Search Images Maps Play YouTube News Gmail Drive More »
Sign in
Screen reader users: click this link for accessible mode. Accessible mode has the same essential features but works better with your reader.

Patents

  1. Advanced Patent Search
Publication numberUSRE41974 E1
Publication typeGrant
Application numberUS 11/640,428
Publication dateNov 30, 2010
Filing dateDec 15, 2006
Priority dateOct 17, 1990
Also published asCA2053586A1, CA2053586C, DE69133303D1, DE69133303T2, DE69133589D1, DE69133589T2, EP0481791A2, EP0481791A3, EP0481791B1, EP1221476A2, EP1221476A3, EP1221476B1, EP1849862A2, EP1849862A3, US5316938, US5633162, USRE39792
Publication number11640428, 640428, US RE41974 E1, US RE41974E1, US-E1-RE41974, USRE41974 E1, USRE41974E1
InventorsMichael John Keen, Nicholas Timothy Rapson
Original AssigneeGlaxosmithkline Llc
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Method for culturing Chinese hamster ovary cells
US RE41974 E1
Abstract
A biochemically defined culture medium for culturing engineered Chinese hamster ovary (CHO) cell lines, which is essentially free from protein, lipid and carbohydrate isolated from an animal source, having water, an osmolality regulator, a buffer, an energy source, amino acids including L-glutamine, an inorganic or recombinant iron source, and a synthetic or recombinant growth factor, and optionally non-ferrous metal ions vitamins and cofactors. Also cells adapted to grow in such a culture medium.
Images(2)
Previous page
Next page
Claims(41)
1. A method for growing genetically engineered CHO cells which comprises in suspension comprising the step of:
culturing genetically engineered CHO cells in suspension under cell growing conditions effective to support secretion of a product from the genetically engineered CHO cells over multiple passages for at least 20 days, wherein the cell growing conditions are characterized as:
in the absencebeing free of both serum and transferrin; and
incomprising a medium comprising water, an osmolality regulator, a buffer, an energy source. L-glutamine and at least one additional amino acid, an inorganic, organic or recombinant iron source and a recombinant or synthetic growth factor wherein each component of said medium is obtained from a source other than directly from an animal source.
2. A The method for culturing growing CHO cells in accordance with claim 1, wherein the medium further comprises at least one component selected from the group consisting of: non-ferrous metals, vitamins or and cofactors.
3. A The method for culturing growing CHO cells in accordance with claim 1, wherein the osmolality regulator maintains the medium at 200-350 mOsm.
4. A The method for culturing growing CHO cells in accordance with claim 1, wherein the medium is maintained at a pH in the range of about 6.5 to about 7.5 by the buffer.
5. A The method for culturing growing CHO cells in accordance with claim 1, wherein the concentration of the energy source is within the range of 1000-10,000 mg/liter.
6. A The method for culturing growing CHO cells in accordance with claim 5, wherein the energy source is a monosaccharide.
7. A The method for culturing growing CHO cells in accordance with claim 1, wherein the additional amino acids are selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine cysteine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.
8. A The method for culturing growing CHO cells in accordance with claim 1, wherein the concentration of L-glutamine is within the range of 400-600 mg/liter.
9. A The method for culturing growing CHO cells in accordance with claim 2, wherein the medium further comprises a lipid factor in an amount of 0.05-10 mg/liter.
10. A The method for culturing growing CHO cells in accordance with claim 1, wherein the iron source is an inorganic ferric or ferrous salt which is provided in a concentration of from 0.25-5 mg/liter.
11. A The method for culturing growing CHO cells in accordance with claim 1, wherein the growth factor comprises is selected from the group consisting of: recombinant or synthetic insulin, platelet derived growth factor, thyroxine T3, thrombin, interleukin, progesterone, hydrocortisone or and vitamin E.
12. A The method for culturing growing CHO cells in accordance with claim 11 1, wherein the growth factor is recombinant or synthetic insulin.
13. A The method for culturing growing CHO cells in accordance with claim 1, wherein the medium further comprises at least one component selected from the group consisting of: a peptide digest, peptide hydrolysate or and peptide extract.
14. A method for culturing cells in accordance with claim 1, A method for growing genetically engineered CHO cells in suspension, comprising the step of:
culturing genetically engineered CHO cells in suspension under cell growing conditions effective to support secretion of a product from the genetically engineered CHO cells over multiple passages for at least 20 days, wherein the cell growing conditions are characterized as:
being free of both serum and transferring; and
comprising a medium comprising water, an osmolality regulator, a buffer, an energy source, L-glutamine, and at least one additional amino acid, an inorganic, organic or recombinant iron source and a recombinant or synthetic growth factor, wherein each component of said medium is obtained from a source other than directly from an animal source, wherein the medium is essentially free of hypoxanthine and thymidine.
15. A The method for culturing growing CHO cells in accordance with claim 14, wherein the medium further comprises methotrexate.
16. A method for culturing growing genetically engineered CHO cells which comprises in suspension comprising the step of:
culturing and growing Chinese hamster ovary genetically engineered CHO cells in suspension under cell growing conditions effective to support secretion of a product from the genetically engineered CHO cells over multiple passages for at least 20 days, wherein the cell growing conditions are characterized as:
in the absencebeing free of both serum and transferrin;
incomprising a medium comprising:
an osmolality regulator to maintain the osmolality of the medium within the range of about 200-350 mOsm,
a buffer to maintain the pH of the medium within the range of about 6.5 to 7.5,
about 1000-10,000 mg of a monosaccharide,
about 400-600 mg of L-glutamine,
about 10-200 mg of at least one amino acid selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine cysteine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine,
about 0.25-5 mg of an inorganic or recombinant iron source,
about 5 μg-5 mg of a recombinant or synthetic insulin,
and sufficient water to provide one liter of medium.
17. A method for culturing CHO cells which comprises culturing and growing Chinese hamster ovary cells in the absence of serum in a medium comprising
a base medium containing the amino acids, non-ferrous metal ions, vitamins and cofactors essentially as set forth in Table 1,
an osmolality regulator selected from NaCl, KCl, and KNO3 in an amount sufficient to maintain the osmolality of the medium within the range of about 200-350 mOsm,
