U.S. patent application number 14/211245 was filed with the patent office on 2014-09-18 for serum-free cell culture medium.
This patent application is currently assigned to Regeneron Pharmaceuticals, Inc.. The applicant listed for this patent is Regeneron Pharmaceuticals, Inc.. Invention is credited to Amy S. JOHNSON, Shawn M. LAWRENCE, Shadia Abike OSHODI.
Application Number | 20140273095 14/211245 |
Document ID | / |
Family ID | 51528781 |
Filed Date | 2014-09-18 |
United States Patent
Application |
20140273095 |
Kind Code |
A1 |
OSHODI; Shadia Abike ; et
al. |
September 18, 2014 |
Serum-Free Cell Culture Medium
Abstract
The specification describes an improved serum-free animal cell
culture medium, which can used for the production of a protein of
interest. Ornithine, or a combination of ornithine and putrescine
can be added to serum-free media or chemically defined media to
improve viable cell density, to reduce cell doubling time, and to
increase the production of a protein of interest.
Inventors: |
OSHODI; Shadia Abike; (New
York, NY) ; JOHNSON; Amy S.; (Briarcliff Manor,
NY) ; LAWRENCE; Shawn M.; (Valley Cottage,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Regeneron Pharmaceuticals, Inc. |
Tarrytown |
NY |
US |
|
|
Assignee: |
Regeneron Pharmaceuticals,
Inc.
Tarrytown
NY
|
Family ID: |
51528781 |
Appl. No.: |
14/211245 |
Filed: |
March 14, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61790136 |
Mar 15, 2013 |
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Current U.S.
Class: |
435/69.6 ;
435/252.3; 435/254.2; 435/326; 435/328 |
Current CPC
Class: |
C12N 5/0037 20130101;
C12N 2511/00 20130101; C12N 2500/32 20130101; C12N 5/005 20130101;
C12P 21/00 20130101; C07K 16/18 20130101; C12N 2510/02 20130101;
C12N 5/0043 20130101; C12N 2500/46 20130101; C12N 5/0031
20130101 |
Class at
Publication: |
435/69.6 ;
435/326; 435/252.3; 435/254.2; 435/328 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C07K 16/18 20060101 C07K016/18 |
Claims
1. A cell culture medium, which is serum-free, comprising
.gtoreq.0.09 mM.+-.0.014 mM ornithine.
2. The cell culture medium of claim 1 comprising
.gtoreq.0.20.+-.0.03 mM putrescine.
3. The cell culture medium of claim 1 comprising 0.09.+-.0.014 mM
to 0.9.+-.0.14 mM ornithine.
4. The cell culture medium of claim 3 comprising ornithine at
0.09.+-.0.014 mM, 0.3.+-.0.05 mM, 0.6.+-.0.09 mM, or 0.9.+-.0.14
mM.
5. The cell culture medium of claim 2 comprising 0.20.+-.0.03 mM to
0.714.+-.0.11 mM putrescine.
6. The cell culture of claim 5 comprising putrescine at
0.20.+-.0.03 mM, 0.35.+-.0.06, or 0.714.+-.0.11 mM.
7. The cell culture medium of claim 1, wherein the medium is
hydrolysate-free.
8. The cell culture medium of claim 1, wherein the medium is
chemically defined.
9. The cell culture medium of claim 1 comprising .gtoreq.40.+-.6 mM
of a mixture of amino acids or salts thereof.
10. The cell culture medium of claim 9, wherein the mixture of
amino acids consists of alanine, arginine, asparagine, aspartic
acid, cysteine, glutamic acid, glycine, histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, and valine.
11. The cell culture medium of claim 1 comprising one or more fatty
acids.
12. The cell culture medium of claim 11, wherein the one or more
fatty acids are selected from the group consisting of linoleic
acid, linolenic acid, thioctic acid, oleic acid, palmitic acid,
stearic acid, arachidic acid, arachidonic acid, lauric acid,
behenic acid, decanoic acid, dodecanoic acid, hexanoic acid,
lignoceric acid, myristic acid, and octanoic acid.
13. The cell culture medium of claim 1 comprising a mixture of
nucleosides.
14. The cell culture medium of claim 13, wherein the mixture of
nucleosides comprises one or more of adenosine, guanosine,
cytidine, uridine, thymidine, and hypoxanthine.
15. The cell culture medium of claim 14 comprising adenosine,
guanosine, cytidine, uridine, thymidine, and hypoxanthine.
16. The cell culture medium of claim 1 comprising one or more
divalent cations.
17. The cell culture medium of claim 16, wherein the divalent
cation is magnesium, calcium, or both.
18. The cell culture medium of claim 17 comprising Ca.sup.2+ and
Mg.sup.2+.
19. A method for cultivating cells, comprising the steps of: (a)
providing a cell culture medium according to claim 1, and (b)
propagating or maintaining a cell in the cell culture medium to
form a cell culture.
20. The method of claim 19, wherein the cell is selected from the
group consisting of mammalian cell, avian cell, insect cell,
bacterial cell, and yeast cell.
21. The method of claim 20, wherein the cell is a CHO cell.
22. The method of claim 21, wherein the cell expresses a protein of
interest.
23. The method of claim 22, wherein the protein of interest is an
antigen binding protein.
24. The method of claim 23, wherein the protein of interest
comprises an Fc domain.
25. The method of claim 24, wherein the protein of interest is a
receptor-Fc-fusion protein.
26. The method of claim 25, wherein the receptor-Fc-fusion protein
is a trap protein.
27. The method of claim 26, wherein the trap protein is an IL-1
antagonist or a VEGF antagonist.
28. The method of claim 23, wherein the protein of interest is an
antibody or an antibody fragment.
29. The method of claim 28, wherein the antibody or the antibody
fragment is a recombinant human antibody or fragment thereof.
30. The method of claim 19, wherein the cells have an average
doubling time of .ltoreq.30 hours.
31. The method of claim 19, wherein the cells have an average
doubling time of .ltoreq.24 hours.
32. The method of claim 19, wherein the cells have an average
doubling time that is at least one third that of cells grown in a
cell culture medium that contains <0.3.+-.0.045 mM ornithine and
<0.2.+-.0.03 mM putrescine.
33. The method of claim 19, wherein the cell culture is capable of
attaining a viable cell count density that is at least 15% greater
than a similar cell culture in media that contains
<0.09.+-.0.014 mM ornithine and <0.2.+-.0.03 mM
putrescine.
34. The method of claim 19, wherein the cell culture is capable of
attaining a viable cell count density that is at least 3-fold
greater than a similar cell culture in a similar cell culture
medium that contains <0.09.+-.0.014 mM ornithine and
<0.2.+-.0.03 mM putrescine.
35. The method of claim 19 comprising the step of adding one or
more point-of-use additions to the cell culture medium.
36. The method of claim 35, wherein the point-of-use additions
comprise one of more of NaHCO.sub.3, glutamine, insulin, glucose,
CuSO.sub.4, ZnSO.sub.4, FeCl.sub.3, NiSO.sub.4, Na.sub.4 EDTA, and
Na.sub.3 Citrate.
37. The method of claim 36, wherein each of NaHCO.sub.3, glutamine,
insulin, glucose, CuSO.sub.4, ZnSO.sub.4, FeCl.sub.3, NiSO.sub.4,
Na.sub.4 EDTA, and Na.sub.3 Citrate are added to the medium as
point-of-use additions.
38. A method for producing a protein comprising the steps of: (a)
introducing into a cell a nucleic acid comprising a sequence
encoding a protein of interest; (b) selecting a cell carrying the
nucleic acid; (c) culturing the selected cell in a cell culture
medium of claim 1; and (d) expressing the protein of interest in
the cell, wherein the protein of interest is secreted into the
medium.
39. The method of claim 38, wherein the cell is a CHO cell, 293
cell or BHK cell.
40. The method of claim 38, wherein the protein of interest is an
antigen-binding protein.
41. The method of claim 40, wherein the protein of interest
comprises an Fc domain.
42. The method of claim 41, wherein the protein of interest is
selected from the group consisting of receptor-Fc-fusion protein
(TRAP), soluble TCR-Fc fusion protein, antibody, Fc-fusion protein,
and ScFv protein.
43. The method of claim 38, wherein the protein-of-interest is
produced at an average 7-day titer that is at least 7% greater than
the average 7-day titer produced by a similar cell in a cell
culture medium that contains less than 0.09.+-.0.014 mM ornithine
and less than 0.2.+-.0.03 mM putrescine.
44. The method of claim 38, wherein the protein-of-interest is
produced at an average 7-day titer that is at least 14% greater
than the average 7-day titer produced by a similar cell in a cell
culture medium that contains less than 0.09.+-.0.014 mM ornithine
and less than 0.2.+-.0.03 mM putrescine.
45. The method of claim 38, wherein the protein-of-interest is
produced at an average 7-day titer that is at least 80% greater
than the average 7-day titer produced by a similar cell in a cell
culture medium that contains less than 0.09.+-.0.014 mM ornithine
and less than 0.2.+-.0.03 mM putrescine.
