U.S. patent application number 12/941887 was filed with the patent office on 2011-03-03 for cell culture performance with betaine.
This patent application is currently assigned to Immunex Corporation. Invention is credited to Brian D. Follstad, Anne H. Potter.
Application Number | 20110053265 12/941887 |
Document ID | / |
Family ID | 36600406 |
Filed Date | 2011-03-03 |
United States Patent
Application |
20110053265 |
Kind Code |
A1 |
Follstad; Brian D. ; et
al. |
March 3, 2011 |
Cell Culture Performance with Betaine
Abstract
This invention relates generally to the field of cell culture.
More particularly, the invention relates to improving viability of
recombinant cell cultures and the yields of secreted polypeptides
therefrom by the addition of betaine to the tissue culture
medium.
Inventors: |
Follstad; Brian D.;
(Seattle, WA) ; Potter; Anne H.; (Sammamish,
WA) |
Assignee: |
Immunex Corporation
|
Family ID: |
36600406 |
Appl. No.: |
12/941887 |
Filed: |
November 8, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12119206 |
May 12, 2008 |
7829309 |
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12941887 |
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|
11437418 |
May 19, 2006 |
7384765 |
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12119206 |
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|
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|
10226931 |
Aug 23, 2002 |
7067279 |
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11437418 |
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Current U.S.
Class: |
435/334 |
Current CPC
Class: |
C12N 2500/32 20130101;
C12N 2510/02 20130101; C12N 5/0018 20130101 |
Class at
Publication: |
435/334 |
International
Class: |
C12N 5/07 20100101
C12N005/07 |
Claims
1. A method comprising culturing a recombinantly engineered animal
cell line in tissue culture medium, wherein the cell line is
recombinantly engineered to express an antibody to a human EGF
receptor, the medium has an effective amount of betaine and the
medium is not hyperosmotic, whereby cell survival is improved
relative to cells grown without betaine.
2. The method of claim 1, wherein the recombinantly engineered cell
line is a mammalian cell line.
3. The method of claim 2, wherein the mammalian cell line is
selected from the group consisting of CHO, VERO, BHK, HeLa, Cos,
MDCK, 293, 3T3, a myeloma cell line, a hybridoma cell line, and
WI38 cells.
4. The method of claim 3, wherein the tissue culture medium is
serum free.
5. The method of claim 4, wherein the cells are grown during a
proliferative phase in the absence of betaine, and in the presence
of betaine in an induction phase.
6. The method of claim 5, wherein the betaine is selected from the
group consisting of glycine betaine and betaine aldehyde.
7. The method of claim 6, wherein cell survival is improved by
about 20%.
8. The method of claim 4, wherein the betaine is at a concentration
in the medium of from about 1 mM to about 100 mM.
9. The method of claim 8, wherein the cell line is cultured in a
bioreactor.
10. The method of claim 9, wherein the cell line is grown in
suspension culture.
11. The method of claim 10, further comprising collecting the
antibody.
12. A method comprising culturing a recombinantly engineered
mammalian cell line in tissue culture medium, wherein the cell line
is recombinantly engineered to express an antibody to a human EGF
receptor, the medium contains betaine at a concentration of from
about 1 mM to about 100 mM, and the tissue culture medium is not
hyperosmotic, and collecting the antibody.
13. The method of claim 12, wherein the mammalian cell line is a
CHO cell line or a hybridoma, and the tissue culture medium is
serum free.
14. A cell culture comprising a recombinantly engineered animal
cell line in tissue culture medium, wherein the cell line is
recombinantly engineered to express an antibody to a human EGF
receptor, the medium contains betaine at a concentration of about 1
mM to about 100 mM, and the tissue culture medium is not
hyperosmotic.
15. The cell culture of claim 14, wherein the recombinantly
engineered cell line is a mammalian cell line.