at least one buffer selected from CaCl2.2H2O, MgSO4.7H2O, NaH2PO4.2H 2O, sodium pyruvate, N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulphonic acid (HEPES) and 3-[N-morpholino]-propanesulfonic acid (MOPS) in an amount sufficient to maintain the medium within the pH range of about 6.5-7.5,
about 1000-10,000 mg of mannose, fructose, glucose or maltose,
about 5 ml of 200 mM L-glutamine,
about 50 mg each of L-proline, L-threonine, L-methionine, L-cysteine and L-tyrosine,
about 20-50 mg of L-ascorbic acid,
about 0.01-0.5 mg each of Vitamin B6 and Vitamin B12,
about 0.25-5 mg of a ferric or ferrous salt,
about 1 mg of zinc sulfate,
about 2.5 μg of copper sulfate,
about 10,000-100,000 IU of at least one antibiotic selected from the group consisting of polymyxin, neomycin, penicillin and streptomycin,
about 3 μl of ethanolamine,
about 0.01-1.0 mg of putrescine,
about 5 μg-5 mg of recombinant insulin and sufficient water to comprise one liter of medium; wherein each component of said medium is obtained from a source other than directly from an animal source.
18. A The method for growing CHO cells in accordance with claim 1, wherein each component of the medium is selected from at least one source selected from the group consisting of: an inorganic, synthetic, recombinant, plant or and bacterial source.
19. A method for growing genetically engineered CHO cells in suspension, comprising the step of:
culturing genetically engineered CHO cells in suspension at a density greater than 1×10 5 cells/mL under cell growing conditions effective to support secretion of a product from the genetically engineered CHO cells over multiple passages for at least 20 days, wherein the cell growing conditions are characterized as:
being free of both serum and transferrin; and
comprising a medium comprising water, an osmolality regulator, a buffer, an energy source, L-glutamine and at least one additional amino acid, an inorganic, organic or recombinant iron source and a recombinant or synthetic growth factor, wherein each component of said medium is obtained from a source other than directly from an animal source.
20. The method for growing CHO cells in accordance with any one of claims 1 to 16, 18, or 19, wherein said culturing results in secretion of said product from the genetically engineered CHO cells into said medium.
21. The method according to any one of claims 1 to 16, 18 or 19, wherein said method results in growth of said genetically engineered CHO cells in suspension and secretion of said product over multiple passages for at least 30 days.
22. The method according to any one of claims 1 to 16, 18 or 19, wherein said method results in growth of said genetically engineered CHO cells in suspension and secretion of said product over multiple passages for at least 40 days.
23. The method according to any one of claims 1 to 16, 18 or 19, wherein said method results in growth of said genetically engineered CHO cells in suspension and secretion of said product over multiple passages for at least 50 days.
24. The method according to any one of claims 1 to 16, 18 or 19, wherein said method results in growth of said genetically engineered CHO cells in suspension and secretion of said product over multiple passages for at least 60 days.
25. The method according to any one of claims 1 to 16, 18 or 19, wherein said method results in growth of said genetically engineered CHO cells in suspension and secretion of said product over multiple passages for at least 70 days.
26. The method according to any one of claims 1 to 16, 18 or 19, wherein said method results in growth of said genetically engineered CHO cells in suspension and secretion of said product over multiple passages for at least 80 days.
27. The method according to any one of claims 1 to 16, 18 or 19, wherein said method results in growth of said genetically engineered CHO cells in suspension and secretion of said product over multiple passages for at least 6 months.
28. A method for growing genetically engineered CHO cells in suspension, comprising the step of:
culturing genetically engineered CHO cells in suspension at a density greater than 1×10 5 cells/mL under cell growing conditions effective to support secretion of a product from the genetically engineered CHO cells over multiple passages for at least 20 days, wherein the cell growing conditions are characterized as:
being free of both serum and transferrin; and
comprising a medium comprising water, an osmolality regulator, a buffer, an energy source, L-glutamine and at least one additional amino acid, an inorganic, organic or recombinant iron source and a recombinant or synthetic growth factor, wherein each component of said medium is obtained from a source other than directly from an animal source,
wherein said culturing results in secretion of at least 30 mg/L of said product from the genetically engineered CHO cells into said medium.
29. The method according to claim any one of claims 1 to 16, 18, 19, or 28, wherein said genetically engineered CHO cells are dhfr CHO cells.
30. The method for growing CHO cells in accordance with any one of claims 14, 16, 19, or 28 wherein the medium further comprises at least one component selected from the group consisting of: non-ferrous metals, vitamins and cofactors.
31. The method for growing CHO cells in accordance with any one of claims 14, 19, or 28, wherein the osmolality regulator maintains the medium at 200-350 mOsm.
32. The method for growing CHO cells in accordance with any one of claims 14, 19, or 28, wherein the medium is maintained at a pH in the range of about 6.5 to about 7.5 by the buffer.
33. The method for growing CHO cells in accordance with any one of claims 14, 19, or 28, wherein the concentration of the energy source is within the range of 1000-10,000 mg/liter.
34. The method for growing CHO cells in accordance with claim 33, wherein the energy source is a monosaccharide.
35. The method for growing CHO cells in accordance with any one of claims 14, 19, or 28, wherein the additional amino acids are selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.
36. The method for growing CHO cells in accordance with any one of claims 14, 19, or 28, wherein the concentration of L-glutamine is within the range of 400-600 mg/liter.
37. The method for growing CHO cells in accordance with any one of claims 14, 16, 19, or 28, wherein the medium further comprises a lipid factor in an amount of 0.05-10 mg/liter.
38. The method for growing CHO cells in accordance with any one of claims 14, 19, or 28, wherein the iron source is an inorganic ferric or ferrous salt which is provided in a concentration of 0.25-5 mg/liter.
39. The method for growing CHO cells in accordance with any one of claims 14, 19, or 28, wherein the growth factor is selected from the group consisting of: recombinant or synthetic insulin, platelet derived growth factor, thyroxine T3 , thrombin, interleukin, progesterone, hydrocortisone and vitamin E.
40. The method for growing CHO cells in accordance with any one of claims 14, 19, or 28, wherein the growth factor is recombinant or synthetic insulin.
41. The method for growing CHO cells in accordance with any one of claims 14, 16, 19, or 28, wherein the medium further comprises at least one component selected from the group consisting of: a peptide digest, peptide hydrolysate and peptide extract.
Description