46. The method of claim 38, wherein the protein-of-interest is
produced at an average 7-day titer that is at least 2-fold greater
than the average 7-day titer produced by a similar cell in a cell
culture medium that contains less than 0.09.+-.0.014 mM ornithine
and less than 0.2.+-.0.03 mM putrescine.
47. The method of claim 38, wherein the protein-of-interest is
produced at an average 7-day titer that is at least 3-fold greater
than the average 7-day titer produced by a similar cell in a cell
culture medium that contains less than 0.09.+-.0.014 mM ornithine
and less than 0.2.+-.0.03 mM putrescine.
48. The method claim 41, wherein the protein-of-interest is a
recombinant human antibody.
Description
FIELD
[0001] The invention relates to media for the culturing of cells
and for the production of recombinant proteins. The invention
specifically relates to serum-free media for the culturing of
recombinant CHO cells for the production of protein
biotherapeutics.
BACKGROUND
[0002] Cell culture media comprising serum or protein hydrolysate
components (i.e., peptones and tryptones) have a long history of
use in the production of recombinant proteins from cultured cells.
These components contain growth factors and a wide variety of other
uncharacterized elements beneficial to cell growth and culture.
However, they also contain uncharacterized elements that reduce
growth or otherwise negatively impact recombinant protein
production. They can also be an unwelcome potential source of
variability. Despite their drawbacks, the benefits of using sera
and hydrolysates have outweighed some of the disadvantages and they
have been widely used in many cell culture applications.
[0003] Human biological therapeutics (biopharmaceuticals) are
generally produced in mammalian cell culture, particularly CHO cell
culture. The presence of uncharacterized or partly characterized
components in those cell cultures are highly undesirable for the
manufacture of biopharmaceuticals for human use. The use of such
uncharacterized or partly characterized components not only
introduces production and regulatory inconsistencies, it raises the
possibility of viral or fungal infection of the production
culture.
[0004] The reduction of lot-to-lot variability in drug product
yield and composition is another important factor in selecting a
culturing process. Sera, hydrolysates, and other undefined elements
introduce variability in the yield, composition, and quality of
biopharmaceutical production lots. The quality and purity of media
elements may also affect yield, since drug titers often rely in
part on maintaining a particular balance of nutrients. Where the
relative amounts of nutrients vary from one media lot to another,
drug yield can vary and this variance can be unacceptable or
uneconomical.
[0005] Using serum containing or hydrolysate-based media introduces
downstream processing challenges. The concentration of a desired
biopharmaceutical in culture is generally on the order of grams per
liter. The presence of serum and hydrolysates in media can add more
than 10 g/L of uncharacterized peptides and proteins, which must be
removed in subsequent processing steps. Serum and hydrolysates can
also introduce variability in the amount of metals and other trace
elements in the media. The elimination of serum and hydrolysates
from culture media therefore eliminates these variations and
potential encumbrances to the production and processing of drug
substance.
[0006] Among others, the benefits to using serum-free and
hydrolysate-free media include reduction in cost, reduction in
variablity between drug lots, and minimization of the risk of
introducing adventitious agents from undefined and unrefined
components. Furthermore, where media are defined and uniform
between batch runs, qualifying runs to test new media batches
against current media are likewise minimized. Thus, there is a need
in the art for media for culturing mammalian cells, wherein the
media are chemically-defined and free of sera and hydrolysates, or
that are serum-free and contain low manageable levels of
hydrolysates, and yet allow for healthy and robust cell growth and
maintenance, and high-titer production of biopharmaceutical drug
substance.
SUMMARY
[0007] The inventors have made the surprising discovery that the
inclusion of ornithine, either with or without putrescine, in cell
culture media that is free of sera ("OS" media) increases cell
viability and density, reduces cell doubling time, and permits high
titer protein production by those cells. The inventors have also
discovered that OS media that contains low or trace amounts of
protein hydrolysates or is chemically defined (i.e., contains no
protein hydrolysates) in particular provides restored cell
viability and density, cell doubling time, and high titer protein
production.
[0008] In one aspect, the invention provides a cell culture medium,
which is serum-free and comprises at least 0.09 mM.+-.0.014 mM
ornithine. In one embodiment the ornithine is present in the medium
at a concentration ranging from 0.09.+-.0.014 mM to 0.9.+-.0.14 mM,
such as 0.09.+-.0.014 mM, 0.3.+-.0.05 mM, 0.6.+-.0.09 mM, or
0.9.+-.0.14 mM ornithine. In some embodiments, the medium also
contains at least 0.20.+-.0.03 mM putrescine. In some embodiments,
the additional putrescine is at a concentration ranging from
0.20.+-.0.03 mM to 0.714.+-.0.11 mM, such as 0.20.+-.0.03 mM,
0.35.+-.0.06, or 0.714.+-.0.11 mM putrescine. In some embodiments,
the medium contains 7.5 g/L hydrolysate. In some embodiments, the
medium is free of any hydrolysate.
[0009] In one embodiment, the medium contains a base medium that is
chemically defined, such as a custom formulation or a commercially
available base medium. In one embodiment, the complete medium is
chemically defined, free of sera and free of hydrolysate.
[0010] In some embodiments, the medium, which is at its useful
concentration (i.e., 1.times.) contains at least 40.+-.6 mM or at
least 70.+-.10.5 mM of a mixture of amino acids or amino acid
salts. In one embodiment, the medium contains at least 40 mM of a
mixture of amino acids. In this or another embodiment, the medium
contains at least 70 mM of a mixture of amino acids. In one
embodiment, the mixture of amino acids (with the notable exception
of glutamine, which may be added back to the medium as a point of
use addition) contains alanine, arginine, asparagine, aspartic
acid, cysteine, glutamic acid, glycine, histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, and valine.
[0011] In some embodiments, the medium contains one or more fatty
acids. In one particular embodiment, the medium contains a mixture
of fatty acids (or fatty acid derivatives) and alpha tocopherol.
Fatty acids or fatty acid derivatives are selected from the group
consisting of linoleic acid, linolenic acid, thioctic acid, oleic
acid, palmitic acid, stearic acid, arachidic acid, acid, lauric
acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid,
lignoceric acid, myristic acid, and octanoic acid.
[0012] In some embodiments, the medium contains a mixture of
nucleosides. In one embodiment, the medium contains adenosine,
guanosine, cytidine, uridine, thymidine, and hypoxanthine.
[0013] In some embodiments, the medium contains a mixture of salts.
Salts include divalent cations, such as calcium and magnesium. In
one embodiment, the medium contains calcium chloride and magnesium
sulfate. Other salts may include those of phosphate.
[0014] In a specific embodiment, the medium (1) contains 7.5 g/L of
a hydrolysate, (2) is serum-free, (3) contains 0.09.+-.0.014 mM,
0.3.+-.0.05 mM, 0.6.+-.0.09 mM, or 0.9.+-.0.14 mM ornithine, (4)
optionally additionally contains 0.20.+-.0.03 mM, 0.35.+-.0.06, or
0.714.+-.0.11 mM putrescine, (5) contains at least about 40 mM or
at least about 70 mM of a mixture of amino acids including alanine,
arginine, asparagine, aspartic acid, cysteine, glutamic acid,
glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine,
and valine, (6) contains tocopherol and a mixture of fatty acids,
(7) contains a mixture of nucleosides including adenosine,
guanosine, cytidine, uridine, thymidine, and hypoxanthine, and (8)
contains salts of calcium, magnesium, and phosphate.
[0015] In another aspect, the invention provides a method for
cultivating cells in a cell culture medium, such as any embodiment
of the medium described in the foregoing aspect. In one embodiment,
the method employs the steps of propagating or maintaining a cell
or cells in a medium that (1) contains 7.5 g/L hydrolysate, or no
hydrolysate, (2) is free of sera, (3) contains ornithine at a
concentration of at least 0.09 mM.+-.0.014 mM, (4) and optionally
contains putrescine, such as at least 0.20.+-.0.03 mM.
[0016] In some embodiments, the cell or cells are mammalian cells,
avian cells, insect cells, yeast cells, or bacteria cells. In one
embodiment, the cells are mammalian cells useful in the production
of recombinant proteins, such as CHO cells or the derivative
CHO-K1. In some embodiments, the cells express a protein of
interest, such as a biotherapeutic protein. The biotherapeutic
protein may be an antigen binding protein, which may contain an Fc
domain. In some embodiments, the protein of interest is a
receptor-Fc-fusion protein, such as an ScFv molecule or a trap
molecule. Trap molecules include the VEGF trap and IL-1 Trap
proteins. In some embodiments, the protein of interest is an
antibody, such as a humanized monoclonal antibody, a bispecific
antibody, or an antibody fragment.
[0017] Given the positive effects on cell growth by including
ornithine or a combination of ornithine and putrescine in
serum-free media, the cells cultured according to this method have
an average doubling time that is no more than 30 hours. In one
embodiment, the cell doubling time is no more than 24 hours. In one
embodiment, when compared to cell growth in media that contains
less than 0.09.+-.0.014 mM ornithine (or less than 0.09.+-.0.014 mM
ornithine and less than 0.2.+-.0.03 mM putrescine), the cells grown
according to this method have an average doubling time that is at
least one third the doubling time of the comparator control
culture.