16. The cell culture of claim 15, wherein the mammalian cell line
is selected from the group consisting of CHO, VERO, BHK, HeLa, Cos,
MDCK, 293, 3T3, a myeloma cell line, a hybridoma cell line, and
WI38 cells.
17. The cell culture of claim 16, wherein the tissue culture medium
is serum free.
18. The cell culture of claim 17, wherein the betaine is selected
from the group consisting of glycine betaine and betaine
aldehyde.
19. The cell culture of claim 17, wherein the cell line is grown in
suspension culture.
20. The cell culture of claim 14, wherein the recombinantly
engineered cell line is a CHO cell line or a hybridoma cell line,
and the tissue culture medium is serum free.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/119,206, filed May 12, 2008, now allowed,
which is a divisional of U.S. patent application Ser. No.
11/437,418, filed May 19, 2006, now U.S. Pat. No. 7,384,765, which
is a divisional of U.S. patent application Ser. No. 10/226,931,
filed Aug. 23, 2002, now U.S. Pat. No. 7,067,279. The
above-identified applications are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] This invention relates generally to the field of cell
culture. More particularly, the invention relates to improving
viability of recombinant cells in culture and the yields of
secreted polypeptides therefrom by the addition of betaine to the
tissue culture medium.
BACKGROUND
[0003] Many commercially important polypeptides are recombinantly
produced in cells that are adapted grown in culture. One of the
limits to growing cell lines in culture is the decreased viability
of the cells over time, which is partially rectified by the
addition of growth factors. However, while this extra step improves
cell viability, it adds significant costs to the production of the
desired recombinant polypeptide.
[0004] Betaine has been shown to counteract the effects of
hyperosmotic conditions in cell cultures in vitro, and also
counteracts the denaturing tendency of urea (Kim et al., (2000)
Biotechnol. Prog., 16:775-781; Ryu et al., (2000) Biotech. Bioeng.,
70:167-175). However, it was also reported that betaine had no
effect on cell culture growth or maximum viable cell concentration
when cultures were at physiological ranges, i.e., 292 mOsm/kg, of
osmolality (Ryu et al., (2000) Biotech. Bioeng., 70:167-175).
[0005] Thus, there is a need in the art for methods of improving
the cell viability of cell cultures so as to reduce cell death, to
reduce the dependence on growth factors to increase recombinant
polypeptide production while not increasing costs. The invention
fulfills this need by providing a simple, easy and inexpensive way
of increasing cell viability and reducing the requirement for
growth factors by cultured cells.
SUMMARY OF THE INVENTION
[0006] In the invention provided herein, betaine is added to medium
used for culturing cells producing recombinant polypeptides in
vitro. Cell cultures grown in such medium demonstrated improved
cell viability and recombinant polypeptide production.
[0007] Accordingly in one aspect, the invention provides a method
comprising culturing cells recombinantly engineered to express a
protein of interest in tissue culture medium wherein the medium has
an effective amount of betaine wherein the tissue culture medium is
not hyperosmotic, and whereby cell survival is improved relative to
cells grown without betaine.
[0008] In a particular aspect, the invention provides a method
comprising culturing animal cells recombinantly engineered to
express a protein of interest in tissue culture medium wherein the
medium has an effective amount of betaine wherein the tissue
culture medium is not hyperosmotic, and whereby the animal cell
survival is improved relative to cells grown without betaine.
[0009] Mammalian cells are advantageously used in the method of the
invention, and particularly CHO cells. The betaine can be, for
example, betaine or any derivative thereof. The invention finds
particular use in the culturing of animal cells that are
genetically engineered to secrete a polypeptide of interest.