This is a continuation of application Ser. No. 07,991,717 filed Dec. 18, 1992, now U.S. Pat. No. 5,316,938 which is a continuation of Ser. No. 07/777,729, filed Oct. 16, 1991, now abandoned.Notice: more than one reissue application has been filed for reissue of U.S. Pat. No. 5,633,162. The reissue applications are application Ser. No. 11/640,428 (the present application), filed on 15 Dec. 2006; and application Ser. No. 10/995,010, filed on 22 Nov. 2004, now RE 39.792.

The present reissue application is a continuation of application Ser. No. 10/995,010, now Reissue No. RE 39,792, filed 22 Nov. 2004, which is a reissue application of U.S. Pat. No. 5,633,162, U.S. Pat. No. 5,633,162, filed as application Ser. No. 08/205,379 on 4 Mar. 1994, is a continuation of U.S. Ser. No. 07/991,717 filed 18 Dec. 1992, now U.S. Pat. No. 5,316,938, which is a continuation of U.S. Ser. No. 07/777,729 filed 16 Oct. 1991, now abandoned.

The present invention relates to a biochemically defined culture medium for culturing Chinese hamster ovary (CHO) cell lines and cells adapted to grow in the culture medium.

Chinese hamster ovary cells (CHO) were first cultured by Puck (J. Exp. Med. 108, 945, 1958) from a biopsy of an ovary from a female Chinese hamster. From these original cells various workers have cloned a number of sub-lines with various deficiencies, one of which, CHO-K1, is proline-requiring and is diploid for the dihydrofolate reduotase (dhfr) gene. From this cell line a dhfrCHO cell line (CHO DUK B11) was developed (PNAS 77, 1980, 4216-4220) which is characterised by the loss of dhfr function as a consequence of a mutation in one dhfr gene and the subsequent loss of the other gene. These cells are functionally dhfr. Other OHO DUK sub-lines have been derived which are also phenotypically dhfr. CHO cells which are dhfrcannot grow without nucleotide precursors such as thymidine, hypoxanthine, or the equivalent nucleosides.

Various proteins have been expressed in such CHO cells including E. coli XGPRT gene (J. Mol. App. Gen. 1981, 1, 165-175), human tissue-type plasminogen activator (Mol. & Cell Biol. 5, 170-1759, 1985), human immune (γ) interferon (PNAS 80 pp 4654-4658), and human beta interferon (Molecular and Cellular Biology 4, 166-172, 1984). A dhfrCHO cell line is transfected with a product gene and a dhfr gene which enables selection of CHO cell transformants of the dhfr+ phenotype. Selection is carried out by culturing the colonies in media devoid of thymidine and hypoxanthine, the absence of which prevents untransformed cells from growing. The transformants usually express low levels of the product gene by virtue of co-integration of both transfected genes. The expression levels for the product gene may be increased by amplification using methotrexate. This drug is a direct inhibitor of the dhfr enzyme and allows isolation of resistant colonies which have amplified their dhfr gene copy number sufficiently to survive under these conditions. Since the dhfr and product genes are usually closely linked in the original transformants, there is normally concomitant amplification resulting in increased expression of the desired product gene.

A different system of selection and amplification is provided by the glutamine synthetase selectable marker (or GS system) which is described in WO87/04462. CHO cells which have been successfully transfected with the gene encoding the GS enzyme and the desired antibody gene can be selected by culturing colonies in media devoid of glutamine and amplifying by the addition of methionine sulphoximine (Msx) as described in PCT published application number WO87/04462.

Engineered CHO cells (those in which a CHO cell line is transfected with a product gene and a selectable marker gene) are routinely grown in culture media containing serum, (References: J. Mol. App. Gen. 1981, 1, 165-175; Mol. & Cell Biol. 1985, 5, 1750-1759, 1985 ; PNAS 1983, 80, pp 4654-4658; Molecular and Cellular Biology 1984, 4, 166-172, 1984 ). Fetal bovine serum (FBS) is probably the most extensively utilised serum for mammalian cell culture, although other mammalian sera are used. However, the use of serum poses a number of problems. Serum is an expensive commodity which is not readily available in amounts required for commercial production. It is also a biochemically undefined material. Serum is known to contain many major components including albumin and transferrin and also minor components many of which have not been fully identified nor their action determined, thus serum will differ from batch to batch possibly requiring testing to determine levels of the various components and their effect on the cells. Frequently, serum is contaminated with microorganisms such as viruses and mycoplasma many of which may be harmless but will represent an additional unknown factor. This problem has become more acute in recent years with the emergence of Bovine Spongiform Encephalopathy (BSE). Despite improvements in screening, regulatory authorities are likely to require the sourcing of bovine products from those areas which are free from (BSE) infections. Furthermore, the presence of animal proteins in culture media can require lengthy purification procedures, in particular the presence of bovine antibodies in bovine serum albumin (BSA) makes purification of the desired antibodies expressed by the recombinant CHO cell line, extremely difficult. Removal of bovine antibody from the medium prior to use is possible but this and the additional product testing required, adds greatly to the everall overall cost of production of the product. Consequently, there has been much research into finding a culture medium devoid of animal components which will support cellular growth, especially of CHO cells. Unfortunately, the problems associated with the provision of such a medium are themselves numerous. CHO cells do not readily grow in serum-free conditions. In addition, the removal of serum may also remove those components that provide cell protection and detoxifying activity.

A culture medium which is serum-free but not free from animal components is described by Mendiaz et al (In Vitro Cellular & Development Biology Vol.22, No.2, 1986) for use in the culture of CHO K1 cells. The medium is a modification of the medium developed by Ham (Microbiology 53 1965 288-293) which is known as “Ham's F12”. Other examples of media have been based on Ham's F12 medium for example as disclosed in EPA390327 and EP325190. These media contain transferrin as the serum substitute, but transferrin is derived from an animal source, so the resulting media do not overcome the contamination problems associated with the use of serum.

A further problem which arises with the use of serum-free media is that of supporting recombinant CHO cells to enable growth and expression of product. Media based on Ham's F12 which are not supplemented with serum are generally not rich enough to support full growth or expression.

Engineered CHO cells are also difficult to grow in suspension. It is highly desirable to achieve growth in suspension when using the cells to express a product such as an antibody. For production of a biological protein on a commercial scale it is preferable to be able to support growth in fermenters fermentors which range from 1 liter glass vessels to multi-thousand liter stainless steel tanks. A suitable medium must be able to support the cells against sheer forces from blade impellers or turbines and from effects of sparging (ie: supplying air, oxygen and CO2 in bubble form directly to the medium).

The present invention therefore provides a biochemically defined culture medium for culturing engineered CHO cells which is essentially free from protein, lipid and carbohydrate isolated from an animal source, comprising water, an osmolality regulator, a buffer, an energy source, amino acids including L-glutamine, an inorganic or recombinant iron source and a recombinant or synthetic growth factor and optionally non-ferrous metal ions, vitamins and cofactors.

The components of the medium are mostly inorganic, synthetic or recombinant and as such are not obtained directly from any animal source. Some components may be obtained from a plant or bacterial source. Recombinant components are prepared under highly pure conditions to minimise the risk of contamination from the parent tissue passing to the cells used to produce the components. Further purification steps may be employed to remove cell proteins. Thus, a medium which is essentially free from all protein, lipid and carbohydrate isolated from an animal source, can be achieved. The preferred culture medium of the invention contains no protein, lipid and carbohydrate isolated from an animal source.

It is advantageous to maintain osmolality in the range 200-30 milli-Osmols (mOsm) preferably in the range 290-350 mOsm. Osmolality regulators are generally salts. Those which may be used in the medium include NaCl, KCl, KNO3.

Buffers of use in the medium to maintain the pH in the range 6.5-7.5 most preferably around pH 7.0. Buffers of use in the medium include carbonates such as NaHCO3; also chlorides, sulphates and phosphates such as CaCl22H2O, MgSO47H2O, NaH2PO42H2O, or sodium pyruvate, such buffers are generally present in an amount 50-500 mg/liter. Other buffers, such as N-[2-hydroxyethyl]piperazine-N′-[2-ethanesul-phonic acid] otherwise known as HEPES and 3-[N-Morpholino]-propanesul-fonic acid otherwise known as MOPS are generally present in an amount 1000-10,000 mg/liter.