[0018] Likewise, the inclusion of ornithine alone or a combination
of ornithine and putrescine in serum-free media allows cultured
cells to reach a higher viable cell count density than without the
inclusion of ornithine or the combination of ornithine and
putrescine. In one serum-free and hydrolysate-free embodiment of
the OS medium, the cell culture is capable of attaining a viable
cell count density that is at least 15% greater than a similar cell
culture in a similar cell culture medium that contains less than
0.09.+-.0.014 mM ornithine (or less than 0.09.+-.0.014 mM ornithine
and less than 0.2.+-.0.03 mM putrescine). In another serum-free and
hydrolysate-free embodiment of the OS medium, the cell culture is
capable of attaining a viable cell count density that is at least
3-fold greater than a similar cell culture in a similar cell
culture medium that contains less than 0.09.+-.0.014 mM ornithine
(or less than 0.09.+-.0.014 mM ornithine and less than 0.2.+-.0.03
mM putrescine).
[0019] In another embodiment, the method includes the step of
adding one or more point-of-use additions to the cell culture
medium. In some embodiments, the point-of-use addition is any one
or more of NaHCO.sub.3, glutamine, insulin, glucose, CuSO.sub.4,
ZnSO.sub.4, FeCl.sub.3, NiSO.sub.4, Na.sub.4 EDTA, and Na.sub.3
Citrate. In one embodiment, the method employs the step of adding
each of the following point-of-use chemicals to the cell culture
medium: NaHCO.sub.3, glutamine, insulin, glucose, CuSO.sub.4,
ZnSO.sub.4, FeCl.sub.3, NiSO.sub.4, Na.sub.4 EDTA, and Na.sub.3
Citrate. In some embodiments, the point-of-use additions can be
included in the medium at the outset.
[0020] In a specific embodiment, the aspect provides a method for
cultivating cells in a serum-free medium which contains (1)
ornithine at either 0.09.+-.0.014 mM, 0.3.+-.0.05 mM, 0.6-.+-.0.09
mM, or 0.9.+-.0.14 mM cell culture medium; (2) optionally
additionally putrescine at either 0.20.+-.0.03 mM, 0.35.+-.0.06, or
0.714.+-.0.11 mM; (3) at least about 40 mM or at least about 70 mM
of a mixture of amino acids including alanine, arginine,
asparagine, aspartic acid, cysteine, glutamic acid, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
proline, serine, threonine, tryptophan, tyrosine, and valine; (4)
tocopherol and a mixture of fatty acids; (6) a mixture of
nucleosides including adenosine, guanosine, cytidine, uridine,
thymidine, and hypoxanthine, and (9) salts of calcium, magnesium,
and phosphate, wherein the cells cultured according to this method
have an average doubling time that is no more than 24 hours or at
least one third the doubling time of the comparator control
culture; and the cells cultured are capable of attaining a viable
cell count density that is at least 15% greater or at least 3-fold
greater than a similar cell culture in a similar cell culture
medium that contains less than 0.09.+-.0.015 mM ornithine (or less
than 0.09.+-.0.014 mM ornithine and less than 0.2.+-.0.03 mM
putrescine). In another embodiment, the cell culture is capable of
attaining a viable cell count density that is at least 3-fold
greater than a similar cell culture in a similar cell culture
medium that contains less than 0.09.+-.0.014 mM ornithine (or less
than 0.09.+-.0.014 mM ornithine and less than 0.2.+-.0.03 mM
putrescine). In one embodiment, the medium contains 7.5 g/L
hydrolysate; and in another embodiment, free of hydrolysates.
[0021] In another aspect, the invention provides a method for
producing a protein of interest by employing the steps of (1)
introducing into a cell a nucleic acid sequence that encodes a
protein of interest; (2) selecting a cell carrying that nucleic
acid sequence; (3) culturing the selected cell in an embodiment of
the serum-free cell culture medium described in the first aspect or
according to any embodiment of the method described in the second
aspect; and (4) expressing the protein of interest in the cell,
wherein the protein of interest is secreted into the medium. In
some embodiments, the cell used in the production of the protein is
a mammalian cell capable of producing a biotherapeutic, such as
CHO, 293, and BHK cell, or any derivatives of them. In one
embodiment, the cell is a CHO cell, such as a CHO-K1 cell.
[0022] In some embodiments the protein of interest is an antigen
binding protein. In some embodiments, the protein of interest is a
protein that has an Fc domain. In some cases, those two proteins of
interest may overlap, such as in the case of a receptor-Fc-fusion
protein, an antibody, and a ScFv protein for example. Thus, in some
embodiments, the protein of interest is an antibody, such as a
human antibody or a humanized antibody, an antibody fragment, such
as an Fab or F(ab').sub.2, a bispecific antibody, a trap molecule,
such as a VEGF-Trap or an IL-1-Trap, an ScFv molecule, a soluble
TCR-Fc fusion protein, or the like.
[0023] In one embodiment, the protein of interest is capable of
being produced at an average seven day titer that is at least 7%
greater, at least 14% greater, at least 80% greater, at least two
fold greater, or at least three fold greater than the average seven
day titer produced by a similar cell in a serum-free cell culture
medium that contains less than 0.09.+-.0.014 mM ornithine (or less
than 0.09.+-.0.014 mM ornithine and less than 0.2.+-.0.03 mM
putrescine) ("non-OS" media).
[0024] In a specific embodiment, the protein of interest is
produced by (1) introducing into a CHO cell a nucleic acid sequence
that encodes a protein of interest, such as an antibody or other
antigen-binding protein; (2) selecting a cell carrying that nucleic
acid sequence; (3) culturing the selected cell in a serum-free cell
culture medium which contains (a) ornithine at either
0.09.+-.0.014, 0.3.+-.0.05 mM, 0.6.+-.0.09 mM, or 0.9.+-.0.14 mM;
(b) optionally additionally putrescine at either 0.20.+-.0.03 mM,
0.35.+-.0.06, or 0.714.+-.0.11 mM; (c) at least 40 mM or at least
70 mM of a mixture of amino acids including: alanine, arginine,
asparagine, aspartic acid, cysteine, glutamic acid, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
proline, serine, threonine, tryptophan, tyrosine, and valine; (d)
tocopherol and a mixture of fatty acids; (e) a mixture of
nucleosides including adenosine, guanosine, cytidine, uridine,
thymidine, and hypoxanthine, and (f) salts of calcium, magnesium,
and phosphate; and (d) expressing the protein of interest in the
CHO cell, wherein the protein of interest is secreted into the
medium. In some embodiments, the serum-free cell culture medium may
include 7.5 g/L hydrolysates; or in other embodiments no
hydrolysates at all.
DETAILED DESCRIPTION
[0025] The applicants have made the surprising discovery that the
addition of ornithine, or a combination of ornithine and putrescine
("OS medium") improves viable cell density, cell doubling time, and
protein production by a cell in a cell culture relative to a
serum-free medium that contains very little or no ornithine or
little or none of a combination of ornithine and putrescine
("non-OS medium").
[0026] Before the present cell cultures and methods are described,
it is to be understood that this invention is not limited to
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.
[0027] 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 SI 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". 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, 3.sup.rd 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, 2.sup.nd 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
[0028] 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. Peptides, polypeptides, and proteins include,
among other things, antibodies and chimeric or fusion proteins.
Peptides, polypeptides, and proteins are produced by recombinant
animal cell lines using cell culture methods.
[0029] The term "heterologous polynucleotide 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 heterologous
polynucleotide sequence may be manufactured by genetic engineering
techniques (e.g., such as a sequence encoding a chimeric protein,
or a codon-optimized sequence, an intronless sequence, et cetera)
and introduced into the cell, where it may reside as an episome or
be intergrated into the genome of the cell. The heterologous
polynucleotide sequence may be a naturally occurring sequence that
is introduced into an ectopic site within the production cell
genome. The heterologous polypeptide sequence may be a naturally
occurring sequence from another organism, such as a sequence
encoding a human ortholog.
[0030] "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 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 amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes
reference to 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 to
express the antibody. The term antibody also includes bispecific
antibody, which includes a heterotetrameric immunoglobulin that can
bind to more than one different epitope. Bispecific antibodies are
generally described in US Patent Application Publication No.
2010/0331527, which is incorporated by reference into this
application.
[0031] The term "antigen-binding portion" of an antibody (or
"antibody fragment"), refers to one or more fragments of an
antibody that retain the ability to specifically bind to an
antigen. Examples of 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. (1989)
Nature 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. (1993) PNAS USA 90:6444-6448; Poljak et al.
(1994) Structure 2:1121-1123).
[0032] 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. Examples of
such immunoadhesion molecules include use of the streptavidin core
region to make a tetrameric scFv molecule (Kipriyanov et al. (1995)
Human Antibodies and Hybridomas 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.
(1994) Mol. Immunol. 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).
[0033] The term "human antibody" is intended to include antibodies
having variable and constant regions derived from human germline
immunoglobulin sequences. The human antibodies of the invention 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. However, the term
"human antibody", as used herein, is not intended to include
antibodies in which CDR sequences derived from the germline of
another mammalian species, such as a mouse, have been grafted onto
human framework sequences.