BRIEF DESCRIPTION OF THE FIGURE
[0010] FIG. 1. Addition of betaine to a recombinant cell culture
(expressing huTNFR-Fc) increases cell viability. The viable cells
are shown as a percentage of living cells over total cells when
measured by trypan blue exclusion staining during the course of the
culture.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The invention is based, in part, on the discovery that
betaine can be used to improve the performance and growth of cell
cultures. Specifically, betaine acts to increase cell viability in
essentially serum free media and thus increases recombinant
polypeptide production of cell cultures, thereby enhancing culture
robustness, and also reduces the reliance on the addition of growth
factors to the medium, thereby reducing costs associated with
recombinant polypeptide production.
[0012] In illustrative, non-limiting examples described below,
betaine was used as a model compound to increase cell viability and
increase polypeptide production in a serum free recombinant
mammalian cell culture system where osmolality was near
physiological conditions. In particular, it is shown that addition
of betaine to the medium increases cell viability and production of
recombinant polypeptides. These results demonstrate that betaine
can be used to improve the performance of in vitro animal cell
culture systems.
[0013] While the working examples provide a description of animal
cells grown in the presence of betaine in non-hyperosmotic
conditions that demonstrate improved viability concomitant with
improved production of recombinant polypeptides, it will be
understood by one of skill in the art that non-animal cells such as
prokaryotic cells, e.g., E. coli, insect cells, e.g., Sf9, plant
cells, yeast cells or the like can also be grown in the presence of
betaine in normal osmotic conditions such that viability is
improved and recombinant production of polypeptides is enhanced.
However, the invention is particularly advantageous for growing
industrially important animal cell lines that have been adapted to
grow in long-term culture and are producing recombinant
polypeptides of interest.
[0014] By animal cell is meant a cell whose progenitors were
derived from a multicellular animal. Preferably, the animal cell
lines are mammalian cell lines. A wide variety of animal cell lines
suitable for growth in culture are available from, for example, the
American Type Culture Collection (ATCC, Manassas, Va.) and NRRL
(Peoria, Ill.). Some of the more established cell lines typically
used in the industrial or academic laboratory and which are
preferred are CHO, VERO, BHK, HeLa, Cos, CV1, MDCK, 293, 3T3, PC12,
hybridoma, myeloma, and WI38 cell lines, to name but a few
examples. The dihydrofolate reductase (DHFR)-deficient mutant cell
line (Urlaub et al., 1980, Proc Natl Acad Sci USA 77:4216-4220),
DXB11 and DG-44, are the CHO host cell lines of choice because the
efficient DHFR selectable and amplifiable gene expression system
allows high level recombinant polypeptide expression in these cells
(Kaufman R. J., 1990, Meth Enzymol 185:527-566). In addition, these
cells are easy to manipulate as adherent or suspension cultures and
exhibit relatively good genetic stability. In addition, new animal
cell lines can be established using methods well known by those
skilled in the art (e.g., by transformation, viral infection,
and/or selection, etc.).
[0015] By in vitro cell culture is meant the growth and propagation
of cells outside of a multicellular organism or tissue. Typically,
in vitro cell culture is performed under sterile, controlled
temperature and atmospheric conditions in containers such as tissue
culture plates (e.g., 10 cm plates, 96 well plates, etc.), or other
adherent culture (e.g., on microcarrier beads) or in suspension
culture such as in roller bottles. Cultures can be grown in
containers such as shake flasks, small scale bioreactors, and/or
large-scale bioreactors. A bioreactor is a device used to culture
animal cells in which environmental conditions such as temperature,
atmosphere, agitation, osmolality and/or pH can be monitored and
adjusted. A number of companies (e.g., ABS Inc., Wilmington, Del.;
Cell Trends, Inc., Middletown, Md.) as well as university and/or
government-sponsored organizations (e.g., The Cell Culture Center,
Minneapolis, Minn.) offer cell culture services on a contract
basis.