The energy source of use in the medium is generally present in an amount 1000-10,000 mg/liter and is preferably a monosaccharide such as manose, fructose, galactose or maltose most preferably glucose, particularly D-glucose.

The non-ferous non-ferrous metal ions optionally of use in the medium include magnesium, copper and zinc; also sodium, potassium and selenium. The ions are generally added to the medium in the form of salts such as chlorides and sulphates sulfates. The amounts are typically similar to those provided in the ISCOVES medium set out in Table 1 but clearly may be varied.

Vitamins and enzyme co-factor vitamins (co-factors) optionally of use in the medium include Vitamin B6 (pyridoxine). Vitamin B12 (cyanocobalamin) and Vitamin K (biotin) present in an amount 0.01-0.5 mg/liter; Vitamin C (ascorbic acid) present in an amount 10-30 mg/liter. Vitamin B2 (riboflavin) present in an amount 0.1-1.0 mg/liter and Vitamin B1 (thiamine), nicotin amide. Vitamin B5 (D calcium pentothenate), folic acid, i-inositol generally present in an amount 0.2-8.0 mg/liter.

It is preferable to include in the basal medium a lipid factor such as choline chloride, lipoic acid, oleic acid, phosphatidylcholine or methyl lineoleate, generally in an amount 0.05-10 mg/liter. Compounds involved in lipid production for example alcoholamines such as ethanolamine may also be added.

It is preferable to include additional amino acids in the medium selected from:

Amino Acid Preferred mg/liter
L-Alanine 20-50
L-Arginine (HCl)  50-100
L-Asparagine (H2O) 20-50
L-Aspartic Acid 20-50
L-Cystine (disodium salt)  50-100
L-Glutamic acid  50-100
L-Glutamine 400-600
Glycine 20-50
L-Histidine (HCl•H2O) 30-60
L-Isoleucine  50-150
L-Leucine  50-150
L-Lysine (HCl) 100-200
L-Methionine 20-50
L-Phenylalanine 40-80
L-Proline 30-60
L-Serine 30-60
L-Threonine  50-120
L-Tryptophan 10-20
L-Tyrosine (disodium salt)  50-120
L-Valine  80-120

The bracketed forms are preferred.

The amino acids are preferably of synthetic origin. The amounts which are usually included vary for each amino acid but are generally in the range 10-150 mg/ml. However, L-glutamine is generally present at much higher concentration preferably in the range 400-600 mg/ml mg/liter.

It may be advantageous to include in the medium a pH indicator for example Phenol red sodium salt for example at 5-50 mg/liter.

Medium A as set out in Table 1, is an example of a medium which provides the preferred quantities of water, osmolality regulator, buffer, energy source, amino acids, non-ferrous metal ions, vitamins and co-factors as a basis for a culture medium according to the invention. This medium does not contain any hypoxanthine or thymidine and is commercially available from GIBCO Ltd., Unit 4, Cowley Mill Td. Est., Uxbridge UB8 2YG. It is similar to a published culture medium (Iscoves and Melcher (1978) J. Exp. Med. 1, 47,923) but does not contain any bovine serum albumin, pure human transferrin or soyabean lecithin.

TABLE 1
Medium A (modification of Iscoves' DMEM lacking albumin,
transferrin and lecithin)
Ingredient mg/liter
L-Alanine 25.00
L-Arginine HCl 84.00
L-Asparagine H2O 28.40
L-Aspartic Acid 30.00
L-Cystine 70.00
L-Glutamic acid 75.00
L-Glutamine 584.00
Glycine 30.00
L-Histidine HCl•H2O 42.00
L-Isoleucine 105.00
L-Leucine 105.00
L-Lysine HCl 146.00
L-Methionine 30.00
L-Phenylalanine 66.00
L-Proline 40.00
L-Serine 42.00
L-Threonine 95.00
L-Tryptophan 16.00
L-Tyrosine disodium salt 104.00
L-Valine 94.00
Biotin 0.013
D.Calcium Pantothenate 4.00
Choline chloride 4.00
Folic acid 4.00
i-Inositol 7.20
Nicotinamide 4.00
Pyridoxal HCl 4.00
Riboflavin 0.40
Thiamin HCl 4.00
Vitamin B 12 0.013
CaCl22H2O 219.00
KCl 330.00
KNO3 0.076
MgSO47H2O 200.00
NaCl 4505.00
NaHCO3 3024.00
NaH2PO42H2O 141.30
D-Glucose 4500.00
HEPES 5958.00
Phenol red sodium salt 15.00
Sodium pyruvate 110.00
Sodium selenite 0.017

DMEM modification of Iscoves N and Melcher (1978), J. Exp. Med. 1, 47, 923.

It is preferable to add to the medium, selenium (optionally in the form of sodium selenite) generally in an amount 0.01-0.2 mg/liter or L-Ascorbic acid generally in an amount 20-50 mg/liter to help minimise the potential toxic effects of ferrous or ferric ions, and oxygen. Further use of chelating agents such as citrate or Ethylenediaminetetraacetic acid (EDTA) or a free radical scavenger such as α-Tocepherol (vitamin E) are advantageous in reducing free radical damage.

Antibiotics such as polymyxin, neomycin, penicillin or streptomycin may be added to-the medium to prevent bacterial contamination. These are usually included in an amount 10,000-100,000 Iu/liter.

Growth factors which may be added to the basal medium are synthetic or recombinant and include insulin. Other factors such as platelet-derived growth factor (PDGF), thyroxtne thyroxine T3, thrombin, interleukins such as IL2 and IL6, progesterone, hydrocortisone and vitamin E may be included. Folic acid, vitamin B6 and vitamin B12 which are involved in the folate pathway may be added to enhance the growth of cells.

The peptide hormone insulin (which in the present context includes analogues thereof such as Nucellin® (recombinant insulin, Eli Lilly) is advantageously obtained by recombinant DNA techniques but is not isolated from an animal source. It is preferably added to the medium in an amount 5 μg-5 mg/liter. Nucellin is the preferred form of insulin for use in the invention.

The non-animal derived iron source to supplement the medium, is preferably inorganic and present in an amount 0.25-5 mg/liter. Examples include ferric and ferrous salts such as ferric citrate or ferrous sulphate. The chelated salts such as ferric citrate and ferric ammonium citrate are preferred. However, any iron source may be used which is not isolated from an animal source, for example, chemical iron chelators or recombinant protein iron carriers.

The concentration of ferric or ferrous ions should be carefully controlled as these may help generate superoxides and free radicals in the medium, which may damage not only the cells themselves, but medium components and the desired end product.

It is also preferable to add to the medium, a compound such as putrescine, advantageously as a salt such as HCl, which is known to play a role in maintaining the structure of the endoplasmic reticulum and to be required by certain CHO cell lines to support growth. Putrescine or a salt thereof is preferably added in an amount 0.01-1.0 mg/liter.