[0034] 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. (1992) Nucl.
Acids Res. 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.
[0035] "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., Proc. Natl. Acad. ScL USA 88: 10535, 1991; Byrn
et al., Nature 344:677, 1990; and Hollenbaugh et al., "Construction
of Immunoglobulin Fusion Proteins", in Current Protocols in
Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992. "Receptor Fc
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). For example, an Fc-fusion protein is a trap, such as for
example an IL-1 trap (e.g., rilonacept, which contains the IL-1
RAcP ligand binding region fused to the IL-1R1 extracellular region
fused to Fc of hIgG1; 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
fused to Fc of hIgG1; see U.S. Pat. Nos. 7,087,411 and
7,279,159).
Media
[0036] The present invention provides a serum-free medium that is
useful in culturing cells and producing a biopharmaceutical drug
substance. "Serum-free" applies to a cell culture medium that does
not contain animal sera, such as fetal bovine serum. The serum-free
media may contain 7.5 g/L of hydrolysates, such as soy hydrolysate.
The present invention also provides chemically defined media, which
is not only serum-free, but also hydrolysate-free.
"Hydrolysate-free" applies to cell culture media that contains no
exogenous protein hydrolysates such as animal or plant protein
hydrolysates such, for example peptones, tryptones and the
like.
[0037] The elimination of serum and reducing or eliminating
hydrolysates from cell culture media, while reducing lot-to-lot
variability and enhancing downstream processing steps,
unfortunately diminishes cell growth, viability and protein
expression. Thus, chemically defined serum-free and low to no
hydrolysate media requires additional ingredients to improve cell
growth and protein production. The cell culture media of the
invention may be supplemented with additional ingredients such as
polyamines or increased concentrations of components like amino
acids, salts, sugars, vitamins, hormones, growth factors, buffers,
antibiotics, lipids, trace elements and the like, depending on the
requirements of the cells to be cultured or the desired cell
culture parameters. Specifically, the cell culture medium here is
supplemented with ornithine, putrescine, or both ("OS media") to
improve cell growth, cell viability, and recombinant protein
production.
[0038] In some embodiments, the OS medium contains ornithine at a
concentration (expressed in micromoles per liter) of at least about
90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 540, 545, 550,
555, 560, 565, 568, 567, 568, 569, 570, 571, 572, 573, 574, 575,
576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588,
589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601,
602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614,
615, 616, 617, 618, 620, 625, 630, 635, 640, 645, 650, 700, 750,
800, 850, or 900 .mu.M.
[0039] In some embodiments, the media contains ornithine at a
concentration of about 85, 90, 95, 100, 105, 110, 113, or 115
.mu.M. In one embodiment, the medium contains 100 .mu.M.+-.15 .mu.M
ornithine. In one embodiment, the medium contains 15 mg/L.+-.2.25
mg/L ornithine.HCl.
[0040] In some embodiments, the media contains ornithine at a
concentration of about 255, 260, 265, 270, 275, 280, 285, 290, 295,
300, 305, 310, 315, 320, 325, 330, 335, 340, or 345 .mu.M. In one
embodiment, the medium contains 300 .mu.M.+-.45 .mu.M ornithine. In
one embodiment, the medium contains 50 mg/L.+-.7.5 mg/L
ornithine.HCl.
[0041] In some embodiments, the media contains ornithine at a
concentration of about 510, 515, 520, 525, 530, 535, 540, 545, 550,
555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615,
620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680,
685, or 690 .mu.M. In one embodiment, the medium contains 600
.mu.M.+-.90 .mu.M ornithine. In one embodiment, the medium contains
100 mg/L.+-.15 mg/L ornithine.HCl.
[0042] In some embodiments, the media contains ornithine at a
concentration of 765, 770, 775, 780, 785, 790, 795, 800, 805, 810,
815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875,
880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940,
945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, 1,000,
1,005, 1,010, 1,015, 1,020, 1,025, 1,030, or 1,035 .mu.M. In one
embodiment, the medium contains 900 .mu.M.+-.135 .mu.M ornithine.
In one embodiment, the medium contains 150 mg/L.+-.22.5 mg/L
ornithine.HCl.
[0043] Putrescine may optionally be added to the ornithine
supplemented media. Putrescine has been included, at very low
concentrations, as a component in some cell culture media
formulations; see for example WO 2005/028626, which describes
0.02-0.08 mg/L putrescine; U.S. Pat. No. 5,426,699 (0.08 mg/L);
U.S. Pat. No. RE30,985 (0.16 mg/L); U.S. Pat. No. 5,811,299 (0.27
mg/L); U.S. Pat. No. 5,122,469 (0.5635 mg/L); U.S. Pat. No.
5,063,157 (1 mg/1); WO 2008/154014 (.about.100 .mu.M-.about.1000
.mu.M); US Pat. App. No. 2007/0212770 (0.5-30 mg/L polyamine; 2
mg/L putrescine; 2 mg/L putrescine+2 mg/L ornithine; 2 mg/L
putrescine+10 mg/L ornithine).
[0044] In some embodiments, the media contains a combination of
ornithine and putrescine, wherein the putrescine can be at a
concentration of at least about 150, 155, 160, 165, 170, 175, 180,
185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,
250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,
315, 320, 325, 330, 335, 340, 345, 350, 355, 260, 365, 370, 375,
380, 385, 390, 395, 400, 405, or 410 .mu.M.
[0045] In some embodiments, the media contains putrescine at a
concentration of about 170, 175, 180, 185, 190, 195, 200, 205, 210,
215, 220, 225, or 230 .mu.M. In one embodiment, the medium contains
200 .mu.M.+-.30 .mu.M putrescine in addition to 90 .mu.M.+-.14
.mu.M ornithine. In one embodiment, the medium contains 30
mg/L.+-.4.5 mg/L putrescine.2HCl in addition to 15 mg/L.+-.2.25
mg/L ornithine.HCl.
[0046] In some embodiments, the media contains putrescine at a
concentration of about 295, 300, 305, 310, 315, 320, 325, 330, 335,
340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, or
405 .mu.M. In one embodiment, the medium contains 350 .mu.M.+-.52.5
.mu.M putrescine in addition to 90 .mu.M.+-.14 .mu.M ornithine. In
one embodiment, the medium contains 57 mg/L.+-.8.55 mg/L
putrescine.2HCl in addition to 15 mg/L.+-.2.25 mg/L
ornithine.HCl.
[0047] In some embodiments, the media contains putrescine at a
concentration of about 595, 600, 605, 610, 615, 620, 625, 630, 635,
640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700,
705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765,
770, 775, 780, 785, 790, 795, 800, or 805 .mu.M. In one embodiment,
the medium contains 714 .mu.M.+-.105 .mu.M putrescine in addition
to 90 .mu.M.+-.14 .mu.M ornithine. In one embodiment, the medium
contains 115 mg/L.+-.17.25 mg/L putrescine.2HCl in addition to 15
mg/L.+-.2.25 mg/L ornithine.HCl.
[0048] In some embodiments, the media contains a pair-wise
combination of any concentration of putrescine and ornithine listed
above. In some embodiments, the media contains any pair-wise
combination of about 595, 600, 605, 610, 615, 620, 625, 630, 635,
640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700,
705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765,
770, 775, 780, 785, 790, 795, 800, or 805 .mu.M putrescine, and
about 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565,
570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630,
635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, or 690 .mu.M
ornithine. For example, the media in one embodiment contains about
700 .mu.M putrescine plus any one of 510, 511, 512 .mu.M, et
sequens ornithine; or 701 .mu.M putrescine plus any one of 510,
511, 512 .mu.M, et sequens ornithine; et cetera. Also for example,
the media in one embodiment contains about 600 .mu.M ornithine plus
any one of 700, 701, 702 .mu.M, et sequens putrescine; or 601 .mu.M
ornithine plus any one of 700, 701, 702 .mu.M, et sequens
putrescine; et cetera. In some embodiments, the media contains 702
.mu.M.+-.106 .mu.M purescine+593 .mu.M.+-.89 .mu.M ornithine. In
one particular embodiment, the media contains about 714 .mu.M
putrescine and 593 .mu.M ornithine. In one embodiment, the media
contains 115 mg/L.+-.17 mg/L putrescine.2HCl and 100 mg/L.+-.15
mg/L ornithine.HCl. In one particular embodiment, the media
contains 115 mg/L putrescine.2HCl and 100 mg/L ornithine.HCl.
