mg/L,
3.0 mg/L to 5.0 mg/L, 3.5 mg/L to 5.0 mg/L, 4.0 mg/L to 5.0 mg/L or
4.5 mg/L to 5.0 mg/L.
[0063] In specific embodiments, the desired concentration of
ornithine or putrescine in a batch of soy hydrolysate is at least
0.5 mg/L, 0.6 mg/L, 0.7 mg/L, 0.8 mg/L, 0.9 mg/L, 1.1 mg/L, 1.2
mg/L, 1.3 mg/L, 1.4 mg/L, 1.5 mg/L, 1.6 mg/L, 1.7 mg/L, 1.8 mg/L,
1.9 mg/L, 2.0 mg/L, 2.1 mg/L, 2.2 mg/L, 2.3 mg/L, 2.4 mg/L, 2.5
mg/L, 2.6 mg/L, 2.7 mg/L, 2.8 mg/L, 2.9 mg/L, 3.0 mg/L, 3.1 mg/L,
3.2 mg/L, 3.3 mg/L, 3.4 mg/L, 3.5 mg/L, 3.6 mg/L, 3.7 mg/L, 3.8
mg/L, 3.9 mg/L, 4.0 mg/L, 4.1 mg/L, 4.2 mg/L, 4.3 mg/L, 4.4 mg/L,
4.5 mg/L, 4.6 mg/L, 4.7 mg/L, 4.8 mg/L, 4.9 mg/L, or is 5.0 mg/L
ornithine or putrescine.
[0064] In other embodiments, the desired concentration of ornithine
or putrescine in a batch of soy hydrolysate is not more than 0.67
mg ornithine per g soy. In still other embodiments, the desired
concentration of ornithine or putrescine in a batch of soy
hydrolysate is not more than 0.27 mg ornithine per g soy. In
another embodiment, the desired concentration of ornithine or
putrescine in a batch of soy hydrolysate is not more than 0.24 mg
ornithine or putrescine per g soy. In some embodiments, the desired
concentration of ornithine or putrescine in a batch of soy is from
0.067 mg to 0.67 mg ornithine or putrescine per g soy. In still
other embodiments, the desired concentration of ornithine or
putrescine in a batch of soy hydrolysate falls with the range of
0.067 mg to 0.27 mg ornithine per g soy. In yet another embodiment,
the desired concentration of ornithine or putrescine in a batch of
soy hydrolysate falls with the range of 0.067 mg to 0.24 mg
ornithine or putrescine per g soy.
[0065] In one embodiment, the relative amount by mass of ornithine
or putrescine (% w/w) in the selected soy hydrolysate (w/w=mass of
ornithine or putrescine/total mass of hydrolysate) is
.ltoreq.0.067%, such as 0.0001%, 0.0002%, 0.0003%, 0.0004%,
0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.0015%,
0.002%, 0.0025%, 0.003%, 0.0035%, 0.004%, 0.0045%, 0.005%, 0.0055%,
0.006%, 0.0061%, 0.0062%, 0.0063%, 0.0064%, 0.0065%, 0.0066%, all
by w/w.
[0066] In one embodiment, the plant protein hydrolysate is selected
on the basis of producing a glycoprotein with a specific quality
attribute. The quality of glycoprotein may be determined by
assessing the level of one or more specific N-glycans on the
glycoprotein, or by assessing the level of one or more specific
sugars on the glycoprotein, or a combination of multiple
attributes. For example, a glycoprotein having a specific fucose
level, e.g., 5-10 moles of fucose per mole of glycoprotein, can be
a quality attribute criterion; or a specific sialic acid level,
e.g., 5-15 moles of sialic acid per mole of glycoprotein; or a
specific ratio of A1 N-glycan per total of all N-glycans, e.g.,
10-17% (w/w) can be considered to have the requisite quality
attribute. A plant protein hydrolysate enabling the production of
said glycoprotein would be considered selectable.
[0067] In one embodiment, the plant protein hydrolysate is selected
by producing a glycoprotein in a cell cultured in a medium
containing the potential selected (potentially selectable) plant
protein hydrolysate (e.g., soy hydrolysate), purifying the
glycoprotein, subjecting the glycoprotein to oligosaccharide
fingerprinting, and determining the relative amount of A1 N-glycan
by calculating the area under the peak associated with the A1
N-glycan and dividing that value by the total area under the peaks
of all N-glycans, and selecting a plant protein hydrolysate that
enabled the production of a glycoprotein with a relative amount of
A1 N-glycan of .gtoreq.10%, .gtoreq.10.5%, 10-17%, 10%, 10.5%, 11%,
11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%,
17%, 17.5%, or 18%.
[0068] Cell Culture
[0069] The present disclosure provides a method for culturing cells
expressing a protein of interest in a cell culture medium using a
selected batch of soy hydrolysate as described above. The instant
disclosure has found, for the first time, that the use of selected
batches of soy hydrolysate comprising 5.0 mg/L ornithine or less in
cell culture media reduces lot-to-lot variability and improves
protein product quality. The instant disclosure has found, for the
first time, that the use of selected batches of soy hydrolysate
comprising 5.0 mg/L putrescine or less in cell culture media
reduces lot-to-lot variability and improves protein product
quality.
[0070] "Cell culture" or "culture" means the growth and propagation
of cells outside of a multicellular organism or tissue. Suitable
culture conditions for mammalian cells are known in the art. See,
e.g., Animal cell culture: A Practical Approach, D. Rickwood, ed.,
Oxford University Press, New York (1992). Mammalian cells may be
cultured in suspension or while attached to a solid substrate.
Fluidized bed bioreactors, hollow fiber bioreactors, roller
bottles, shake flasks, or stirred tank bioreactors, with or without
microcarriers, and operated in a batch, fed batch, continuous,
semi-continuous, or perfusion mode are available for mammalian cell
culture. Cell culture media or concentrated feed media may be added
to the culture continuously or at intervals during the culture. For
example, a culture may be fed once per day, every other day, every
three days, or may be fed when the concentration of a specific
medium component, which is being monitored, falls outside a desired
range.
[0071] As used herein, the terms "cell culture media", "media",
"cell media", "cell culture medium" or "culture medium" refers to
any nutrient solution used for growing cells, e.g., animal or
mammalian cells, and which generally provides at least one or more
components from the following: an energy source (usually in the
form of a carbohydrate such as glucose); one or more of all
essential amino acids, and generally the twenty basic amino acids,
plus cysteine; vitamins and/or other organic compounds typically
required at low concentrations; lipids or free fatty acids; and
trace elements, e.g., inorganic compounds or naturally occurring
elements that are typically required at very low concentrations,
usually in the micromolar range. In some embodiments, a cell
culture media is formed by combining a soy or other plant protein
hydrolysate with an additional ingredient.
[0072] As used herein, "additional ingredient" includes any one or
more of cell culture media components including but not limited to
water, an energy source, one or more of all essential amino acids,
and generally the twenty basic amino acids, plus cysteine; vitamins
and/or other organic compounds typically required at low
concentrations, lipids or free fatty acids, and trace elements.
[0073] In specific embodiments, the cell culture media is
supplemented with an amount of a selected batch of soy hydrolysate.