[0016] Further, the recombinant, mammalian cell cultures of the
invention (adherent or non-adherent and growing or growth
arrested), can be small scale cultures, such as for example in 100
ml containers having about 30 ml of media, 250 ml containers having
about 80 to about 90 ml of media, 250 ml containers having about
150 to about 200 ml of media. Alternatively, the cultures can be
large scale such as for example 1000 ml containers having about 300
to about 1000 ml of media, 3000 ml containers having about 500 ml
to about 3000 ml of media, 8000 ml containers having about 2000 ml
to about 8000 ml of media, and 15000 ml containers having about
4000 ml to about 15000 ml of media.
[0017] Optimal periods for which the cultures are in contact with
betaine are for longer than the typical period for one normal
growth cycle (e.g., for Chinese hamster ovary cells (CHO cells),
where one growth cycle has been reported to be approximately 20-22
hours (Rasmussen et al., (1998) Cytotechnology, 28:31-42)). As
such, in a preferred embodiment, the cultures comprise betaine
preferably for at least about 20 hours, more preferably for about
22 hours, more preferably for about one day, more preferably for
about 2 days, more preferably for about 3 days, more preferably for
about 4 days, more preferably for about 5 days and even more
preferably for about 7 days.
[0018] Additionally, the methods of the invention can be applied to
perfused cell cultures. Perfused cell cultures are typically
cultured continuously and can be grown for as little as about five
days and for long as about nine months or longer, but are typically
cultured for about 25 days. Thus, it is contemplated that betaine
can be included in perfused culture media either continuously, or
intermittently over the course of the perfused culture run.
[0019] For the purposes of the invention, the cells may be growing
or induced to stop growing, e.g., senescent, by a method or reagent
commonly used in the art, such as for example radiation or drugs.
Alternatively, the cells may become growth arrested by virtue of
overgrowth and crowding in the container. Thus, the culture medium
can comprise betaine during either growth or senescence of the
cells, including during passaging of cells, amplification of cells,
growth of cells during recombinant polypeptide production stages,
feeding of cell cultures during any of the foregoing, and/or during
freezing and storage of cells. Further, in feeding of growing
cultures, betaine can be added in variable concentrations to pulse
the culture with high concentrations, followed by a removal of the
betaine. For example, the cells can be grown during a proliferative
phase in the absence of betaine, and then in an induction phase in
the presence of betaine. Alternatively, betaine can be present
during both proliferative and induction phases.
[0020] Further, the methods of the invention can be used in
combination with known or yet to be discovered methods of inducing
the production of recombinant proteins. By "inducing conditions" is
meant a technique to increase the relative production per cell of a
desired recombinant protein. Such techniques include cold
temperature shift, and additions of chemicals such as alkanoic acid
(including butyrate compounds, as described in U.S. Pat. No.
5,705,364 to Etcheverry et al., incorporated herein by reference),
DMSO, DMF, DMA, TNF-alpha, phorbol 12-myristate 13-acetate, PMA,
propionate, forskolin, dibutyryl cAMP, 2-aminopurine, adenine,
adenosine, okadaic acid, and combinations of any of these
techniques, to name just a few examples, as well as any yet to be
described and/or discovered induction techniques. Typically, a
batch culture of cells at high density is induced to produce the
recombinant protein. Often, other cell processes (such as growth
and division) are inhibited so as to direct most of the cells'
energy into recombinant protein production.
[0021] The invention finds particular use because it increases
production of secreted recombinant polypeptides, in part because
the cell cultures have increased numbers of viable cells. In
addition, in some embodiments, the methods and compositions of the
invention result in an increase of the desired folding of the
secreted recombinant polypeptide. And in additional embodiments,
the methods and compositions of the invention result in an increase
in the amount of secreted recombinant polypeptide that is active in
the desired activity, for example, recombinantly expressed TNFR-Fc
that binds with high affinity to tumor necrosis factor.
[0022] Tissue culture medium is defined, for purposes of the
invention, as a medium suitable for growth of animal cells, and
preferably mammalian cells, in in vitro cell culture. Typically,
tissue culture medium contains a buffer, salts, energy source,
amino acids, vitamins and trace essential elements. In addition,
the medium can oftentimes require additional components such as
growth factors, lipids, and/or other serum components (e.g.,
transferrin).