[0049] In one embodiment and in addition to the inclusion of
ornithine or putrescine, the media contains a mixture of
nucleosides in a cumulative concentration of at least 50 .mu.M, at
least 60 .mu.M, at least 70 .mu.M, at least 80 .mu.M, at least 90
.mu.M, at least 100 .mu.M, at least 110 .mu.M, at least 115 .mu.M,
at least 120 .mu.M, at least 125 .mu.M, at least 130 .mu.M, at
least 135 .mu.M, at least 140 .mu.M, at least 145 .mu.M, at least
150 .mu.M, at least 155 .mu.M, at least 160 .mu.M, at least 165
.mu.M, or at least 170 .mu.M. In one embodiment, the media contains
about 174 .mu.M.+-.26 .mu.M nucleoside. In one embodiment, the
media contains purine derivatives in a cumulative concentration of
at least 40 .mu.M, at least 45 .mu.M, at least 50 .mu.M, at least
55 .mu.M, at least 60 .mu.M, at least 65 .mu.M, at least 70 .mu.M,
at least 75 .mu.M, at least 80 .mu.M, at least 85 .mu.M, at least
90 .mu.M, at least 95 .mu.M, at least 100 .mu.M, or at least 105
.mu.M. In one embodiment, the media contains about 106 .mu.M.+-.5
.mu.M of purine derivatives. Purine derivatives include
hypoxanthine and the nucleosides adenosine and guanosine. In one
embodiment, the media contains pyrimidine derivatives in a
cumulative concentration of at least 30 .mu.M, at least 35 .mu.M,
at least 40 .mu.M, at least 45 .mu.M, at least 50 .mu.M, at least
55 .mu.M, at least 60 .mu.M, or at least 65 .mu.M. In one
embodiment, the media contains about 68 .mu.M.+-.5 .mu.M of
pyrimidine derivatives. Pyrimidine derivatives include the
nucleosides thymidine, uridine, and cytidine. In one particular
embodiment, the media contains adenosine, guanosine, cytidine,
uridine, thymidine and hypoxanthine.
[0050] In addition to the inclusion of ornithine or putrescine, in
one embodiment, the media also contains amino acids in a cumulative
concentration of at least 40 mM, wherein the amount of glutamine is
not included in the calculation of the cumulative total. In one
embodiment, glutamine is not included in the media, but may be
supplied as a "point-of-use addition" to the media during the
culturing of cells such as during the production of protein. Thus,
in some embodiments, such as in the method to culture cells or the
method to produce a protein of interest, the media may be
supplemented with glutamine as a point-of-use addition. In one such
embodiment, glutamine is added in an amount less than about 40 mM,
less than about 35 mM, less than about 30 mM, less than about 25
mM, less than about 20 mM, less than about 15 mM, less than about
10 mM, less than about 8 mM, less than about 7 mM, less than about
6 mM, less than about 5 mM, less than about 4 mM, less than about 3
mM, or less than about 2.5 mM. In one embodiment the amount of
glutamine in the media that was supplemented with glutamine is
about 2 mM.+-.0.5 mM.
[0051] In one embodiment, in addition to the inclusion of ornithine
or a combination of both ornithine and putrescine, the media also
contains amino acids having a non-polar side group in a
concentration of at least 15 mM, at least 24 mM, at least 25 mM, at
least 26 mM, at least 27 mM, at least 28 mM, at least 29 mM, or at
least 30 mM. In one embodiment, the media contains about 30 mM of
amino acids having a non-polar side group. In one embodiment, of
the total amount of amino acids by mole contained within the media,
at least 32%, at least 33%, at least 34%, at least 35%, at least
36%, at least 37%, at least 38%, at least 39%, at least 40%, or at
least 41% are amino acids having non-polar side groups. In one
embodiment, about 42%.+-.1% by mole of the amino acids in the media
are amino acids having a non-polar side group. Amino acids having a
non-polar side group include alanine, valine, leucine, isoleucine,
proline, phenylalanine, tryptophan, and methionine.
[0052] In one embodiment, in addition to the inclusion of ornithine
or a combination of both ornithine and putrescine, the media also
contains amino acids having an uncharged polar side group in a
concentration of about 10 mM to 34 mM, about 11 mM to 33 mM, about
12 mM to 32 mM, about 13 mM to 31 mM, about 14 mM to 30 mM, about
15 mM to 29 mM, about 16 mM to 28 mM, about 17 mM to 27 mM, about
18 mM to 26 mM, about 19 mM to 25 mM, about 20 mM to 24 mM, about
21 mM to 23 mM, or about 22 mM. In one embodiment, the medium
contains about 22 mM of amino acids having an uncharged polar side
group. In another embodiment, the medium contains about 12 mM amino
acids having an uncharged polar side group. In one embodiment, of
the total amount by mole of amino acids contained within the media,
about 14% to 46%, about 15% to 45%, about 16% to 44%, about 17% to
43%, about 18% to 42%, about 19% to 41%, about 20% to 40%, about
21% to 39%, about 22% to 38%, about 23% to 37%, about 24% to 36%,
about 25% to 35%, about 26% to 34%, about 27% to 33%, about 28% to
32%, about 29% to 31%, or about 30% are amino acids having
uncharged polar side groups. In one embodiment, about 30%.+-.3% by
mole of the amino acids in the media are amino acids having an
uncharged polar side group. Amino acids having an uncharged polar
side group include glycine, serine, threonine, cysteine, tyrosine,
asparagine, and glutamine.
[0053] In one embodiment, in addition to the inclusion of ornithine
or a combination of both ornithine and putrescine, the media also
contains amino acids having a negative charge at pH 6 (i.e., acidic
amino acids) in a concentration of about 4 mM to 14 mM, about 5 mM
to 13 mM, about 6 mM to 12 mM, about 7 mM to 11 mM, about 8 mM to
10 mM, about 9 mM, or about 4 mM. In one embodiment, the media
contains about 9 mM of acidic amino acids. In one embodiment, the
media contains 9 mM.+-.1 mM of acidic amino acids. In one
embodiment, of the total amount by mole of amino acids contained
within the media, about 8% to 18%, about 9% to 17%, about 10% to
16%, about 11% to 15%, about 12% to 14%, or about 13% are acidic
amino acids. In one embodiment, about 12.6%.+-.1% by mole of the
amino acids in the media are acidic amino acids. Acidic amino acids
include aspartic acid and glutamic acid.
[0054] In one embodiment, in addition to the inclusion of ornithine
or a combination of both ornithine and putrescine, the media also
contains amino acids having a positive charge at pH 6 (i.e., basic
amino acids) in a concentration of at least 3.5 mM, at least 4 mM,
at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at
least 9 mM, at least 10 mM, or at least 11 mM. In one embodiment,
the media contains about 11 mM of basic amino acids. In one
embodiment, the media contains about 11.42 mM.+-.1 mM of basic
amino acids. In one embodiment, of the total amount by mole of
amino acids contained within the media, at least 5%, at least 6%,
at least 7%, at least 8%, at least 9%, at least 10%, at least 11%,
at least 12%, at least 13%, at least 14%, or at least 15% are basic
amino acids. In one embodiment, about 16% by mole of the amino
acids in the media are basic amino acids. In one embodiment, about
15.8%.+-.2.4% by mole of the amino acids in the media are basic
amino acids. In one embodiment, about 21%.+-.3.2% by mole of the
amino acids in the media are basic amino acids. Basic amino acids
include lysine, arginine, and histidine.
[0055] In one embodiment, in addition to the inclusion of ornithine
or a combination of both ornithine and putrescine, the media also
contains about 30 mM non-polar amino acids, about 22 mM uncharged
polar amino acids, about 9 mM acidic amino acids, and about 11 mM
basic amino acids. In one embodiment, of the amino acids in the
media about 42% by mole are non-polar amino acids, about 30% by
mole are uncharged polar amino acids, about 13% by mole are acidic
amino acids, and about 16% by mole are basic amino acids.
[0056] In addition to the inclusion of ornithine or a combination
of both ornithine and putrescine, in one embodiment, the media
contains micromolar amounts of fatty acids (or fatty acid
derivatives) and tocopherol. In one embodiment, the fatty acids
include any one or more of linoleic acid, linolenic acid, thioctic
acid, oleic acid, palmitic acid, stearic acid, arachidic acid,
arachidonic acid, lauric acid, behenic acid, decanoic acid,
dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and
octanoic acid. In one embodiment, the media contains tocopherol,
linoleic acid, and thioctic acid.
[0057] In one embodiment, the media also contains a mixture of
vitamins, which includes other nutrients and essential nutrients,
at a cumulative concentration of at least about 700 .mu.M or at
least about 2 mM. In one embodiment, the mixture of vitamins
contains one or more of D-biotin, choline chloride, folic acid,
myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid
(hemiCa), riboflavin, thiamine HCl, vitamin B12, and the like. In
one embodiment, the mixture of vitamins includes all of D-biotin,
choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine
HCl, D-pantothenic acid (hemiCa), riboflavin, thiamine HCl, and
vitamin B12.
[0058] Various embodiments of the media of the invention include
any of the combinations of the above described embodiments,
including chemically defined, hydrolysate-free serum-free media
comprising ornithine or putrescine in the indicated amounts, plus
inter alia (a) amino acids; (b) optionally nucleosides; (c) salts
of divalent cations; (d) fatty acids and tocopherol; and (e)
vitamins. In some embodiments, all small amounts of hydrolysates
may be added to the OS media.