In certain embodiments, the cell culture medium is supplemented
with about 0.5 g/L to about 25 g/L of a selected soy hydrolysate.
In some embodiments, the cell culture medium is supplemented with
about 0.5 g/L, 1 g/L, 1.5 g/L, 2 g/L, 2.5 g/L, 2 g/L, 2.5 g/L, 3
g/L, 3.5 g/L, 4 g/L, 4.5 g/L, 5 g/L, 5.5 g/L, 6 g/L, 6.5 g/L, 7
g/L, 7.5 g/L, 8 g/L, 8.5 g/L, 9 g/L, 9.5 g/L, 10 g/L, 10.5 g/L, 11
g/L, 11.5 g/L, 12 g/L, 12.5 g/L, 13 g/L, 13.5 g/L, 14 g/L, 14.5
g/L, 15 g/L, 15.5 g/L, 16 g/L, 16.5 g/L, 17 g/L, 17.5 g/L, 18 g/L,
18.5 g/L, 19 g/L, 19.5 g/L, 20 g/L, 20.5 g/L, 21 g/L, 21.5 g/L, 22
g/L, 22.5 g/L, 23 g/L, 23.5 g/L, 24 g/L, 24.5 g/L, or about 25 g/L
of a selected batch of soy hydrolysate.
[0074] In one embodiment, the concentration of ornithine or
putrescine in the cell culture media after addition of the plant
protein hydrolysate is .ltoreq.5 mg/L, 0.6-3 mg/L, 0.01 mg/L, 0.02
mg/L, 0.03 mg/L, 0.04 mg/L, 0.05 mg/L, 0.06 mg/L, 0.07 mg/L, 0.08
mg/L, 0.09 mg/L, 0.010 mg/L, 0.015 mg/L, 0.02 mg/L, 0.025 mg/L,
0.03 mg/L, 0.035 mg/L, 0.04 mg/L, 0.045 mg/L, 0.05 mg/L, 0.055
mg/L, 0.06 mg/L, 0.065 mg/L, 0.07 mg/L, 0.075 mg/L, 0.08 mg/L,
0.085 mg/L, 0.09 mg/L, 0.095 mg/L, 0.1 mg/L, 0.15 mg/L, 0.2 mg/L,
0.25 mg/L, 0.3 mg/L, 0.35 mg/L, 0.4 mg/L, 0.45 mg/L, 0.5 mg/L, 0.55
mg/L, 0.6 mg/L, 0.65 mg/L, 0.7 mg/L, 0.75 mg/L, 0.8 mg/L, 0.85
mg/L, 0.9 mg/L, 0.95 mg/L, 1 mg/L, 1.5 mg/L, 2 mg/L, 2.5 mg/L, 3
mg/L, 3.5 mg/L, 4 mg/L, 4.5 mg/L, or 5 mg/L.
[0075] In one embodiment, the cells being cultured are cells of a
cell line capable of producing a biotherapeutic protein.
Non-limiting examples of cell lines that are used to produce
protein biotherapeutics include inter alia primary cells, BSC
cells, HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS
cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells,
RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK
cells, BHK cells, BHK-21 cells, CHO cells, CHO-K1 cells, NS-1
cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells,
RK cells, Per.C6 cells and chicken embryo cells. In one embodiment,
the cell line is a CHO cell line or one or more of several specific
CHO cell variants optimized for large-scale protein production,
e.g., CHO-K1, or the CHO-K1-derived EESYR.RTM. (enhanced expression
and stability regions) cells (U.S. Pat. No. 7,771,997).
[0076] In one embodiment, the cells that are cultured and express
the heterologous glycoprotein are a population of cells obtained by
clonal expansion of a cell (i.e., the progenitor cell) that harbors
and expresses a polynucleotide encoding the glycoprotein or a
subunit of the glycoprotein, where the glycoprotein is a complex
multi-subunit protein like an antibody. In some embodiments at
least 50%, at least 60%, at least 70%, at least 80%, at least 90%,
at least 95%, at least 98%, at least 99%, or about 100% of the
constituent cells of the population of cells obtained or descended
by clonal expansion from the progenitor cell contain the
glycoprotein-encoding polynucleotide and express the
glycoprotein.
[0077] Mammalian cells, such as CHO cells, may be cultured in small
scale cell culture containers, such as in 125 ml containers having
about 25 ml of media, 250 ml containers having about 50 to 100 ml
of media, 500 ml containers having about 100 to 200 ml of media.
Alternatively, the cultures can be large scale such as for example
1000 ml containers having about 300 to 1000 ml of media, 3000 ml
containers having about 500 ml to 3000 ml of media, 8000 ml
containers having about 2000 ml to 8000 ml of media, and 15000 ml
containers having about 4000 ml to 15000 ml of media. Cultures for
manufacturing (i.e., production cell cultures) can contain 10,000 L
of media or more. Large scale cell cultures or "production cell
cultures", such as for clinical manufacturing of protein
therapeutics, are typically maintained for days, or even weeks,
while the cells produce the desired protein(s). During this time
the culture can be supplemented with a concentrated feed medium
containing components, such as nutrients and amino acids, which are
consumed during the course of the culture.
[0078] In certain embodiments, a concentrated feed medium is used.
Concentrated feed medium may be based on any cell culture media
formulation. Such a concentrated feed medium can contain most of
the components of a cell culture medium described herein at, for
example, about 5.times., 6.times., 7.times., 8.times., 9.times.,
10.times., 12.times., 14.times., 16.times., 20.times., 30.times.,
50.times., 100.times., 200.times., 400.times., 600.times.,
800.times., or even about 1000.times. of their normal useful
amount. Concentrated feed media are often used in fed batch culture
processes.
[0079] In some embodiments, the cell culture media is supplemented
with "point-of-use additions", also known as additions,
point-of-use ingredients, or point-of-use chemicals, during the
course of cell growth or protein production. Point-of-use additions
include any one or more of a growth factor or other proteins, a
buffer, an energy source, a salt, an amino acid, a metal, and a
chelator. Other proteins include transferrin and albumin. Growth
factors, which include cytokines and chemokines, are generally
known in the art and are known to stimulate cell growth, or in some
cases, cellular differentiation. A growth factor is usually a
protein (e.g., insulin), a small peptide, or a steroid hormone,
such as estrogen, DHEA, testosterone, and the like. In some cases,
a growth factor may be a non-natural chemical that promotes cell
proliferation or protein production, such as e.g., tetrahydrofolate
(THF), methotrexate, and the like. Non-limiting examples of protein
and peptide growth factors include angiopoietins, bone
morphogenetic proteins (BMPs), brain-derived neurotrophic factor
(BDNF), epidermal growth factor (EGF), erythropoietin (EPO),
fibroblast growth factor (FGF), glial cell line-derived
neurotrophic factor (GDNF), granulocyte colony-stimulating factor
(G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF),
growth differentiation factor-9 (GDF9), hepatocyte growth factor
(HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like
growth factor (IGF), migration-stimulating factor, myostatin
(GDF-8), nerve growth factor (NGF) and other neurotrophins,
platelet-derived growth factor (PDGF), thrombopoietin (TPO),
transforming growth factor alpha (TGF-.alpha.), transforming growth
factor beta (TGF-.beta.), tumor necrosis factor-alpha(TNF-.alpha.),
vascular endothelial growth factor (VEGF), wnt signaling pathway
agonists, placental growth factor (P1GF), fetal Bovine
somatotrophin (FBS), interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, and the like. In one embodiment, the cell culture media
is supplemented with the point-of-use addition growth factor
insulin. In one embodiment, the concentration of insulin in the
media, i.e., the amount of insulin in the cell culture media after
addition is from about 0.1 .mu.M to 10 .mu.M. One or more the
point-of-use additions can also be included in the media
formulation of some embodiments.