Serum-free media disclosed to date contain hypoxanthine or thymidine. This could bypass the selection pressure placed on the dhfr selection and amplification system as previously disclosed. The result may be loss of genetic material specifying the product and the dhfr genes. Therefore, In another aspect of the invention there is provided a culture medium for the growth of engineered dhfrCHO cells in accordance with the invention, essentially free from hypoxanthine and/or thymidine.

The culture medium of the present invention supports CHO cell growth and when supplemented with an appropriate agent such as methotrexate for the dhfr system usually in an amount 0.1-5.0 μM, (or MSX for the GS system), allow full selection pressure to be exerted on the cells. It will be understood that hypoxanthine and thymidine at concentrations which are insufficient to bypass selection of the dhfr system may be present in the medium, but the presence of these two nucleotide precursors is not preferred for use with the present invention.

In large scale fermenters fermentors, mammalian cells are particularly susceptible to sheer forces arising from the sparging of the vessel with gases and the mixing with the impeller. To minimise minimize the occurrence of cellular damage it is advantageous for the medium to contain a cell protectant such as polyethylene glycol, polyvinyl alcohols or pluronic polyols. Of these, Pluronic® (polyol, BASF Wyandotte Corp.) polyol F68 is preferred since unlike polyvinyl alcohols this is a non-toxic substance and unlike polyethylene glycols does not interfere with downstream purification.

Further improvements in CHO cell growth may be obtained by supplementing the medium with a peptide digest, hydrolysates or extracts, such as Tryprone, casein hydrolysate, yeast extract, or preferably papain digested soya peptone. The preferred amounts are 1%-0.025% w/v, most preferably 0.25% w/v.

The media of the invention for culturing recombinant CHO cells are capable of supporting the growth and secretion of product from such cells in suspension in small and large scale fermenters fermentors, static cultures and/or spinners. The culture medium according to the invention is also capable of supporting growth of cells at high cell density namely greater than 1×105 cells/ml up to or greater than 1.5×106 cells/ml and product secretion of 30 mg/l up to greater than 150 mg/l. The medium according to the invention is also capable of supporting this growth and product secretion over multiple passages lasting upto or greater than 6 months.

The medium is preferred for the production of all types of antibodies natural and altered. The invention therefore includes production of human antibodies wherein the amino acid sequences of the heavy and light chains are homologous with those sequences of antibodies produced by human lymphocytes in vivo or in vitro by hybridomas. Also provided are hybrid antibodies in which the heavy and light chains are homologous to a natural antibody but are combined in a way that would not occur naturally. For example, a bispecific antibody has antigen binding sites specific to more than one antigen. The constant region of the antibody may relate to one or other of the antigen binding regions or may be from a further antibody. Altered antibodies, for example chimaeric antibodies have variable regions from one antibody and constant regions from another. Thus, chimaeric antibodies may be species/species chimaeras or class/class chimaeras. Such chimaeric antibodies may have one or more further modifications to improve antigen binding ability or to alter effector functioning. Humanised Humanized or CDR-grafted antibodies (EP 239400) are embraced within the invention, in particular Campath 1H (EP328404) (Campath is a TM of The Wellcome Foundation) also composite antibodies, wherein parts of the hypervariable regions in addition to the CDRs are tranferred transferred to the human framework. Additional amino acids in the framework or constant regions of such antibodies may be altered. The invention further includes the production of Feb Fab fragments which are roughly equivalent to the Y branch portions of the heavy and light chains; this includes incomplete fragments or fragments including part of the Fc region.

In a further aspect of the invention there is provided an engineered CHO cell adapted to grow in a medium according to the invention. In particular a CHO cell engineered to express proteins such as tissue plasminogen activator or antibodies as defined above. In particular the invention provides a dhfr- CHO cell line transfected with a gene encoding a biologically active protein and a dhfr selectable marker gene, adapted to grow in a culture medium according to the invention. The protein is preferably an antibody as defined above.

The ingredients of the culture medium may be added in any order but it is preferable to add the iron source and when used, tyrosine, last to avoid precipitation.

Accompanying Figures are for illustration only.

FIG. 1 shows growth of C1H 3D11* 44 in WCM5 (protein-free medium) in a 1 liter fermenterfermentor measured as cell count/ml over 90 days.

FIG. 2 shows antibody production from C1H 3D11* 44 cells in WCM5 in a 1 liter formenterfermentor measured as micrograms of antibody/ml over 80 days.

EXAMPLE 1

Formulation for medium WCM4.

Medium A: (Iscoves modification of DMEM without BSA, transferrin and lecithin as set out in Table 1).

5 ml/liter+5 ml/liter 200 mM L glutamine
+50 mg/liter L proline
+50 mg/liter L threonine
+50 mg/liter L methionine
+50 mg/liter L cysteine
+50 mg/liter L tyrosine
+25 mg/liter ascorbic acid
+0.062 mg · liter vitamin B6
+1.36 mg · liter vitamin B12
+0.2 mg/liter lipoic acid
+0.088 mg/liter methyl linoleate
+1 mg · lit methotrexate
+1 IU/liter FeSO4
+1 IU/liter ZnSO4
+0.0025 μl/liter CuSO4
+5 mg/liter recombinant insulin (Nucellin)
+50,000 Iu/liter polymyxin
+20,000 Iu/liter neomycin
+0.16 mg/liter putrescine-2 HCL

This medium does not contain hypoxanthine, thymidine or folinic acid which can bypass methotrexate selection. The medium does contain glycine which cannot by itself bypass selection. Therefore, this medium maintains full selection for methotrexate resistance.

EXAMPLE 2

Formulation for Medium WGM5 WCM5

MedmiumMedium A: (Iscoves modification of DMEM without BSA, transferrin or lecithin).

+ 5 ml/liter 200 mM L glutamine
+ 50 mg/liter L proline
+ 50 mg/liter L threonine
+ 50 mg/liter L methionine
+ 50 mg/liter L cysteine
+ 50 mg/liter L tyrosine
+ 25 mg/liter L ascorbic acid
+ 0.062 mg · liter Vitamin B6
+ 1.36 mg · liter Vitamin B12
+ 2 mg/liter Ferric citrate
+ 1 mg/liter Zinc sulphate
+ 0.0025 mg · lit Copper sulphate
+ 50,000 IU/liter Polymyxin
+ 20,000 IU/liter Neomycin
+ 3 μl/liter Ethanolamine
+ 0.16 mg/liter Putrescine
+ 5 mg/liter Recombinant Insulin (Nucellin ®)

EXAMPLE 3

Growth of and Production from C1H 3D11* 44 in WCM4

C1H 3D11* cells are genetically engineered CHO DUK B11 cells (Urlaub and Chasin PNAS ( 1980,) PNAS 77, 7 pp 4216-4220). CHO DUK B11 cells cannot produce dihydrofolate reductase (dhfr). These cells were engineered to produce a humanised humanized IgG antibody, Campath 1H (Winter et al., Nature, 1988, 322, 323-327), using plasmid constructs to express heavy and light antibody chains and the mouse dhfr. Expression is amplified and maintained using the folate antagonist methotrate methotrexate. C1H 3D11* cells growing as a mono-layer in Isover+10% FBS Flow, non-essential amino acids, 10−6M Methotrexate and antibiotics were approximately 90% confluent. These cells were removed from the plastic with trypsin/versene, washed in Iscoves medium without supplements, centrifuged and resuspended at 5×104/ml in WCM4 medium+0.25% peptone+0.1% polyethylene glycol (PEG) 10,000+0.5% fetal bovine serum (FBS) without methotrexate (MTX). Three 25 cm 2 cm 3 flasks were set up with 10 ml of cell suspension+hypoxanthine (H), thymidine (T) or HT. These flasks were incubated at 36.5° C. in 5% CO2 incubator.