[0059] The applicants envision that in the practice of this
invention any one or more of a variety of base media or
combinations thereof, to which the ornithine or a combination of
both ornithine and putrescine are added, may be used. Base media
are generally known in the art and include inter alia Eagle's MEME
(minimal essential media) (Eagle, Science, 1955,
112(3168):501-504), Ham's F12 (Ham, Proc. Nat'l. Acad. Sci. USA,
1965, 53:288-293), F-12 K medium, Dulbecco's medium, Dulbecco's
Modified Eagle Medium (Proc. Natl. Acad. Sci. USA., 1952 August;
38(8): 747-752), DMEM/Ham's F12 1:1, Trowell's T8, A2 media Holmes
and Wolf, Biophys. Biochem. Cytol., 1961, 10:389-401), Waymouth
media (Davidson and Waymouth, Biochem. J., 1945, 39(2):188-199),
Williams E media (William's et al., Exp. Cell Res., 1971, 69:105 et
seq.), RPMI 1640 (Moore et al., J. Amer. Med. Assoc., 1967,
199:519-524), MCDB 104/110 media (Bettger et al., Proc. Nat'l.
Acad. Sci. USA, 1981, 78(9):5588-5592), Ventrex HL-1 media,
albumin-globulin media (Orr et al., Appl. Microbiol., 1973,
25(1):49-54), RPM I-1640 Medium, RPMI-1641 Medium, Iscove's
Modified Dulbecco's Medium, McCoy's 5 A Medium, Leibovitz's L-15
Medium, and serum-free media such as EX-CELL.TM. 300 Series (JRH
Biosciences, Lenexa, Kans.), protamine-zinc-insulin media (Weiss et
al., 1974, U.S. Pat. No. 4,072,565), biotin-folate media (Cartaya,
1978, US Re30,985), Transferrin-fatty acid media (Baker, 1982, U.S.
Pat. No. 4,560,655), transferrin-EGF media (Hasegawa, 1982, U.S.
Pat. No. 4,615,977; Chessebeuf, 1984, U.S. Pat. No. 4,786,599), and
other media permutations (see Inlow, U.S. Pat. No. 6,048,728;
Drapeau, U.S. Pat. No. 7,294,484; Mather, U.S. Pat. No. 5,122,469;
Furukawa, U.S. Pat. No. 5,976,833; Chen, U.S. Pat. No. 6,180,401;
Chen, U.S. Pat. No. 5,856,179; Etcheverry, U.S. Pat. No. 5,705,364;
Etcheverry, U.S. Pat. No. 7,666,416; Ryll, U.S. Pat. No. 6,528,286;
Singh, U.S. Pat. No. 6,924,124; Luan, U.S. Pat. No. 7,429,491; and
the like).
[0060] In a particular embodiment, the media is chemically defined
and contains in addition to the ornithine or combination of both
ornithine and putrescine: CaCl.sub.2 2H.sub.2O; HEPES buffer, KCl;
MgSO.sub.4; NaCl; Na.sub.2HPO.sub.4 or other phosphate salts;
pyruvate; L-alanine; L-arginine HCl; L-asparagine H.sub.2O;
L-aspartic acid; L-cysteine HCl H.sub.2O; L-glutamic acid; Glycine;
L-histidine HCl H.sub.2O; L-isoleucine; L-leucine; L-lysine HCl;
L-methionine; L-ornithine HCl; L-phenylalanine; L-proline;
L-serine; L-threonine; L-tryptophan; L-tyrosine 2Na 2H.sub.2O;
L-valine; D-biotin; choline chloride; folic acid; myo-inositol;
niacinamide; pyridoxine HCl; D-pantothenic acid; riboflavin;
thiamine HCl; vitamin B12; .rho.-aminobenzoic acid; ethanolamine
HCl; Pluronic F68; DL-a-tocopherol phosphate; linoleic acid;
Na.sub.2SeO.sub.3; thioctic acid; and glucose; and optionally
adenosine; guanosine; cytidine; uridine; thymidine; and
hypoxanthine 2Na.
[0061] In one embodiment, the starting osmolarity of the media of
the invention is 200-500, 250-400, 275-350, or about 300 mOsm.
During growth of the cells in the media of the invention, and in
particular following any feedings according to a fed batch
protocol, the osmolarity of the culture may increase up to about
350, 400, 450, or as high as 500 mOsm.
[0062] In some embodiments wherein the osmolarity of the defined
medium is less than about 300, the osmolarity is brought to about
300 with the addition of one or more salts in excess of the amount
specified. In one embodiment, osmolarity is increased to a desired
level by adding one or more of an osmolyte selected from sodium
chloride, potassium chloride, a magnesium salt, a calcium salt, an
amino acid salt, a salt of a fatty acid, sodium bicarbonate, sodium
carbonate, potassium carbonate, a chelator that is a salt, a sugar
(e.g., galactose, glucose, sucrose, fructose, fucose, etc.), and a
combination thereof. In one embodiment, the osmolyte is added over
and above its concentration in a component already present in the
defined medium (e.g., a sugar is added over and above the
concentration specified for a sugar component).
[0063] Each and every embodiment of the media described above, as
well as any other serum-free media containing at least about 90
.mu.M ornithine (or containing a combination of at least about 100
.mu.M ornithine plus at least about 200 .mu.M putrescine) are
hereinafter referred to as ornithine supplemented ("OS") media.
Conversely, media containing no ornithine (or no
ornithine/putrescine combination), or media containing less than
100 .mu.M ornithine (or media containing less than 100 .mu.M
ornithine and less than 200 .mu.M putrescine), are hereinafter
refered to as non-ornithine supplemented ("non-OS") media.
Cell Culture
[0064] The present invention provides a cell culture comprising a
cell line expressing a protein of interest in an OS medium as
described above. In one embodiment, the cell culture contains
insulin, which can be added as a point-of-use ingredient to the
media, or can be included in the media formulation. In one
embodiment, the cell line comprises cells capable of producing a
biotherapeutic protein. Examples of cell lines that are routinely
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.
[0065] "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.
[0066] Animal cells, such as CHO cells, may be cultured in small
scale cultures, 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 can
contain 10,000 L of media or more. Large scale 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. Concentrated feed medium may be based on any
cell culture media formulation. Such a concentrated feed medium can
contain most of the components of the cell culture medium 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.
[0067] 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 differentialtion. 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 (PIGF), 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.
[0068] 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 NaHCO.sub.3. In one embodiment, the point-of-use addition
buffer comprises NaHCO.sub.3. In another embodiment, the buffer is
HEPES.
[0069] 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.
In some cases, glucose can be added at high levels up to 10
g/L.
[0070] 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 Na.sub.3C.sub.6H.sub.5O.sub.7.
[0071] In one embodiment, the cell culture may be supplemented with
one or more point-of-use addition amino acids, such as e.g.,
glutamine. In one embodiment, the cell culture media is
supplemented with the point-of-use addition glutamine at a final
concentration of about 1 mM to 13 mM.
[0072] 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 chlroide; and nickel
sulfate.
[0073] In one embodiment, the cell culture media is supplemented
with any one or more or all of the following point-of-use
additions: about 29.8 mM NaHCO3, about 2 mM glutamine, about 0.86
.mu.M insulin, about 11.1 mM glucose, about 6.54 .mu.M zinc
sulfate, about 0.168 .mu.M copper sulfate, about 75 .mu.M ferric
chloride, about 0.639 .mu.M nickel sulfate, about 85 .mu.M EDTA,
and about 50 .mu.M citrate.
[0074] 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 Y. M. Huang et al., Biotechnol Prog. 2010
September-October; 26(5): 1400-10).
[0075] Cell viability, viable cell density, and cell doubling are
improved relative to cells grown in culture without ornithine or
putrescine. Regarding cell viability, cells grown in OS media show
a viability that is at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 45%,
at least 50%, at least, 55%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 99%, at least 100%, or at least 3-fold greater
than the viability of similar or identical cells grown in non-OS
media.
[0076] In some embodiments, the doubling rate of viable mammalian
cells in OS media is at least 5%, at least 6%, at least 7%, at
least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at
least 13%, at least 14%, at least 15%, at least 16%, at least 17%,
at least 18%, at least 19%, at least 20%, at least 21%, at least
22%, at least 23%, at least 24%, at least 25%, at least 26%, at
least 27%, at least 28%, at least 29%, at least 30%, at least 35%,
at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 70%, at least 80%, at least 90%, at least 100%, or at
least 3-fold greater than the doubling rate of mammalian cells
cultured in non-OS media. In some embodiments, the doubling rate of
viable mammalian cells in OS media is about 10%, 11%, 12%, 13%,
14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,
27%, 28%, 29%, or 30% greater than the doubling rate of mammalian
cells in non-OS media.
[0077] In some embodiments, the doubling time of actively cycling
mammalian cells is less than 30 hours, less than 29 hours, less
than 28 hours, less than 27 hours, less than 26 hours, less than 25
hours, less than 24 hours, less than 23 hours, less than 22 hours,
less than 21 hours, less than 20 hours, less than 19 hours, or less
than 18 hours in OS media. In some embodiments, the doubling time
of actively growing mammalian cells is less than 28 hours in OS
media. In some embodiments, the doubling time of mammalian cells is
about 27.+-.1 hours, about 26.+-.1 hours, about 25.+-.1 hours,
about 24.+-.1 hours, about 23.+-.1 hours, about 22.+-.1 hours, or
about 21.+-.1 hours in OS media. In some embodiments, the doubling
time of actively cycling mammalian cells is about 24.+-.1 hours in
OS media. In some embodiments, the doubling time of actively
dividing cells cultured in OS media is at least 15%, at least 16%,
at least 17%, at least 18%, at least 19%, at least 20%, or at least
25% shorter than the doubling time of actively cycling cells
cultured in a non-OS media.