[0080] Buffers are generally known in the art. The invention is not
restricted to any particular buffer or buffers, and any one of
ordinary skill in the art can select an appropriate buffer or
buffer system for use with a particular cell line producing a
particular protein. In one embodiment, a point-of-use addition
buffer is NaHCO3/CO2 system. In one embodiment, the point-of-use
addition buffer comprises NaHCO.sub.3. In another embodiment, the
buffer is HEPES.
[0081] Energy sources for use as a point-of-use addition in cell
culture are also well known in the art. Without limitation, in one
embodiment, the point-of-use addition energy source is glucose.
Given the particular and specific requirements of a particular cell
line and the protein to be produced, in one embodiment the glucose
can be added to a concentration of about 1 to 20 mM in the
media.
[0082] Chelators are likewise well known in the art of cell culture
and protein production. Tetrasodium EDTA dehydrate and citrate are
two common chelators used in the art, although other chelators may
be employed in the practice of this invention. In one embodiment, a
point-of-use addition chelator is tetrasodium EDTA dihydrate. In
one embodiment, a point-of-use addition chelator is citrate, such
as Na3C6H5O7.
[0083] In one embodiment, the cell culture may be supplemented with
one or more point-of-use addition amino acids such as, glutamine.
Other point-of-use additions include one or more of various metal
salts, such as salts of iron, nickel, zinc and copper. In one
embodiment, the cell culture media is supplemented with any one or
more of copper sulfate, zinc sulfate, ferric chloride; and nickel
sulfate.
[0084] In one embodiment, the media is supplemented at intervals
during cell culture according to a fed-batch process. Fed-batch
culturing is generally known in the art and employed to optimized
protein production. See, e.g., Y. M. Huang et al., Biotechnol Prog.
(2010) 26(5) pp. 1400-1410.
[0085] In another aspect of the present disclosure, cells cultured
in medium comprising soy hydrolysate containing ornithine or
putrescine at a desired concentration (i.e., less than or equal to
5.0 mg/L, e.g., from 0.5 mg/L to 5.0 mg/L or from 0.5 mg/L to 2.0
mg/L) produce a protein of interest with improved quality, as
compared to cells cultured in medium comprising soy hydrolysate
containing ornithine or putrescine at a concentration of greater
than 5 mg/L. In certain embodiments, improved protein quality is
measured by: the presence or absence of glycosylation at one or
more amino acids on the protein of interest, the amount of glycan
on the protein of interest, the presence of sialic acid at one or
more glycosylation sites on the protein of interest, or a
combination thereof. As used herein "enhanced quality", "improved
quality" or "high quality" protein product can also refer to the
more consistent quality, for example, post-translational
modifications observed in a biotherapeutic protein production lot.
Consistent quality includes having, for example, a repeatable
desired glycosylation profile after replicate production lines.
Consistency, with respect to quality, refers to a degree of
uniformity and standardization, whereas replicate production
batches are essentially free from variation.
[0086] In certain embodiments, the protein product (protein of
interest) is an antibody, a human antibody, a humanized antibody, a
chimeric antibody, a monoclonal antibody, a multispecific antibody,
a bispecific antibody, an antigen binding antibody fragment, a
single chain antibody, a diabody, triabody or tetrabody, a Fab
fragment or a F(ab')2 fragment, an IgD antibody, an IgE antibody,
an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2
antibody, an IgG3 antibody, or an IgG4 antibody. In one embodiment,
the antibody is an IgG1 antibody. In one embodiment, the antibody
is an IgG2 antibody. In one embodiment, the antibody is an IgG4
antibody. In one embodiment, the antibody is a chimeric IgG2/IgG4
antibody. In one embodiment, the antibody is a chimeric IgG2/IgG1
antibody. In one embodiment, the antibody is a chimeric
IgG2/IgG1/IgG4 antibody.
[0087] In some embodiments, the antibody is selected from the group
consisting of an anti-Programmed Cell Death 1 antibody (e.g., an
anti-PD1 antibody as described in U.S. Pat. Appln. Pub. No.
US2015/0203579A1), an anti-Programmed Cell Death Ligand-1 (e.g., an
anti-PD-L1 antibody as described in U.S. Pat. Appln. Pub. No.
US2015/0203580A1), an anti-D114 antibody, an anti-Angiopoetin-2
antibody (e.g., an anti-ANG2 antibody as described in U.S. Pat. No.
9,402,898), an anti-Angiopoetin-Like 3 antibody (e.g., an
anti-AngPt13 antibody as described in U.S. Pat. No. 9,018,356), an
anti-platelet derived growth factor receptor antibody (e.g., an
anti-PDGFR antibody as described in U.S. Pat. No. 9,265,827), an
anti-Erb3 antibody, an anti-Prolactin Receptor antibody (e.g.,
anti-PRLR antibody as described in U.S. Pat. No. 9,302,015), an
anti-Complement 5 antibody (e.g., an anti-05 antibody as described
in U.S. Pat. Appln. Pub. No US2015/0313194A1), an anti-TNF
antibody, an anti-epidermal growth factor receptor antibody (e.g.,
an anti-EGFR antibody as described in U.S. Pat. No. 9,132,192 or an
anti-EGFRvIII antibody as described in U.S. Pat. Appln. Pub. No.
US2015/0259423A1), an anti-Proprotein Convertase Subtilisin Kexin-9
antibody (e.g. an anti-PCSK9 antibody as described in U.S. Pat. No.
8,062,640 or U.S. Pat. Appln. Pub. No. US2014/0044730A1), an
anti-Growth And Differentiation Factor-8 antibody (e.g., an
anti-GDF8 antibody, also known as anti-myostatin antibody, as
described in U.S. Pat. No. 8,871,209 or 9,260,515), an
anti-Glucagon Receptor (e.g., anti-GCGR antibody as described in
U.S. Pat. Appln. Pub. Nos. US2015/0337045A1 or US2016/0075778A1),
an anti-VEGF antibody, an anti-IL1R antibody, an interleukin 4
receptor antibody (e.g., an anti-IL4R antibody as described in U.S.
Pat. Appln. Pub. No. US2014/0271681A1 or U.S. Pat. No. 8,735,095 or
8,945,559), an anti-interleukin 6 receptor antibody (e.g., an
anti-IL6R antibody, as described in U.S. Pat. No. 7,582,298,
8,043,617 or 9,173,880), an anti-IL1 antibody, an anti-IL2
antibody, an anti-IL3 antibody, an anti-IL4 antibody, an anti-IL5
antibody, an anti-IL6 antibody, an anti-IL7 antibody, an
anti-interleukin 33 (e.g., anti-IL33 antibody as described in U.S.