[0023] Any medium capable of supporting growth of animal cells in
culture can be used; the invention is broadly applicable to animal
cells in culture, particularly mammalian cells, and the choice of
medium is not crucial to the invention. Tissue culture media
suitable for use in the invention are commercially available from
ATCC (Manassas, Va.). For example, any one or combination of the
following media can be used: RPMI-1640 Medium, Dulbecco's Modified
Eagle's Medium, Minimum Essential Medium Eagle, F-12K Medium,
Iscove's Modified Dulbecco's Medium. Often, depending upon the
requirements of the particular cell line used, medium also contains
a serum additive such as Fetal Bovine Serum, or a serum
replacement. Examples of serum-replacements (for serum-free growth
of cells) are TCH.TM., TM-235.TM., and TCH.TM.; these products are
available commercially from Celox (St. Paul, Minn.). When defined
medium that is serum-free and/or peptone-free is used, the medium
is usually highly enriched for amino acids and trace elements (see,
for example, U.S. Pat. No. 5,122,469 to Mather et al., and U.S.
Pat. No. 5,633,162 to Keen et al.).
[0024] Serum adds to the expense of cell culture, and problems
arise from variance between serum lots and serum quality, in
addition, there are serious regulatory concerns about viral
contamination in serum and further, removing serum proteins from
downstream processing is burdensome, as such, in a preferred
embodiment the medium is serum free or essentially free of serum
and the recombinant polypeptide producing cell lines have been
selected for growth without serum (Rasmussen et al., (1998)
Cytotechnology 28:31-42). Essentially serum free media is meant to
include very low amounts of serum in the culture media. This
includes less than about 2% serum, more preferably less than about
1% serum, more preferably less than about 0.5% serum, and even more
preferably less than about 0.25% serum. In another preferred
embodiment, the recombinant cell line is a dihydrofolate reductase
negative, CHO cell line, adapted for growth without serum.
[0025] Added to the medium is an effective amount of betaine. An
effective amount of betaine is that amount that is capable of
increasing cell survival of a culture wherein osmotic conditions
are approximately physiological, i.e., 292 mOsm. In further
embodiments, the osmolality is within 100 mOsm of physiological,
more preferably within 75 mOsm of physiological, still more
preferably within 50 mOsm of physiological, yet more preferably
within 40 mOsm of physiological, even more preferably within 30
mOsm of physiological, still even more preferably within 20 mOsm of
physiological and most preferably within 10 mOsm of physiological
osmolality. An improvement in cell survival is measured as an
increase in cells in a betaine treated culture relative to the
number of cells in untreated cultures, wherein cell survival is
increased 5%, more preferably cell survival is increased 10%, more
preferably cell survival is increased 15%, more preferably cell
survival is increased 20%, more preferably cell survival is
increased 25%, more preferably cell survival is increased 30%, more
preferably cell survival is increased 35%, more preferably cell
survival is increased 40%, more preferably cell survival is
increased 45%, and even more preferably cell survival is increased
50%.
[0026] Betaine can be produced in different forms including as a
glycine betaine. Other preferred betaines include but are not
limited to betaine aldehyde and all betaine derivatives therefrom
and therein.
[0027] The concentration of such compounds to use in the invention
can be determined by those skilled in the art by, for example,
comparing the cell death inhibitory activity of glycine betaine
against that of another isoform of betaine, and extrapolating
appropriate concentrations therefrom. The extrapolated
concentrations can then be used as a starting point to determine
the range of effective amounts of compound that should be added to
a culture medium, which amounts can then be determined using small
scale experiments such as those described herein. In preferred
embodiments, the betaine is glycine betaine and is in the culture
medium at about 1 to 100 mM betaine, more preferably 5 to 75 mM
betaine, more preferably about 10 to 60 mM betaine, and more
preferably about 20 to 40 mM betaine.