Protein Production
[0078] In addition to chemically defined OS media and methods of
culturing cells in OS media, the present invention provides methods
of producing a protein, such as a therapeutically effective
antibody or other biopharmaceutical drug substance, in a cell
cultured in OS media.
[0079] In some embodiments, the rate of production of protein by
mammalian cells cultured in OS media is at least 5%, 10%, 15%, or
20% greater than the rate of production of protein by an identical
mammalian cell cultured in non-OS media. In some embodiments the
rate of production of protein in cells cultured in OS media is at
least 1 .rho.g/cell/day ("PCD"), at least 2 PCD, at least 3 PCD, at
least 4 PCD, at least 5 PCD, at least 6 PCD, at least 7 PCD, at
least 8 PCD, at least 9 PCD, at least 10 PCD, at least 15 PCD, at
least 20 PCD, at least 25 PCD, at least 30 PCD, at least 35 PCD, at
least 40 PCD, at least 45 PCD, at least 50 PCD, at least 75 PCD, or
at least 100 PCD.
[0080] In some embodiments the protein production yield or titer,
which can be expressed in grams of protein product per liter of
culture media, from cells cultured in OS media is at least 100
mg/L, at least 1 g/L, at least 1.2 g/L, at least 1.4 g/L, at least
1.6 g/L, at least 1.8 g/L, at least 2 g/L, at least 2.5 g/L, at
least 3 g/L, at least, 3.5 g/L, at least 4 g/L, at least 4.5 g/L,
at least 5 g/L, at least 5.5 g/L, at least 6 g/L, at least 6.5 g/L,
at least 7 g/L, at least 7.5 g/L, at least 8 g/L, at least 8.5 g/L,
at least 9 g/L, at least 9.5 g/L, at least 10 g/L, or at least 20
g/L.
[0081] In some 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.
[0082] In some 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
of 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, such
as for example an IL-1 trap (e.g., rilonacept, which contains the
IL-1 RAcP ligand binding region fused to the II-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), 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
fused to Fc of hIgG1; see U.S. Pat. Nos. 7,087,411 and
7,279,159).
[0083] The present invention is not limited to any particular type
of cell for protein production. Examples of cell types suitable for
protein production include mammalian cells, insect cells, avian
cells, bacterial cells, and yeast cells. The cells may be stem
cells or recombinant cells transformed with a vector for
recombinant gene expression, or cells transfected with a virus for
producing viral products. The cells may contain a recombinant
heterologous polynucleotide construct that encodes a protein of
interest. That construct can be an episome or it can be an element
that is physically integrated into the genome of the cell. The
cells may also produce a protein of interest without having that
protein encoded on a heterologous polypeptide construct. In other
words, the cell may naturally encode the protein of interest, such
as a B-cell producing an antibody. The cells may also be primary
cells, such as chicken embryo cells, or primary cell lines.
Examples of useful cells include BSC 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-21 cells, chicken embryo cells, NS-1 cells,
MRC-5 cells, WI-38 cells, BHK cells, 293 cells, RK cells, Per.C6
cells and CHO cells. In various embodiments, the cell line is a CHO
cell derviative, such as CHO-K1, CHO DUX B-11, CHO DG-44,
Veggie-CHO, GS-CHO, S-CHO, or CHO Iec mutant lines.
[0084] In one embodiment, the cell, which is a CHO cell,
ectopically expresses a protein. In one embodiment, the protein
comprises an immunoglobulin heavy chain region, such as a CH1, CH2,
or CH3 region. In one embodiment, the protein comprises a human or
rodent immunoglobulin CH2 and CH3 region. In one embodiment, the
protein comprises a human or rodent immunoglobulin CH1, CH2, and
CH3 region. In one embodiment, the protein comprises a hinge region
and a CH1, CH2, and CH3 region. In a specific embodiment, the
protein comprises an immunoglobulin heavy chain variable domain. In
a specific embodiment, the protein comprises an immunoglobulin
light chain variable domain. In a specific embodiment, the protein
comprises an immunoglobulin heavy chain variable domain and an
immunoglobulin light chain variable domain. In a specific
embodiment, the protein is an antibody, such as a human antibody, a
rodent antibody, or a chimeric human/rodent antibody (e.g.,
human/mouse, human/rat, or human hamster).
[0085] A production phase can be conducted at any scale of culture,
from individual flasks and shaker flasks or wave bags, to one-liter
bioreactors, and to large scale industrial bioreactors. A large
scale process can be conducted in a volume of about 100 liters to
20,000 liters or more. One or more of several means may be used to
control protein production, such as temperature shift or chemical
induction. A growth phase may occur at a higher temperature than a
production phase. For example, a growth phase may occur at a first
temperature of about 35.degree. C. to 38.degree. C., and a
production phase may occur at a second temperature of about
29.degree. C. to 37.degree. C., optionally from about 30.degree. C.
to 36.degree. C. or from about 30.degree. C. to 34.degree. C. In
addition, chemical inducers of protein production, such as
caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline,
and hexamethylene bisacetamide (HMBA), may be added concurrent
with, before, or after a temperature shift. If inducers are added
after a temperature shift, they can be added from one hour to five
days after the temperature shift, such as from one to two days
after the temperature shift. Production cell cultures may be run as
continuous feed culture system, as in a chemostat (see C.
Altamirano et al., Biotechnol Prog. 2001 November-December;
17(6):1032-41), or according to a fed-batch process (Huang,
2010).
[0086] The invention is useful for improving protein production via
cell culture processes. The cell lines used in the invention can be
genetically engineered to express a polypeptide of commercial or
scientific interest. Genetically engineering the cell line involves
transfecting, transforming or transducing the cells with a
recombinant polynucleotide molecule, or otherwise altering (e.g.,
by homologous recombination and gene activation or fusion of a
recombinant cell with a non-recombinant cell) so as to cause the
host cell to express a desired recombinant polypeptide. Methods and
vectors for genetically engineering cells 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); Sambrook et al.,
Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory
Press, 1989); Kaufman, R. J., Large Scale Mammalian Cell Culture,
1990, pp. 15-69. A wide variety of cell lines suitable for growth
in culture are available from the American Type Culture Collection
(Manassas, Va.) and commercial vendors. Examples of cell lines
commonly used in the industry include VERO, BHK, HeLa, CVI
(including Cos), MDCK, 293, 3T3, myeloma cell lines (e.g., NSO,
NSI), PC12, WI38 cells, and Chinese hamster ovary (CHO) cells. CHO
cells are widely used for the production of complex recombinant
proteins, such as cytokines, clotting factors, and antibodies
(Brasel et al. (1996), Blood 88:2004-2012; Kaufman et al. (1988),
J. Biol Chem 263:6352-6362; McKinnon et al. (1991), J Mol
Endocrinol 6:231-239; Wood et al. (1990), J Immunol.
145:3011-3016). The dihydrofolate reductase (DHFR)-deficient mutant
cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:
4216-4220), DXBI 1 and DG-44, are desirable CHO host cell lines
because the efficient DHFR selectable and amplifiable gene
expression system allows high level recombinant protein expression
in these cells (Kaufman RJ. (1990), Meth Enzymol 185:537-566). In
addition, these cells are easy to manipulate as adherent or
suspension cultures and exhibit relatively good genetic stability.
CHO cells and the proteins recombinantly expressed by them have
been extensively characterized and have been approved for use in
clinical and commercial manufacturing by regulatory agencies. In
some embodiments, the CHO cell lines are cell lines as described in
U.S. Patent Application Publications No. 2010/0304436 A1,
2009/0162901 A1 and 2009/0137416 A1, and U.S. Pat. Nos. 7,455,988
B2, 7,435,553 B2, and 7,105,348 B2.
[0087] The present invention is not limited in scope by the
specific embodiments described herein, which are intended as
illustrations of individual aspects or embodiments of the
invention. Functionally equivalent methods and components are
within the scope of the invention. Various modifications of the
invention, in addition to those described here, are apparent to
those skilled in the art from the foregoing description and
accompanying drawings. Such modifications fall within the scope of
the invention.
[0088] The invention is based, in part, on the discovery that
addition of ornithine or a combination of ornithine and putrescine
to serum free cell culture media results in increased cell growth,
viability and polypeptide production from a recombinantly
engineered animal cell line (or natural cell) expressing a protein
of interest, thereby enhancing culture robustness, improving the
yield of the polypeptide of interest.
Example 1
Improved Viable Cell Culture Density
[0089] A 250 mL shake flask was inoculated from a seed culture of a
recombinant antibody producing cell line derived from CHO K1. The
inoculated cells were grown at 36.5.degree. C. for seven days and
fed glucose on days three and five. Cells were grown in each of two
separate chemically defined (hydrolysate-free and serum-free)
media. The first medium contained about 75 mM amino acids (Medium
1), the second medium contained about 40 mM amino acids (Medium 2),
and both formulations contained no more than 2.5 .mu.M (0.4 mg/L)
putrescine. Another group of medium conditions was generated by
adding soy hydrolysate at a concentration of 7.5 g/L to Medium 2.