Pat. Appln. Pub. Nos. US2014/0271658A1 or US2014/0271642A1), an
anti-Respiratory syncytial virus antibody (e.g., anti-RSV antibody
as described in U.S. Pat. Appln. Pub. No. US2014/0271653A1), an
anti-Cluster of differentiation 3 (e.g., an anti-CD3 antibody, as
described in U.S. Pat. Appln. Pub. Nos. US2014/0088295A1 and
US20150266966A1, and in U.S. Application No. 62/222,605), an
anti-Cluster of differentiation 20 (e.g., an anti-CD20 antibody as
described in U.S. Pat. Appln. Pub. Nos. US2014/0088295A1 and
US20150266966A1, and in U.S. Pat. No. 7,879,984), an anti-CD19
antibody, an anti-CD28 antibody, an anti-Cluster of
Differentiation-48 (e.g., anti-CD48 antibody as described in U.S.
Pat. No. 9,228,014), an anti-Fel d1 antibody (e.g., as described in
U.S. Pat. No. 9,079,948), an anti-Middle East Respiratory Syndrome
virus (e.g., an anti-MERS antibody as described in U.S. Pat. Appln.
Pub. No. US2015/0337029A1), an anti-Ebola virus antibody (e.g., as
described in U.S. Pat. Appln. Pub. No. US2016/0215040), an
anti-Zika virus antibody, an anti-Lymphocyte Activation Gene 3
antibody (e.g., an anti-LAG3 antibody, or an anti-CD223 antibody),
an anti-Nerve Growth Factor antibody (e.g., an anti-NGF antibody,
as described in U.S. Pat. Appln. Pub. No. US2016/0017029 and U.S.
Pat. Nos. 8,309,088 and 9,353,176) and an anti-Activin A antibody.
In some embodiments, the bispecific antibody is selected from the
group consisting of an anti-CD3.times.anti-CD20 bispecific antibody
(as described in U.S. Pat. Appln. Pub. Nos. US2014/0088295A1 and
US20150266966A1), an anti-CD3.times.anti-Mucin 16 bispecific
antibody (e.g., an anti-CD3.times.anti-Muc16 bispecific antibody),
and an anti-CD3.times.anti-Prostate-specific membrane antigen
bispecific antibody (e.g., an anti-CD3.times.anti-PSMA bispecific
antibody). In some embodiments, the protein of interest is selected
from the group consisting of alirocumab, sarilumab, fasinumab,
nesvacumab, dupilumab, trevogrumab, evinacumab, and rinucumab. All
publications mentioned throughout this disclosure are incorporated
herein by reference in their entirety.
[0088] In other embodiments, the protein of interest is a
recombinant protein that contains an Fc moiety and another domain,
(e.g., an Fc-fusion protein). In some embodiments, an Fc-fusion
protein is a receptor Fc-fusion protein, which contains one or more
extracellular domain(s) of a receptor coupled to an Fc moiety. In
some embodiments, the Fc moiety comprises a hinge region followed
by a CH2 and CH3 domain of an IgG. In some embodiments, the
receptor Fc-fusion protein contains two or more distinct receptor
chains that bind to either a single ligand or multiple ligands. For
example, an Fc-fusion protein is a trap protein, such as for
example an IL-1 trap (e.g., rilonacept, which contains the IL-1RAcP
ligand binding region fused to the Il-1R1 extracellular region
fused to Fc of hIgG1; see U.S. Pat. No. 6,927,004, which is herein
incorporated by reference in its entirety), a VEGF trap (e.g.,
aflibercept or ziv-aflibercept, which contains the Ig domain 2 of
the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF
receptor Flk1 fused to Fc of hIgG1; see U.S. Pat. Nos. 7,087,411
and 7,279,159; or conbercept, which contains the Ig domain 2 of the
VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor
Flk1 fused to the Ig domain 4 of the VEGF receptor Flk1 fused to Fc
of hIgG1; see U.S. Pat. No. 8,216,575), or a TNF trap (e.g.,
etanercept, which contains the TNF receptor fused to Fc of hIgG1;
see U.S. Pat. No. 5,610,279). In other embodiments, an Fc-fusion
protein is a ScFv-Fc-fusion protein, which contains one or more of
one or more antigen-binding domain(s), such as a variable heavy
chain fragment and a variable light chain fragment, of an antibody
coupled to an Fc moiety.
[0089] Protein Production
[0090] A protein of interest can be expressed by a host cell using
methods known by those of ordinary skill in the art. Generally, any
protein of interest suitable for expression in mammalian cells can
be produced by the instant methods, however glycoproteins will
especially benefit from the present methods. For example, in
specific embodiments the protein of interest is an antibody or
antigen-binding fragment thereof, a bispecific antibody or fragment
thereof, a chimeric antibody or fragment thereof, an ScFv or
fragment thereof, an Fc-tagged protein (e.g., Trap protein) or
fragment thereof, a growth factor or a fragment thereof, a cytokine
or a fragment thereof, or an extracellular domain of a cell surface
receptor or fragment thereof.
[0091] Glycoproteins with asparagine-linked (N-linked) glycans are
ubiquitous in eukaryotic cells. Biosynthesis of these glycans and
their transfer to polypeptides takes place in the endoplasmic
reticulum (ER). N-glycan structures are further modified by a
number of glycosidases and glycosyl-transferases in the ER and the
Golgi complex. Protein production using the present methods is
directed at improving consistency of desired N-glycan structure in
order to eliminate immunogenic epitopes ("glycotopes"). Detailed
structural analysis of glycan-linked proteins may be correlated to
functional features of the protein. Such analysis characterizing
protein glycosylation typically involves several steps: i) an
enzymatic or chemical release of the attached glycans; ii)
derivatization of the released glycans via reductive amination with
aromatic or aliphatic amines or permethylation; iii) analysis of
the glycans. Many variations of analyzing glycosylation patterns in
known to the skilled person. Glycoproteins may carry several types
of glycoforms occupying various sites in specific quantities, and
therefore their complexity may make it difficult to reproduce in
certain production methods. Consistency of type and quantity of
glycoform is measurable and represents a desirable outcome for
therapeutic protein production.
[0092] The present disclosure shows that producing numerous batches
of a protein of interest in batch or fed-batch culture, by
culturing cells expressing the protein of interest in media
comprising soy hydrolysate with specific concentrations of
ornithine or putrescine result in increased quality of the proteins
being produced and improved consistency from batch-to-batch.
Therefore, another aspect of the present disclosure provides a
plurality of protein preparations that have each been produced by
culturing cells in media comprising separate batches of soy
hydrolysate containing a predetermined amount of ornithine or
putrescine. In certain embodiments, each batch of soy hydrolysate
selected for use in cell culture has a concentration of 0.67 mg
ornithine or putrescine per g soy or less, particularly from 0.0067
mg to 0.67 mg ornithine or putrescine per g soy, or from 0.0067 to
0.27 mg ornithine or putrescine per g soy.