[0028] The invention finds particular utility in improving the
production of recombinant polypeptides via cell culture processes.
The cell lines used in the invention can be genetically engineered
to express a polypeptide of commercial or scientific interest. By
genetically engineered is meant that the cell line has been
transfected, transformed or transduced with a recombinant
polynucleotide molecule, and/or otherwise altered (e.g., by
homologous recombination and gene activation or fusion of a
recombinant cell with a non-recombinant cell) so as to cause the
cell to express a desired recombinant polypeptide. Methods and
vectors for genetically engineering cells and/or cell lines to
express a polypeptide of interest are well known to those of skill
in the art; for example, various techniques are illustrated in
Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley
& Sons, New York, 1988, and quarterly updates) and Sambrook et
al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory
Press, 1989).
[0029] Particularly preferred polypeptides for expression are
polypeptide-based drugs, also known as biologics. Preferably, the
polypeptides are expressed as extracellular products, which can be
either secreted into the culture medium or transmembrane, i.e.,
having a portion of the polypeptide extruding through the cell
membrane into the extracellular milieu. Recombinant polypeptides
that can be produced using the invention include but are not
limited to Flt3 ligand, CD40 ligand, erythropoietin,
thrombopoeitin, calcitonin, Fas ligand, ligand for receptor
activator of NF-kappa B (RANKL), TNF-related apoptosis-inducing
ligand (TRAIL), ORK/Tek, thymic stroma-derived lymphopoietin,
granulocyte colony stimulating factor, granulocyte-macrophage
colony stimulating factor, mast cell growth factor, stem cell
growth factor, epidermal growth factor, RANTES, growth hormone,
insulin, insulinotropin, insulin-like growth factors, parathyroid
hormone, nerve growth factors, glucagon, interleukins 1 through 18,
colony stimulating factors, lymphotoxin-.beta., tumor necrosis
factor, leukemia inhibitory factor, oncostatin-M, and various
ligands for cell surface molecules Elk and Hek (such as the ligands
for eph-related kinases, or LERKS). Descriptions of polypeptides
that can be produced according to the invention may be found in,
for example, Human Cytokines: Handbook for Basic and Clinical
Research, Vol. II (Aggarwal and Gutterman, Eds. Blackwell Sciences,
Cambridge Mass., 1998); Growth Factors: A Practical Approach (McKay
and Leigh, Eds. Oxford University Press Inc., New York, 1993) and
The Cytokine Handbook (AW Thompson, ed.; Academic Press, San Diego
Calif.; 1991).
[0030] Production of the receptors for any of the aforementioned
polypeptides can also be improved using the invention, including
the receptors for both forms of tumor necrosis factor receptor
(referred to as p55 and p75), Interleukin-1 receptors (type 1 and
2), Interleukin-4 receptor, Interleukin-15 receptor, Interleukin-17
receptor, Interleukin-18 receptor, granulocyte-macrophage colony
stimulating factor receptor, granulocyte colony stimulating factor
receptor, receptors for oncostatin-M and leukemia inhibitory
factor, receptor activator of NF-kappa B (RANK), receptors for
TRAIL, and receptors that comprise death domains, such as Fas or
Apoptosis-Inducing Receptor (AIR). A particularly preferred
receptor is a soluble form of the IL-1 receptor type II; such
polypeptides are described in U.S. Pat. No. 5,767,064, incorporated
herein by reference in its entirety.