To each of the three control media, about 593 .mu.M ornithine (as
100 mg/L L-ornithine.HCl), or a combination of about 593 .mu.M
ornithine (as 100 mg/L L-ornithine.HCl) and about 714 .mu.M
putrescine (as 115 mg/L putrescine.2HCl) were added. Aliquots of 3
mL culture were removed on days 3, 5, and 7 and viable cell counts
were conducted using trypan blue exclusion on a BioProfile FLEX.TM.
instrument (Nova Biomedical). At day zero, all cultures contained
0.8.times.10.sup.6 viable cells per mL. For a given medium (Medium
1, Medium 2, or Medium 2+Soy), viable cell counts over a seven-day
period revealed that CHO cells grown in media supplemented with
ornitihine or ornithine plus putrescine had increased viable cell
densities. The effect was especially pronounced in the hydrolysate
free media (i.e., 2-fold to 4-fold or more increase in viable cell
density) during the seven-day period. Hydrolysate free OS Medium 2
performed comparably to soy containing non-OS Medium 2 indicating
that the cell growth benefit of soy hydrolysate can be replicated
by ornithine replacement. Increased cell density by adding
ornithine or ornithine and putrescine to Medium 2+ soy was also
observed. Results are presented in Table 1.
TABLE-US-00001 TABLE 1 AVERAGE VIABLE CELL CULTURE DENSITY
(10.sup.6 CELLS PER MILLILITER) AND X FOLD INCREASE OVER BASELINE*
Ornithine + Supplement: Time Unsupplemented Ornitihine putrescine
Medium 1 3 days 2.4/1X 6.1/2.5X 5.0/2.1X 5 days 3.4/1X 12.6/3.7X
12.4/3.6X 7 days 3.6/1X 7.0/1.9X 6.8/1.9X Medium 2 3 days 1.7/1X
5.1/3.0X 5.2/3.1X 5 days 2.0/1X 7.6/3.8X 8.0/4.0X 7 days 1.6/1X
5.9/3.7X 5.8/3.6X Medium 2 + 3 days 5.2/1X 5.4/1X.sup. 4.7/0.9X soy
5 days 7.7/1X 9.3/1.2X 9.3/1.2X hydrolysate 7 days ND 9.6/ND 9.1/ND
*Base line is unsupplemented media for a given medium formulation
on a given day.
[0090] We also examined the effect of various amounts of
ornithine.HCl (i.e., 50 mg/mL, 100 mg/mL, and 150 mg/mL) on viable
cell density in Medium 3, which contains about 75 mM of amino acids
and 0.4 mg/L putrescine HCl ("Medium 3"). A single seed train
culture of a recombinant antibody producing cell line derived from
CHO K1 was used to inoculate 50 mL TubeSpin.RTM. Bioreactors (TPP)
at 0.4.times.10.sup.6 cells/mL at a 15 mL working volume. The cells
were grown in a 37.degree. C. incubator for three days. Aliquots of
3 mL culture were removed on day 3 and viable cell counts were
conducted using trypan blue exclusion on a BioProfile FLEX.TM.
instrument (Nova Biomedical). All three levels of ornithine
improved cell density on average (N=3) by slightly more than two
fold. The results are depicted in Table 2.
TABLE-US-00002 TABLE 2 VIABLE CELL CULTURE DENSITY (10.sup.6 CELLS
PER MILLILITER) AND X FOLD INCREASE OVER BASELINE* Control
Ornithine HCl Concentration 0 50 100 150 (mg/mL) Viable cell 1.3
3.2 3.1 3.1 density (10.sup.6 cells/mL) Fold increase 1X 2.5X 2.4X
2.4X over control *Base line is unsupplemented Medium 3.
Example 2
Improved Cell Culture Doubling Time
[0091] The doubling time of a recombinant antibody producing cell
line derived from CHO K1 cells in logarithmic growth phase was
determined under various cell culture media conditions. Seed train
cultures were passaged at 36.5.degree. C. in 250 mL shaker flasks
over a period of 14 days in each of three separate media: Medium 1,
Medium 2, and Medium 2 containing soy hydrolysate (Medium 2+Soy).
Aliquots of 1 mL were removed from each condition on Day 0 and at
the time of seed train passage (every 2 or 3 days), and viable cell
counts were conducted using trypan blue exclusion on a CDV.TM.
instrument (Nova Biomedical). Medium 1 was tested unsupplemented or
supplemented with ornithine.HCl at 100 mg/L or both putrescine.2HCl
at 115 mg/L and ornithine.HCl at 100 mg/L. Medium 2 with low
putrescine.2HCl (0.4 mg/L) was tested unsupplemented or
supplemented with ornithine.HCl at 100 mg/L or both putrescine.2HCl
at 115 mg/L and ornithine.HCl at 100 mg/L. The results are depicted
in Tables 3 and 4. Ornithine supplementation, either with or
without putrescine, to Medium 1 was required to achieve significant
growth. Supplementing hydrolysate free Medium 2 with ornithine or
ornithine+putrescine decreased the cell doubling time by about 25%
to 30%. Doubling times were also reduced to a lesser extent upon
the addition of ornithine or ornithine+putrescine to hydrolysate
containing Medium 2.
TABLE-US-00003 TABLE 3 CELL DOUBLING TIME (HOURS) AND APPROXIMATE
PERCENT DOUBLING TIME DECREASE RELATIVE TO BASELINE* IN MEDIUM 1
Supplement Medium 1 *None 75 Ornithine 23 69% Putrescine + 21 72%
ornithine *Baseline is unsupplemented Medium 1.
TABLE-US-00004 TABLE 4 CELL DOUBLING TIME (HOURS) AND APPROXIMATE
PERCENT DOUBLING TIME DECREASE RELATIVE TO BASELINE* IN MEDIUM 2
Supplement Medium 2 Medium 2 + Soy *Unsupplemented 27 22.5
Ornithine 21 22% 20.5 8.9% Putrescine + 19.5 28% 21 6.7% ornithine
*Baseline is unsupplemented Medium 2.
Example 3
Improved Antibody Titers
[0092] Having established that the inclusion of ornithine or
ornitihine+putrescine improves cell proliferation and viable cell
density in culture, we further investigated the effect of those
conditions on recombinant protein production titers. We examined
the expression and secretion of recombinant IgG by a CHO-K1 derived
cell line. In this experiment, the average antibody titer was
determined at day seven in culture under various media formats. As
above, Medium 1 with low putrescine (0.4 mg/L putrescine.2HCl),
ornithine (100 mg/L ornithine.HCl), and both ornithine and
putrescine (100 mg/L ornithine.HCl/115 mg/L putrescine.2HCl) were
tested. Medium 2 and Medium 2+Soy with low putrescine (0.4 mg/L
putrescine.2HCl), ornithine (100 mg/L ornithine.HCl), and both
ornithine and putrescine (100 mg/L ornithine.HCl/115 mg/L
putrescine.2HCl) were also tested. In all cases, the inclusion of
ornithine or ornithine and putrescine at a level above 0.4 mg/L
resulted in a significantly larger protein titer, i.e., at least
about two-fold higher titers. The results are depicted in Table
5.
TABLE-US-00005 TABLE 5 AVERAGE SEVEN-DAY ANTIBODY TITERS AND
APPROXIMATE FOLD INCREASE (X) IN TITER RELATIVE TO BASELINE* Medium
Medium Medium Supplement 1 2 2 + Soy Unsupple- 0.31 g/L .sup. 1X
0.29 g/L .sup. 1X 0.54 g/L 1X mented* Ornithine 0.94 g/L 3.0X 0.65
g/L 2.2X 0.98 g/L 1.8X.sup. Putrescine + 0.95 g/L 3.1X 0.64 g/L
2.2X 1.07 g/L 2X ornithine *Baseline is set at titer in for
unsupplemented media of each type.
[0093] We also examined the effect of various amounts of
ornithine.HCl (i.e., 50 mg/mL, 100 mg/mL, and 150 mg/mL) in Medium
3 on antibody production. A single seed train culture of a
recombinant antibody producing cell line derived from CHO K1 was
used to seed 50 mL TubeSpin.RTM. Bioreactors (TPP) at
0.4.times.10.sup.6 cells/mL at a 15 mL working volume. The cells
were grown in a 37.degree. C. incubator for three days. All three
levels of ornithine supplementation improved antibody titer on the
average (N=3) by slightly more than 50%. The results are depicted
in Table 6.
TABLE-US-00006 TABLE 6 ANTIBODY TITERS (GRAMS PER LITER) AND X FOLD
INCREASE IN TITER OVER BASELINE* Control Ornithine HCl Supplement
concentration 0 50 100 150 (mg/mL) Antibody titer 79 120 127 124
(mg/mL) Fold increase over control 1X 1.5X 1.6X 1.6X *Base line is
unsupplemented Medium 3.
* * * * *