[0093] In other embodiments, the concentration of ornithine or
putrescine in a soy hydrolysate-containing cell culture media
ranges from 0.5 mg/L to 4.5 mg/L, 0.5 mg/L to 4.0 mg/L, 0.5 mg/L to
3.5 mg/L, 0.5 mg/L to 3.0 mg/L, 0.5 mg/L to 2.5 mg/L, 0.5 mg/L to
2.0 mg/L, 0.5 mg/L to 1.5 mg/L or 0.5 mg/L to 1.0 mg/L. In some
embodiments, the concentration of ornithine or putrescine in soy
hydrolysate-containing cell culture media ranges from 1.0 mg/L to
5.0 mg/L, 1.5 mg/L to 5.0 mg/L, 2.0 mg/L to 5.0 mg/L, 2.5 mg/L to
5.0 mg/L, 3.0 mg/L to 5.0 mg/L, 3.5 mg/L to 5.0 mg/L, 4.0 mg/L to
5.0 mg/L or 4.5 mg/L to 5.0 mg/L.
[0094] In specific embodiments, a cell culture media containing soy
hydrolysate includes ornithine or putrescine at an amount of 0.5
mg/L, 0.6 mg/L, 0.7 mg/L, 0.8 mg/L, 0.9 mg/L, 1.1 mg/L, 1.2 mg/L,
1.3 mg/L, 1.4 mg/L, 1.5 mg/L, 1.6 mg/L, 1.7 mg/L, 1.8 mg/L, 1.9
mg/L, 2.0 mg/L, 2.1 mg/L, 2.2 mg/L, 2.3 mg/L, 2.4 mg/L, 2.5 mg/L,
2.6 mg/L, 2.7 mg/L, 2.8 mg/L, 2.9 mg/L, 3.0 mg/L, 3.1 mg/L, 3.2
mg/L, 3.3 mg/L, 3.4 mg/L, 3.5 mg/L, 3.6 mg/L, 3.7 mg/L, 3.8 mg/L,
3.9 mg/L, 4.0 mg/L, 4.1 mg/L, 4.2 mg/L, 4.3 mg/L, 4.4 mg/L, 4.5
mg/L, 4.6 mg/L, 4.7 mg/L, 4.8 mg/L, 4.9 mg/L, or 5.0 mg/L.
[0095] In other embodiments, the desired concentration of ornithine
or putrescine in a batch of media containing soy hydrolysate is not
more than 5.0 mg/L. In still other embodiments, the desired
concentration of ornithine or putrescine in a batch of media
containing soy hydrolysate is not more than 2.0 mg/L. In another
embodiment, the desired concentration of ornithine or putrescine in
a batch of media containing soy hydrolysate is not more than 1.8
mg/L. In some embodiments, the desired concentration of ornithine
or putrescine in a batch of media containing soy is from 0.5 mg/L
to 5.0 mg/L. In still other embodiments, the desired concentration
of ornithine or putrescine in a batch of media containing soy
hydrolysate falls with the range of 0.5 mg/L to 2.0 mg/L. In yet
another embodiment, the desired concentration of ornithine or
putrescine in a batch of media containing soy hydrolysate falls
with the range of 0.5 mg/L to 1.8 mg/L.
[0096] In certain embodiments, quality of the protein of interest
or amount of certain glycans produced in each protein preparation
of a plurality of protein preparations is improved when compared to
a protein preparation produced by a method including culturing
cells in media supplemented with soy hydrolysate containing
ornithine or putrescine at a concentration of greater than 5 mg/L.
In certain embodiments, improved protein quality exhibited by each
protein preparation is measured by: the presence or absence of
glycosylation at one or more amino acids of the protein of
interest, the amount of glycan on the protein of interest, the
presence of sialic acid at one or more glycosylation sites on the
protein of interest, or a combination thereof. In one embodiment,
the protein quality corresponds to the glycosylation state of
individual members of a population of proteins produced in culture.
In certain embodiments, quality is improved by modulating the
glycosylation substitutions present on individual glycoproteins of
a population of proteins produced in culture by culturing the cells
in media supplemented with soy hydrolysate having a concentration
of 5.0 mg/L or less of ornithine or putrescine, from 0.5 mg/L to
5.0 mg/L of ornithine or putrescine, or from 0.5 mg/L to 2.0 mg/L
of ornithine or putrescine.
[0097] In one embodiment, protein quality is determined by
comparing the abundance of at least one glycan molecule in each
batch of proteins from a plurality of protein preparations to the
abundance of the same glycan molecule(s) in another batch of
proteins. The term "abundance" as used herein refers to the
percentage of proteins having a particular glycan molecule in a
particular production lot, or the amount of proteins having a
particular glycan molecule relative to the amount of all types of
glycan molecules in a production lot. In some embodiments, the
glycan molecule is selected from the group consisting of A1, A1F,
A2, A2F, Man5, NA2, NA2F, NA2G1, NA2G1F, NGA2, and NGA2FI. In a
specific embodiment, the glycan molecule is A1 (e.g., Peak 11 of
FIG. 2).
[0098] Proteins of interest produced by the cell culture methods of
the instant disclosure display favorable quality characteristics.
Protein quality can be measure, for example, by using methods well
known to those skilled in the art, such as weak cation exchange
chromatography, capillary isoelectric focusing, size-exclusion
chromatography, High Performance Liquid Chromatography (HPLC),
ELISA, and/or western blot analysis. In some embodiments, protein
quality is measured by mass spectrometry, such as capillary
electrophoresis mass spectrometry (CE-MS). In specific embodiments,
protein quality is determined by comparing mass spectrometry read
outs of each batch of proteins from a plurality of protein
preparations.
[0099] High Performance Liquid Chromatography (HPLC) with
fluorescent detection of exemplary production lots show that the
proteins of interest (glycoproteins) produced from cells cultured
in media including soy hydrolysate with an ornithine or putrescine
concentration from 0.5 mg/L to 5.0 mg/L have more consistent glycan
expression and glycosylation patterns, as exemplified in Tables
2-4, herein.
[0100] Oligosaccharide Profiling
[0101] The extent and distribution of specific N-linked sugar
chains on glycoproteins can be ascertained by oligosaccharide
profiling. In one embodiment, the glycoprotein is deglycosylated
with peptide:N-glycosidase F (PNGase F) to cleave and remove the
N-linked oligosaccharides from asparagine side chains. The
oligosaccharides are then derivatized with a fluorescent reagent,
such as anthranilic acid. The sugar chains are then separated by
normal phase anion-exchange HPLC and detected with a fluorescence
detector, generating an HPLC chromatogram.
[0102] In another embodiment, as part of the overall carbohydrate
characterization analysis, individual glycopeptides are isolated
following trypsin digestion of reduced and alkylated glycoprotein.