[0031] Other polypeptides that can be produced using the invention
include cluster of differentiation antigens (referred to as CD
polypeptides), for example, those disclosed in Leukocyte Typing VI
(Proceedings of the VIth International Workshop and Conference;
Kishimoto, Kikutani et al., Eds. Kobe, Japan, 1996), or CD
molecules disclosed in subsequent workshops. Examples of such
molecules include CD27, CD30, CD39, CD40; and ligands thereto (CD27
ligand, CD30 ligand and CD40 ligand). Several of these are members
of the tumor necrosis factor (TNF) receptor family, which also
includes 41BB and OX40; the ligands are often members of the TNF
family (as are 41BB ligand and OX40 ligand); accordingly, members
of the TNF and TNF receptor (TNFR) families can also be produced
using the present invention.
[0032] Polypeptides that are enzymatically active can also be
produced according to the instant invention. Examples include
metalloproteinase-disintegrin family members, various kinases,
glucocerebrosidase, alpha-galactosidase A, superoxide dismutase,
tissue plasminogen activator, Factor VIII, Factor IX,
apolipoprotein E, apolipoprotein A-I, globins, an IL-2 antagonist,
alpha-1 antitrypsin, TNF-alpha Converting Enzyme, and numerous
other enzymes. Ligands for enzymatically active polypeptides can
also be produced by applying the instant invention.
[0033] The inventive compositions and methods are also useful for
production of other types of recombinant polypeptides, including
immunoglobulin molecules or portions thereof, and chimeric
antibodies (i.e., an antibody having a human constant region
couples to a murine antigen binding region) or fragments thereof.
Numerous techniques are known by which DNA encoding immunoglobulin
molecules can be manipulated to yield DNAs capable of encoding
recombinant polypeptides such as single chain antibodies,
antibodies with enhanced affinity, or other antibody-based
polypeptides (see, for example, Larrick et al., 1989, Biotechnology
7:934-938; Reichmann et al., 1988, Nature 332:323-327; Roberts et
al., 1987, Nature 328:731-734; Verhoeyen et al., 1988, Science
239:1534-1536; Chaudhary et al., 1989, Nature 339:394-397).
Recombinant cells producing fully human antibodies (such as are
prepared using transgenic animals, and optionally further modified
in vitro), as well as humanized antibodies can also be used in the
invention. The term humanized antibody also encompasses single
chain antibodies. See, e.g., Cabilly et al., U.S. Pat. No.
4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss
et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No.
0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M.
S. et al., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No.
5,225,539; Winter, European Patent No. 0,239,400 B1; Queen et al.,
European Patent No. 0 451 216 B1; and Padlan, E. A. et al., EP 0
519 596 A1. For example, the invention can be used to induce the
expression of human and/or humanized antibodies that
immunospecifically recognize specific cellular targets, e.g., any
of the aforementioned polypeptides, the human EGF receptor, the
her-2/neu antigen, the CEA antigen, Prostate Specific Membrane
Antigen (PSMA), CD5, CD11a, CD18, NGF, CD20, CD45, Ep-cam, other
cancer cell surface molecules, TNF-alpha, TGF-b1, VEGF, other
cytokines, alpha 4 beta 7 integrin, IgEs, viral polypeptides (for
example, cytomegalovirus), etc., to name just a few.
[0034] Various fusion polypeptides can also be produced using the
invention. A fusion polypeptide is a polypeptide, or domain or a
polypeptide (e.g. a soluble extracellular domain) fused to a
heterologous polypeptide or peptide. Examples of such fusion
polypeptides include polypeptides expressed as a fusion with a
portion of an immunoglobulin molecule, polypeptides expressed as
fusion polypeptides with a zipper moiety, and novel polyfunctional
polypeptides such as a fusion polypeptides of a cytokine and a
growth factor (i.e., GM-CSF and IL-3, MGF and IL-3). WO 93/08207
and WO 96/40918 describe the preparation of various soluble
oligomeric forms of a molecule referred to as CD40L, including an
immunoglobulin fusion polypeptide and a zipper fusion polypeptide,
respectively; the techniques discussed therein are applicable to
other polypeptides. Another fusion polypeptide is a recombinant
TNFR:Fc, also known as "entanercept." Entanercept is a dimer of two
molecules of the extracellular portion of the p75 TNF alpha
receptor, each molecule consisting of a 235 amino acid TNFR-derived
polypeptide that is fused to a 232 amino acid Fc portion of human
IgG1. In fact, any of the previously described molecules can be
expressed as a fusion polypeptide including but not limited to the
extracellular domain of a cellular receptor molecule, an enzyme, a
hormone, a cytokine, a portion of an immunoglobulin molecule, a
zipper domain, and an epitope.