After six days, the flasks were pooled and added to an equal volume of WCM4+MTX without peptone or PEG, and were transferred to a 75 cm2 flask.

These cells were used to seed a 500 ml Techner spinner, incubated at 36.5° C. spinning at 40 rpm. Cells continued growing serum free for a period of over five months and although it was-found that the cells needed a period of adaptation, the growth rate and viability steadily improved. The population doubling time was calculated to be 73.1 hours over approximately 7 weeks; this decreased to 47.4 hours over the subsequent 20 days then stabilised. Antibody secretion remained high at levels in excess of 60 μg/ml. It was determined that the gene copy number in these cells did not decrease according to band intensity using Northern blot analysis.

In fermenters fermentors, these cells produced antibody in excess of 70 μg/ml and regularly achieved levels of 100 μg/ml or more. The cells are denoted C1H 3D11* 44.

EXAMPLE 4

Growth and Production of CIH C1H 3D11* 44 in WCM5 in a 1 liter fermenter fermentor.

C1H 3D11*44 cells from Example 3 which had been growing serum-free for over 2 months were transferred to a SGi 1 liter fermenter fermentor with a stainless steel angled paddle turning at 70 rpm. The temperature was set at 37° C., dO2 at 10% and pH control to 7-7.2. The fermenter fermentor was seeded on day 0 with 0.22×106 cells/ml in WCM4 (Example 1) with 0.1% polyethylene glycol (PEG) 10,000 and 0.25% soy peptone, and was top gassed with O2.The cells were routinely passaged using fresh medium and a split rate typically between 1 to 2 and 1 to 4.

On day 33 the top gassing was replaced with deep sparging which is can be expected to cause more physical damage to the cells.

On day 50 onwards WCM5 (Example 2) was used together with peptone and PEG instead of WCM4.

On day 53 the PEG was replaced with 0.1% Pluronic F68. The resulting growth and antibody levels achieved are shown in the the attached graphs (FIGS. 1 and 2), and demonstrate the capacity of the invention to allow protein-free production of antibody in excess of 100 μg/ml in fermenters fermentors.

EXAMPLE 5

Growth of CHO AJ19 MCB1 in WCM4 and compared to CHO AJ19 MCB1 grown in serum containing medium

Chinese hamster ovary cells, CHO AJ19 MCB1, derived from CHO DUK cells. (Urlaub & Chasin PNAS, 1980, 77, 7, pp 4216-4220, 1980 ), were genetically engineered to produce tPA under methotrexate selection. This cell line had been routinely grown in a fermenter fermentor as a suspension culture using normal growth medium consisting of RPMI 1640 medium (GIBCO), 2.5% acid hydrolysed hydrolyzed adult bovine serum (Imperial), 0.5% Tryptone, 50 IU/ml polymycin, 20 IU/ml neomycin, 500 nM methotrexate (MTX).

Medium WCM4 was formulated to which was added:

    • 46B 0.25% w/v N-Z Soy Peptone (Sigma P1265), 0.1% w/v Polyethylene glycol (PEG) 20,000 (Serva, Carbowax® 20M). 1 uM MTX.
    • 46C 0.25% w/v Yeast extract (Sigma Y0500), 0.1% w/v PEG 20,000 1 uM MTX. In this medium the Iscoves' in CM4 was replaced by RPMI 1640 medium (ICN FLOW).
    • 46D 0.25% w/v Yeast extract, 0.1% w/v PEG 20,000, 1 uM MTX.
    • 46E 0.25% w/v Yeast extract, 0.1% w/v PEG 20,000, 0.25% Fetal bovine serum (Imperial), 1 uM MTX.

The yeast extract, Peptone and PEG were made up as 10% w/v solutions with water (Wellcome media production unit) and filtered through a 0.2 um disposable filter (Gelman, Supor Vac), then diluted for use. The cells were incubated at 37° C. in a humidified incubator containing 5% CO2.

Cells growing in normal growth medium were pelleted by centrifugation at 1200 g +4° C. for 5 minutes, were washed in RPMI 1640 without supplements and pelleted again. The cells were then resuspended at 105 cell/ml in normal growth medium (46A) and the other media (46B, 46C, 46D or 46E). 24 well plates (Costar 16 mm wells) were seeded with 1 ml/well and incubated, at 37° C. in an incubator containing 5% CO2. On days 3, 4, 5 and 6 one well of each was counted using a haemcytometer and trypan blue exclusion. Two further wells of each were harvested, pooled and pelleted at 1200 g +4° C. 5 minutes. The supernatant was separated and stored at −20° C. These samples were subsequently assayed for tPA. On day 6 samples from 46A and 46D only were harvested.

RESULTS

tPA specific activities in various crude harvests

Crude material produced in the five different media were tested using a QA validated ELISA assay to measure the tPA antigen concentrations μg/ml using binding to a polyclonal antibody against tPA, and clot lysis assay to measure tPA activity in IU/ml. From these results (Table 2), the specific activities were calculated.

TABLE 2
MEAN tPA MEAN tPA
DAYS ACTIVITY CONTENT SPECIFIC
IN CELLCOUNT ×10−5 IU/ml ug/ml ACTIVITY
EXPERIMENT CULTURE VIABLE NONVIABLE (n = 3) (n = 3) MegIU/mg
46A 3 3.5 0.1 3051 10.51 0.290
46A 4 3.7 0.3 4841 14.85 0.326
46A 5 4.1 0.2 5306 15.52 0.335
46A 6 5.8 0.5 8235 23.22 0.355
46B 3 5.2 0.1 2552 10.44 0.244
46B 4 7.2 0.3 5310 18.58 0.286
46B 5 7.8 0.2 6230 22.19 0.281
46C 3 3.8 0.2 2779 9.61 0.289
46C 4 4.9 0.3 3536 16.54 0.214
46C 5 5.6 0.3 4639 19.88 0.233
46D 3 7.5 0.2 4650 17.66 0.263
46D 4 8.3 0.8 7369 25.99 0.285
46D 5 7.4 1.0 7882 24.26 0.325
46D 6 6.1 2.0 8095 27.06 0.299
46E 3 6.4 0.1 6262 23.85 0.263
46E 4 7.3 0.5 10180 29.70 0.343
46E 5 6.1 1.3 9080 34.25 0.265

From the above table there was no change of the specific activity in the five different crudes. The yield of tPA from protein free medium B, C and D was nearly equal to the yield of tPA from standard growth medium in group A and E.