Individual tryptic glycopeptides are separated by reverse phase
HPLC, coupled with a subsequent C18 column for increased resolution
as needed. The oligosaccharides are released from each of the
separated glycopeptides by PNGase F digestion, derivatized with
anthranilic acid, and analyzed by fluorescence HPLC to obtain a
site-specific oligosaccharide profile of the glycoprotein. In one
embodiment where the glycoprotein is rilonacept (SEQ ID NO: 1),
asparagine residues at N37, N87, N91, N98, optionally N176, N189,
N279, N418, N511, N551, N567, N581, N615, and N730 are
glycosylated. In one embodiment, any one or more of residues N37,
N98, N418, and N511 of rilonacept (residue positions correlating to
SEQ ID NO: 1) contain an A1 oligosaccharide. In one embodiment
where the glycoprotein is aflibercept (SEQ ID NO: 2), asparagine
residues at N36, N68, N123, N196, and N282 are glycosylated. In one
embodiment, any one or both of residues N123 and N196 of
aflibercept (residue positions correlating to SEQ ID NO: 2) contain
an A1 oligosaccharide.
[0103] In another embodiment, oligosaccharide pools from the
glycoprotein are generated by deglycosylation of the proteins with
PNGase F, followed by anthranilic acid derivatization and
subsequent solid phase extraction (SPE). The masses of
oligosaccharides are then measured using MALDI-TOF in a negative
linear mode with 2, 4, 6-trihydroxyacetophenone (THAP) as
matrix.
[0104] Each observed mass is assigned to a unique oligosaccharide
structure based on the masses of commonly observed N-linked glycans
in recombinant proteins. The expected mass assignments of all the
peaks are summarized in Table 1. The expected masses are the
average mass calculated based on the proposed N-linked sugar chain
structures with addition of anthranilic acid residue mass. The
monosaccharide compositions are listed based on the proposed
N-linked sugar chain structures as well.
[0105] In another embodiment, a quantitative oligosaccharide
fingerprint assay using capillary electrophoresis is used to
characterize the N-glycan (oligosaccharide) structure of the
subject glycoprotein. The glycoprotein is denatured and then
deglycosylated by treatment with PNGase F. Released
oligosaccharides are then isolated by precipitation following
removal of the protein. Isolated oligosaccharide pools are labeled
with the fluorophore 8-aminopyrene 1,3,6-trisulfonate (APTS).
Labeled oligosaccharides are then separated by capillary
electrophoresis and monitored with a laser induced fluorescence
detector using an excitation wavelength of 488 nm and emission
wavelength of 520 nm.
[0106] An electropherogram is generated, as depicted in FIG. 2 for
the aflibercept glycoprotein, with all quantifiable peaks numbered
(total of 21 peaks in this example). The complete integrated peak
area (total peak area) for the oligosaccharide fingerprint is
determined. The relative amount of each oligosaccharide can be
determined by dividing the peak area for that particular
oligosaccharide (e.g., A1 peak area) by the total peak area.
[0107] In some embodiments, the quality of the subject glycoprotein
is assessed by determining the level of sialylation (the amount of
sialic residues per glycoprotein) or fucosylation (the amount of
fucose residues per glycoprotein). In one embodiment, the total
number of sialic acids on a glycoprotein are determined using a
quantitative HPLC assay. In this assay, the sialic acids are
released from the glycoprotein using mild acid hydrolysis, then
derivatized with o-Phenylenediamine, separated by HPLC, and
detected with either UV or fluorescence detectors. Quantitation of
sialic acid can be assessed relative to a standard curve using
e.g., sialyllactose. The sialic acid content is calculated from the
moles of sialic acid released and the moles of the glycoprotein
used in the reaction.
[0108] In one embodiment, the sialic acid content of the rilonacept
glycoprotein is about 30-70 moles sialic acid per 1 mole of
glycoprotein (mol/mol), about 35-65 mol/mol, 30 mol/mol, 31
mol/mol, 32 mol/mol, 33 mol/mol, 34 mol/mol, 35 mol/mol, 36
mol/mol, 37 mol/mol, 38 mol/mol, 39 mol/mol, 40 mol/mol, 41
mol/mol, 42 mol/mol, 43 mol/mol, 44 mol/mol, 45 mol/mol, 46
mol/mol, 47 mol/mol, 48 mol/mol, 49 mol/mol, 50 mol/mol, 51
mol/mol, 52 mol/mol, 53 mol/mol, 54 mol/mol, 55 mol/mol, 56
mol/mol, 57 mol/mol, 58 mol/mol, 59 mol/mol, 60 mol/mol, 61
mol/mol, 62 mol/mol, 63 mol/mol, 64 mol/mol, 65 mol/mol, 66
mol/mol, 67 mol/mol, 68 mol/mol, 69 mol/mol, or 70 mol/mol.
[0109] In one embodiment, the sialic acid content of the
aflibercept glycoprotein is about 5-15 moles sialic acid per 1 mole
of glycoprotein (mol/mol), about 8-12 mol/mol, 4 mol/mol, 5
mol/mol, 6 mol/mol, 7 mol/mol, 8 mol/mol, 9 mol/mol, 10 mol/mol, 11
mol/mol, 12 mol/mol, 13 mol/mol, 14 mol/mol, 15 mol/mol, 16
mol/mol, 17 mol/mol, 18 mol/mol, 19 mol/mol, or 20 mol/mol.
[0110] In one embodiment, oligosaccharide profiling is employed to
determine the extent and distribution of sialylation of N-linked
sugar chains on the glycoprotein. The glycoprotein is
deglycosylated with PNGase F, and then derivatized with the
fluorescent reagent, anthranilic acid. The oligosaccharides are
then separated by normal phase anion-exchange HPLC and detected
with a fluorescence detector to generate an HPLC chromatogram of
the oligosaccharide profile. The Z number (which measures the
average degree of sialylation) for the glycoprotein is calculated
from the following formula:
(OS A*O)+(ISA*111-(2SA*2)+(3SA*3)+ . . . (nSA*n)1/(OSA+15A+2SA+35A+
. . . n5A)
[0111] To determine the Z number, the area of each peak from the
oligosaccharide profile is integrated. The total sialic acid is
calculated as the sum of the areas of the 0 sialic acid/chain peaks
multiplied by 0, the 1 sialic acid/chain peaks multiplied by 1, the
2 sialic acid/chain peaks multiplied by 2, and the 3 sialic
acid/chain peaks multiplied by 3, etc. The total number of sugar
chains is generated as the sum of the areas of all of the peaks.
The Z number is the total sialic acid area divided by the total
sugar chain area.
[0112] In one embodiment, the sialic acid Z number of the
rilonacept glycoprotein is about 1.3-1.6, 1.4-1.5, 1.41-1.48, 1.3,
1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41,
1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52,
1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, or 1.60.
[0113] In one embodiment, the sialic acid Z number of the
aflibercept glycoprotein is about 0.5-2, 1-1.5, 1-1.2, 0.5, 0.51,
0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62,
0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73,
0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84,
0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96,
0.97, 0.98, 0.99, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07,
1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18,
1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, or
1.3.
EXAMPLES
[0114] The following examples are put forth so as to provide those
of ordinary skill in the art how to make and use the methods and
compositions described herein, and are not intended to limit the
scope of what the inventors regard as their invention. Efforts have
been made to ensure accuracy with respect to numbers used (e.g.,
amount, temperature, etc.) but some experimental error and
deviation should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is average molecular
weight, temperature is in degrees Centigrade, and pressure is at or
near atmospheric.