[0035] The resulting expressed polypeptide can then be collected.
In addition the polypeptide can purified, or partially purified,
from such culture or component (e.g., from culture medium or cell
extracts or bodily fluid) using known processes. By "partially
purified" means that some fractionation procedure, or procedures,
have been carried out, but that more polypeptide species (at least
10%) than the desired polypeptide is present. By "purified" is
meant that the polypeptide is essentially homogeneous, i.e., less
than 1% contaminating polypeptides are present. Fractionation
procedures can include but are not limited to one or more steps of
filtration, centrifugation, precipitation, phase separation,
affinity purification, gel filtration, ion exchange chromatography,
hydrophobic interaction chromatography (HIC; using such resins as
phenyl ether, butyl ether, or propyl ether), HPLC, or some
combination of above.
[0036] The invention also optionally encompasses further
formulating the polypeptides. By the term "formulating" is meant
that the polypeptides can be buffer exchanged, sterilized,
bulk-packaged and/or packaged for a final user. For purposes of the
invention, the term "sterile bulk form" means that a formulation is
free, or essentially free, of microbial contamination (to such an
extent as is acceptable for food and/or drug purposes), and is of
defined composition and concentration. The term "sterile unit dose
form" means a form that is appropriate for the customer and/or
patient administration or consumption. Such compositions can
comprise an effective amount of the polypeptide, in combination
with other components such as a physiologically acceptable diluent,
carrier, or excipient. The term "physiologically acceptable" means
a non-toxic material that does not interfere with the effectiveness
of the biological activity of the active ingredient(s).
[0037] The invention having been described, the following examples
are offered by way of illustration and not limitation.
Example: Betaine in Culture Medium
[0038] TNFR-Fc production in some CHO cell lines results in greater
quantities of recombinant protein product but with lower viability
than other cell lines during the final days of the run. The
decreased viability correlates with increased protease activity in
the culture supernatant, a result of dying cells releasing
proteases into the media, which decreases stability of the final
purified product. The current study was performed to determine if
betaine could prolong viability at production osmolality (250-280
mOsm initial value).
[0039] The bioreactors used for this study were stirred vessels
with a 1 liter working volume, with temperature, pH and dO.sub.2
control. The medium was serum-free with bicarbonate, IGF-1 (LongR3;
GroPep, Australia), intralipids and peptones added. Betaine was
added to 1 L bioreactor cultures of TNFR-Fc producing cells at 0,
10, 20, 30, and 40 mM with the osmolality of each culture adjusted
to 270 to 280 mOsm with an appropriate amount of NaCl. The reactors
were induced to express recombinant protein on day 2 by reducing
the temperature and adding butyrate to 0.5 mM. Daily samples were
taken for analysis of metabolic and productivity parameters.
[0040] Based on the viable cell density (VCD) profiles, betaine has
a positive effect on cell viability, which is most noticeable after
day 5 (FIG. 1). There was a titration effect up to 30 mM, after
which higher concentrations did not increase viability as much. The
ability of induced cells to produce TNFR:Fc was not decreased in
the presence of betaine. Furthermore, in the presence of betaine,
the percentage of the secreted recombinant polypeptide having the
desired conformation increased by 17.7%, i.e., native conformation
and dimerized, as measured by running the samples through
hydrophobic interaction chromatography (HIC) and comparing to known
controls.
[0041] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying drawing. Such modifications are intended to fall
within the scope of the appended claims.
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