Example 1: Screening Soy Hydrolysate to Determine Amino Acid
Concentration
[0115] A soy hydrolysate sample was weighed and a 20 gram portion
thereof was dissolved in 1 L water to a starting concentration of
20 g/L. The resulting soy hydrolysate solution was then further
diluted in water to a desired concentration for use in cell culture
and the molecular composition of the resulting soy hydrolysate
solution was determined using by chromatography.
[0116] The concentration of amino acids in the soy hydrolysate
sample was measured by chromatography on an ion exchange column
with post column ninhydrin detection. See, e.g., Moore and Stein.
J. Biol. Chem. (1954) Vol. 211 pp. 907-913. Soy hydrolysate samples
were diluted to permit sensitive separation and resolution of
individual peaks (amino acids) as eluted from the HPLC column and
compared to a standard. Each peak area of the chromatogram, as
shown in FIGS. 1A and 1B, was compared to a standard to determine
concentration of each eluate.
[0117] To determine whether a batch of soy hydrolysate powder
contains less than 0.67 milligram ornithine or putrescine per gram
soy, the chromatogram of each representative sample is compared to
a standard. For example, FIG. 1A shows a batch of soy hydrolysate
with an eluate containing ornithine at retention time 89.02, which
reveals a peak area equivalent to 1.57 mg ornithine per g soy of
ornithine, as compared to the standard. FIG. 1B illustrates a batch
of soy hydrolysate having an ornithine concentration of less than
0.67 mg ornithine per g soy. Batches of soy hydrolysate with
between 0.067 and 0.67 mg ornithine per g soy were selected for use
in cell culture methods to produce biotherapeutic proteins with
more consistent protein glycosylation from lot-to lot. However, soy
hydrolysate batches containing ornithine at a concentration greater
than less than 0.67 mg ornithine per g soy were employed in further
experiments, as described below, to determine the effects of soy
hydrolysate ornithine concentration on protein production.
Example 2: Expression and Glycosylation Profile of a Protein of
Interest
[0118] CHO cells expressing a trap protein (receptor-Fc fusion
protein, VEGF-trap) were cultured in proprietary medium including
soy hydrolysate containing varying amounts of ornithine, putrescine
and citrulline, or a combination thereof, in order to determine
which amino acid components affect the quality of proteins
produced. Table 2, shows that the levels of ornithine in the
hydrolysate correlate negatively with the quality of protein
production lots, as indicated by the increased area under the curve
for a key N-glycan for protein lots produced as a result of
culturing CHO cells in media supplemented with soy hydrolysate
containing ornithine at a concentration less than 5.0 mg/L
independent of citrulline concentration.
[0119] As shown in Table 2 and depicted in FIG. 3, VEGF-trap
protein product lots produced by cells cultured in media including
soy hydrolysate with an ornithine concentration of 2.0 mg/L or less
produce higher quality protein product, when compared to cells
cultured in media comprising more than 5.0 mg/L of ornithine,
citrulline or putrescine.
TABLE-US-00002 TABLE 2 A1 N-Glycan relative amount Soy amino acid
concentration (% area under the curve) 1.6 mg/L ornithine 12.5 6.6
mg/L ornithine 9.8 31.6 mg/L ornithine 9.3 36.6 mg/L putrescine 9.0
1.6 mg/L ornithine; 12.0 0 mg/L citrulline 1.6 mg/L ornithine; 11.5
30 mg/L citrulline 31.6 mg/L ornithine; 8.8 0 mg/L citrulline
[0120] Detailed glycan analysis was performed using chromatography
based on well-known methods for HPLC and fluorescent anthranilic
acid (AA) tags (Anumula, and Dhume, Glycobiology (1998) 8(7) pp.
685-694) for each lot of glycoprotein to determine whether
ornithine had an impact on protein glycosylation profiles. As shown
in Table 3, culturing cells in media that includes soy hydrolysate
comprising less than or equal less than 0.67 mg ornithine per g soy
results in more consistent protein production from lot-to-lot. More
specifically, .about.90% of production lots cultured in medium
comprising selected soy hydrolysate meet FDA production criteria.
In contrast, only 57% of production lots cultured in medium
comprising soy hydrolysate having more than 5 mg/L ornithine meet
FDA production criteria (area under the curve for a specific
N-glycan peak). As shown in Table 3, VEGF-trap protein product lots
produced by cells cultured in medium including soy hydrolysate with
an ornithine concentration of 0.67 mg ornithine per g soy or less
exhibit increased product quality and more consistent quality from
lot-to-lot.
TABLE-US-00003 TABLE 3 Lots with glycan amount mg ornithine greater
than 10.5% Suitable protein Failed protein per g soy (quality
parameter) product lots product lots .ltoreq.0.67 21 19/21 2/21
>0.67 4 4/7 3/7
[0121] Each production lot was also compared (with respect to
glycan profile) to a reference standard which represents a
therapeutically acceptable batch of protein for the exemplary
VEGF-trap protein. Representative glycan analysis is shown in Table
4 for protein lots produced from cells cultured in medium
supplemented with soy hydrolysate resulting in final concentration
of ornithine between 0.5 mg/L and 2.0 mg/L. Compared to the
reference, each produced trap protein comprises a consistent glycan
profile having peaks within an acceptable range (75% of lots
analyzed). In contrast, each lot produced by cells cultured in
medium supplemented with soy hydrolysate comprising over 5.0 mg/L
ornithine failed to meet FDA acceptance criteria. As shown in Table
4, Protein product lots produced by cells cultured in media
including soy hydrolysate with an ornithine concentration from 0.5
mg/L to 2.0 mg/L or less produce higher quality lots, as
demonstrated by A1 N-glycan levels falling below product acceptance
criteria, than cells cultured in media comprising more soy
hydrolysate having an ornithine concentration greater than 5.0
mg/L.
TABLE-US-00004 TABLE 4 omithine Glycan A2 A2F A1 A1F NGA2F NA2G1F
NA2 NA2F (mg/L) Product lot 4-9 10-23 10-17 11-19 5-17 8-13 4-11
2-8 -- acceptance criteria (% area under curve) Soy 6.4 15.2 12.5
14.1 9.9 9.6 6.7 4.4 1.8 hydrolysate batch # 1 Soy 7.0 16.8 13.2
13.9 9.6 9.9 6.7 4.1 0.5 hydrolysate batch # 2 Soy 7.0 18.5 11.9
13.5 9.5 10.2 5.9 4 1.5 hydrolysate batch # 3 Soy 9.7 18.1 14.7
11.0 8.5 9.5 5.5 3.6 0.6 hydrolysate batch # 4 Soy 6.0 16.6 9.8
13.5 11.6 9.9 5.8 4.3 13.6 hydrolysate batch # 5 Soy 5.6 15.7 9.2
14.2 12.1 10.5 6.3 4.5 28.6 hydrolysate batch # 6
[0122] FIG. 4 shows the strong negative correlation between the
level of ornithine in soy hydrolysate and glycoprotein
(aflibercept) quality as demonstrated by A1 N-glycan levels.
Example 3: Glycoprotein Production Titer
[0123] 16 soy hydrolysate lots were tested for their ability to
affect the metabolomics of CHO cell production of rilonacept.
Approximately 426 soy hydrolysate analytes were measured and
compared to final glycoprotein titer and lactate metabolism. FIG. 5
depicts the loading plots of correlations between the soy
hydrolysate analytes and maximum lactate and final glycoprotein
titer. The determinations of lactate and glycoprotein titer
demonstrate a negative correlation of ornithine in the soy
hydrolysate.
Example 4: Marker Confirmation with Spiking Study
[0124] FIGS. 6A and 6B depict CHO cell cultures under control media
and feed conditions that received a spike of either ornithine or
putrescine to demonstrate the effect of ornithine and putrescine,
respectively, on cell growth and glycosylation. Table 6B highlights
the effect at peak 11, which is particularly pronounced.
[0125] Having described embodiments of the invention with reference
to the accompanying drawings, it is to be understood that the
invention is not limited to the precise embodiments, and that
various changes and modifications may be effected therein by those
skilled in the art without departing from the scope or spirit of
the invention as defined in the appended claims.
Sequence CWU 1
1
21879PRTArtificialsynthetic 1Ser Glu Arg Cys Asp Asp Trp Gly Leu
Asp Thr Met Arg Gln Ile Gln1 5 10 15Val Phe Glu Asp Glu Pro Ala Arg
Ile Lys Cys Pro Leu Phe Glu His 20 25 30Phe Leu Lys Phe Asn Tyr Ser
Thr Ala His Ser Ala Gly Leu Thr Leu 35 40 45Ile Trp Tyr Trp Thr Arg
Gln Asp Arg Asp Leu Glu Glu Pro Ile Asn 50 55 60Phe Arg Leu Pro Glu
Asn Arg Ile Ser Lys Glu Lys Asp Val Leu Trp65 70 75 80Phe Arg Pro
Thr Leu Leu Asn Asp Thr Gly Asn Tyr Thr Cys Met Leu 85 90 95Arg Asn
Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro Leu Glu Val Val 100 105
110Gln Lys Asp Ser Cys Phe Asn Ser Pro Met Lys Leu Pro Val His Lys
115 120 125Leu Tyr Ile Glu Tyr Gly Ile Gln Arg Ile Thr Cys Pro Asn
Val Asp 130 135 140Gly Tyr Phe Pro Ser Ser Val Lys Pro Thr Ile Thr
Trp Tyr Met Gly145 150 155 160Cys Tyr Lys Ile Gln Asn Phe Asn Asn
Val Ile Pro Glu Gly Met Asn 165 170 175Leu Ser Phe Leu Ile Ala Leu
Ile Ser Asn Asn Gly Asn Tyr Thr Cys 180 185 190Val Val Thr Tyr Pro
Glu Asn Gly Arg Thr Phe His Leu Thr Arg Thr 195 200 205Leu Thr Val
Lys Val Val Gly Ser Pro Lys Asn Ala Val Pro Pro Val 210 215 220Ile
His Ser Pro Asn Asp His Val Val Tyr Glu Lys Glu Pro Gly Glu225 230
235 240Glu Leu Leu Ile Pro Cys Thr Val Tyr Phe Ser Phe Leu Met Asp
Ser 245 250 255Arg Asn Glu Val Trp Trp Thr Ile Asp Gly Lys Lys Pro
Asp Asp Ile 260 265 270Thr Ile Asp Val Thr Ile Asn Glu Ser Ile Ser
His Ser Arg Thr Glu 275 280 285Asp Glu Thr Arg Thr Gln Ile Leu Ser
Ile Lys Lys Val Thr Ser Glu 290 295 300Asp Leu Lys Arg Ser Tyr Val
Cys His Ala Arg Ser Ala Lys Gly Glu305 310 315 320Val Ala Lys Ala
Ala Lys Val Lys Gln Lys Val Pro Ala Pro Arg Tyr 325 330 335Thr Val
Glu Lys Cys Lys Glu Arg Glu Glu Lys Ile Ile Leu Val Ser 340 345
350Ser Ala Asn Glu Ile Asp Val Arg Pro Cys Pro Leu Asn Pro Asn Glu
355 360 365His Lys Gly Thr Ile Thr Trp Tyr Lys Asp Asp Ser Lys Thr
Pro Val 370 375 380Ser Thr Glu Gln Ala Ser Arg Ile His Gln His Lys
Glu Lys Leu Trp385 390 395 400Phe Val Pro Ala Lys Val Glu Asp Ser
Gly His Tyr Tyr Cys Val Val 405 410 415Arg Asn Ser Ser Tyr Cys Leu
Arg Ile Lys Ile Ser Ala Lys Phe Val 420 425 430Glu Asn Glu Pro Asn
Leu Cys Tyr Asn Ala Gln Ala Ile Phe Lys Gln 435 440 445Lys Leu Pro
Val Ala Gly Asp Gly Gly Leu Val Cys Pro Tyr Met Glu 450 455 460Phe
Phe Lys Asn Glu Asn Asn Glu Leu Pro Lys Leu Gln Trp Tyr Lys465 470
475 480Asp Cys Lys Pro Leu Leu Leu Asp Asn Ile His Phe Ser Gly Val
Lys 485 490 495Asp Arg Leu Ile Val Met Asn Val Ala Glu Lys His Arg
Gly Asn Tyr 500 505 510Thr Cys His Ala Ser Tyr Thr Tyr Leu Gly Lys
Gln Tyr Pro Ile Thr 515 520 525Arg Val Ile Glu Phe Ile Thr Leu Glu
Glu Asn Lys Pro Thr Arg Pro 530 535 540Val Ile Val Ser Pro Ala Asn
Glu Thr Met Glu Val Asp Leu Gly Ser545 550 555 560Gln Ile Gln Leu
Ile Cys Asn Val Thr Gly Gln Leu Ser Asp Ile Ala 565 570 575Tyr Trp
Lys Trp Asn Gly Ser Val Ile Asp Glu Asp Asp Pro Val Leu 580 585
590Gly Glu Asp Tyr Tyr Ser Val Glu Asn Pro Ala Asn Lys Arg Arg Ser
595 600 605Thr Leu Ile Thr Val Leu Asn Ile Ser Glu Ile Glu Ser Arg
Phe Tyr 610 615 620Lys His Pro Phe Thr Cys Phe Ala Lys Asn Thr His
Gly Ile Asp Ala625 630 635 640Ala Tyr Ile Gln Leu Ile Tyr Pro Val
Thr Asn Ser Gly Asp Lys Thr 645 650 655His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser 660 665 670Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 675 680 685Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 690 695 700Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala705 710
715 720Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val 725 730 735Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr 740 745 750Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr 755 760 765Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu 770 775 780Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys785 790 795 800Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 805 810 815Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 820 825
830Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
835 840 845Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 850 855 860Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly865 870 8752431PRTArtificialsynthetic 2Ser Asp Thr Gly
Pro Arg 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 Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 260 265 270Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val 275 280 285Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 290 295 300Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr305 310 315
320Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
325 330 335Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys 340 345 350Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser 355 360 365Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp 370 375 380Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser385 390 395 400Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 405 410 415Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 420 425 430
* * * * *