Example 6 Continuous growth of CHO AJ19 MCBI in WCM4

CHO AJ19 MCBI in WCM4 cells growing in normal growth medium were pelleted and washed as in Example 5 and were resuspended at 7×104/ml in 500 ml of medium 46B. These cells were transferred to a Techne spinner flask and incubated, as above, stirring at 40 rpm. At various time intervals the cells were counted and subcultured using the same medium. A sample was taken for tPA assay and treated as in Example 5.

The specific activity of tPA in various cell subcultures

The specific activity of supernatants from different pass levels of cells grown in WCM4 with peptone and 0.1% PEG 20K were measured by a combination of ELISA and clot lysis assay. The specific activities of different cell passages are summarised in Table 3.

TABLE 3
tPA present in supernatant
tPA
conc. ACTIVITY SPECIFIC
CELLCOUNT ×10−5 SPLIT ug/ml IU/ml ACTIVITY
DAYS PASS VIABLE NONVIABLE RATE (n = 3) (n = 3) Meg.U/mg
7 1 9.75 0.65  1-10 ND ND ND
10 2 4.95 0.01 1-5 ND ND ND
13 3 6.35 0.0  1-10 22.2 8865 0.399
16 4 3.8 0.0  1-10 7.25 1914 0.264
21 5 7.2 0.8  1-10 15.08 4331 0.287
24 6 4.1 0.3  1-10 8.28 2040 0.246
30 7 5.3 0.4 1-6 7.30 2052 0.281
34 8 5.2 0.32 13.65 3518 0.256
36 8 7.95 0.10 1-8 18.60 5327 0.286
37 8 ND ND 20.68 5526 0.267
38 8 100% 19.10 5474 0.287
38 9 12.00 0.5 1-5 20.85 8348 0.400
43 10 5.5 0.12 1-5 7.38 1888 0.256
48 11 4.4 0.19 1-6 13.4 3143 0.235
12 Experiment terminated
ND = not done.

Over a 48 day period, base on the above split rate, one cell could have divided to give 3.77×108 cells. This is equivalent to 31.8 population doublings with a doubling time of 36 hours.

The results of the experiments conducted in Examples 5 and 6 demonstrate that the serum free media of the present invention is capable of supporting cell growth and tPA yield comparable to that achieved in serum containing media.

Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US4205126Dec 14, 1978May 27, 1980Cartaya Oscar ASerum-free cell culture media
US4657866Sep 13, 1984Apr 14, 1987Sudhir KumarSerum-free, synthetic, completely chemically defined tissue culture media
US4767704Oct 7, 1983Aug 30, 1988Columbia University In The City Of New YorkProtein-free culture medium
US4929706Mar 3, 1989May 29, 1990W. R. Grace & Co.-Conn.Cell growth enhancers and/or antibody production stimulators comprising chemically modified hydrophilic polyurea-urethane prepolymers and polymers
US5019499Apr 14, 1987May 28, 1991Daiichi Seiyaku Co., Ltd.Method of producing peptides by transforming myeloma cells with a recombinant plasmid
US5045468Aug 15, 1989Sep 3, 1991Cell Enterprises, Inc.Protein-free culture medium which promotes hybridoma growth
US5063157Jan 11, 1989Nov 5, 1991Boehringer Mannheim GmbhSerum-free culture medium for mammalian cells
US5122469 *Oct 3, 1990Jun 16, 1992Genentech, Inc.Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
US5135866Mar 3, 1989Aug 4, 1992W. R. Grace & Co.-Conn.Very low protein nutrient medium for cell culture
US5232848Nov 16, 1989Aug 3, 1993W. R. Grace & Co.-Conn.Basal nutrient medium for cell culture
US5316938Dec 18, 1992May 31, 1994Burroughs Wellcome Co.Defined media for serum-free tissue culture
US5545403Nov 23, 1993Aug 13, 1996Burroughs Wellcome Co.Method for treating a mammal by administering a CHO-glycosylated antibody
US5545404Nov 3, 1994Aug 13, 1996Burroughs Wellcome Co.Method for treating a mammal suffering from a T-cell medicated disorder with a CHO-Glycosylated antibody
US5545405Nov 3, 1994Aug 13, 1996Burroughs Wellcome Co.Method for treating a mammal suffering from cancer with a cho-glycosylated antibody
US5633162Mar 4, 1994May 27, 1997Glaxo Wellcome Inc.Method for culturing Chinese hamster ovary cells
US5807715Jun 27, 1994Sep 15, 1998The Board Of Trustees Of The Leland Stanford Junior UniversityMethods and transformed mammalian lymphocyte cells for producing functional antigen-binding protein including chimeric immunoglobulin
US5846534Apr 29, 1994Dec 8, 1998British Technology Group LimitedAntibodies to the antigen campath-1
US5876961Jan 26, 1995Mar 2, 1999Glaxo Wellcome Inc.Production of antibodies
US6100061Jun 19, 1998Aug 8, 2000Immuno AktiengesellschaftRecombinant cell clone having increased stability in serum- and protein-free medium and a method of recovering the stable cell clone and the production of recombinant proteins by using a stable cell clone
US20040259838Dec 6, 2002Dec 23, 2004Joyce Joseph G.Staphylococcus aureus exopolysaccharide and process
USRE40070May 27, 2005Feb 19, 2008Smithkline Beecham CorporationAntibody purification
EP0164813A2Jun 12, 1985Dec 18, 1985Teijin LimitedMethod of cultivating animal or plant cells
EP0229530A2Dec 30, 1986Jul 22, 1987Bass Public Limited CompanyThe propagation of yeast, fermentation employing that yeast and products of that fermentation
EP0239400A2Mar 26, 1987Sep 30, 1987Medical Research CouncilRecombinant antibodies and methods for their production
EP0248656A2Jun 3, 1987Dec 9, 1987Director-General of the Agency of Industrial Science and TechnologyComposition for cell cultivation and use thereof
EP0274394A2Jan 7, 1988Jul 13, 1988Xoma CorporationChimeric antibody with specificity to human B cell surface antigen
EP0307247A2Sep 12, 1988Mar 15, 1989Genentech, Inc.A method for culturing recombinant cells
EP0307248A2Sep 12, 1988Mar 15, 1989Genentech, Inc.Method for increasing the expression of polypeptides in recombinant cell culture
EP0314161A1Oct 28, 1988May 3, 1989Bristol-Myers Squibb CompanyHuman immunoglobulines produced by recombinant DNA techniques
EP0316068A1Oct 7, 1988May 17, 1989Collaborative Research Inc.Modified low molecular weight plasminogen activator and method of preparation
EP0325190A2Jan 16, 1989Jul 26, 1989Boehringer Mannheim GmbhPentosansulfate medium
EP0328404A1Feb 10, 1989Aug 16, 1989British Technology Group LimitedModified antibodies
EP0363703A1Sep 21, 1989Apr 18, 1990Kenneth AlonsoMethod of producing human-human hybridomas, the production of monoclonal and polyclonal antibodies therefrom, and therapeutic use thereof
EP0388151A1Mar 13, 1990Sep 19, 1990Celltech LimitedModified antibodies
EP0389786A1Feb 21, 1990Oct 3, 1990W.R. Grace & Co.-Conn.Very low protein nutrient medium for cell culture
EP0390327A2Feb 22, 1990Oct 3, 1990Eli Lilly And Companyimproved tissue culture method
EP0404003A2Jun 18, 1990Dec 27, 1990Xoma CorporationChimeric mouse-human KM10 antibody with specificity to a human tumor cell antigen
EP0481790A2Oct 17, 1991Apr 22, 1992The Wellcome Foundation LimitedAntibody production
EP0481791A2Oct 17, 1991Apr 22, 1992The Wellcome Foundation LimitedCulture medium for CHO-cells and adapted CHO-cells
EP0513738A2May 12, 1992Nov 19, 1992Boehringer Mannheim GmbhSerum-free medium for mammalian cells cultivation
EP0523949A1Jul 14, 1992Jan 20, 1993The Wellcome Foundation LimitedProduction of antibodies
EP0610447A1Oct 14, 1992Aug 17, 1994The Wellcome Foundation LimitedCDw52 - SPECIFIC ANTIBODY FOR TREATMENT OF T-CELL MEDIATED INFLAMMATION OF THE JOINTS
EP0822255A2Oct 17, 1991Feb 4, 1998The Wellcome Foundation LimitedCHO (Chinese Hamster Ovary) glycosylated antibodies and their use in therapy
FR2543158A1 Title not available
GB2196348A Title not available
JPS637780A Title not available
JPS6125480A Title not available
JPS63192381A Title not available
WO1987000195A1Jun 30, 1986Jan 15, 1987Celltech LimitedAnimal cell culture
WO1987001131A1Aug 18, 1986Feb 26, 1987Gene Labs, Inc.Non-human primate monoclonal antibodies and methods
WO1988000967A1Aug 4, 1987Feb 11, 1988The University Of New South WalesSerum free tissue culture medium containing polymeric cell-protective agent
WO1989000999A1Jul 25, 1988Feb 9, 1989International Genetic Engineering, Inc.Modular assembly of antibody genes, antibodies prepared thereby and use
WO1990003429A1Sep 13, 1989Apr 5, 1990Cetus CorporationLipid microemulsions for culture media
WO1991004336A1Sep 19, 1990Apr 4, 1991Centocor, Inc.Method for improving human monoclonal antibody production
WO1991010722A2Dec 27, 1990Jul 25, 1991Centocor, Inc.Chimeric immunoglobulin for cd4 receptors
WO1992007084A1Oct 17, 1991Apr 30, 1992The Wellcome Foundation LimitedPurified cdw52-specific antibodies
WO1993002108A1Jul 24, 1992Feb 4, 1993Idec Pharmaceuticals CorporationRecombinant antibodies for human therapy
WO1993007899A1Oct 14, 1992Apr 29, 1993The Wellcome Foundation LimitedCDw52 - SPECIFIC ANTIBODY FOR TREATMENT OF T-CELL MEDIATED INFLAMMATION OF THE JOINTS
WO2001051615A1Jan 12, 2001Jul 19, 2001Hypoxi Co. Ltd.Method for increasing survival rate of cells in animal cell culture under hypoxia condition
Non-Patent Citations
Reference
11990 GIBCO BRL Catalogue & Refernce Guide (confirmation of availability attached).
2Aathoon and Birch, Science, (1986) 232: 1390-1395.
3Abstract of the USPTO trademark database regarding the trademark for Nucellin of Eli Lilly.
4Ahmed, S. (2001) "Eating Human Hair by Another Name?," www.albalagh.net/hala/col2.shtml.
5Ahrens, et al., Post Graduate Medicine vol. 80, pp. 181-187 (1988).
6Alfred hahn's CV submitted in Opposition of EP0523949Email of Jan. 11, 2007 from Ingrid Haas submitted in Opposition of EP0523949.
7Alsmadi et al. "Antibody-Dependant Cellular Cytotoxicity Directed against Cells Expreesing Human Immunodeficiency Virus Type 1 Envelope of Primary or Laboratory-Adapted Strains by Human and Chimpanzee Monoclonal Antibodies of Different Eptope Specificities", Journal of Virology, 72(1),:286-293 (1998).
8Anthony Lubinicki, ESACT 9th Meeting, Editors Spier R.E. et al., pp. 85-92 (1989).
9Barnes, Biotechniques, (1987) vol. 5, No. 6, pp. 534-542.
10Bartholomew, et al. "functional analysis of the effects of a fully humanized anti-Cd4 antibody on resting and activated human T cells"Immunology 85:41-48 (1995).
11Bebbington et al., Methods: A companion to Methods in Enzymology, vol. 2(2) pp. 136-145 (1991) Abstract.
12Biech Z., reprinted with permission from MK News and Views, vol. IV (6) 2003.
13Bobbington, et al., Biotechnology, vol. 10 pp. 169-175 (1992).
14Bohak, et al., Biopolymers, (1987) 26:s205-213.
15Broad, et al., In Vitro Cellular and Developmental Biology, (1986) 22 (2):66-74.
16Brogden, et al., Drugs, vol. 34 pp. 350-371 (1987).
17Brown et al., Emerging Infectious Diseases, vol. 7 (1) pp. 6-16 (2001).
18Buck, et al., J. Virol. Meth., (1985) 10:171-184.
19Bulens, et al., Uer. J. Biochem., (1991) 195:235-242.
20 *Bullied, et al., Biochem Journal, vol. 268 pp. 777-781 (1990).
21C6852, Biochemicals and Reagents for Life Science Research, p. 600 (2002-2003) Sigma-Aldrich Company (Current website information also included).
22C7880, Biochemicals and Reagents for Life Science Research, p. 600 (2002-2003) Sigma-Aldrich Company (Current website information also included).
23C8503, Biochemicals and Reagents for Life Science Research, p. 508 (2002-2003) Sigma-Aldrich Company (Current website information also included).
24Carter et al., PNAS, vol. 89 pp. 4285-4289 (May 1992).
25Castro, et al., Bitoech Appl. BioChem (1985) 21:57-100.
26Chandler, Josseph P., Cultivation of mammalian cells in serum-free medium, American Biotechology Journal 8(1), pp. 18-28, Jan. 1990.
27Chotigeat, et al., Cytotechnology (1994) 15:217-221.
28Cini, et al., "Molecular Basic for the Isozymes of bovine Glucose-6-Phosphate Isomerase," Arch Biochem. Biophys. 263(1) 96-106.
29Colcher et al., Cancer Research, vol. 49 pp. 1738-1745 (1989).
30Colomb, et al., Boochem. J., vol. 145 pp. 177-183 (1985).
31Curling, Biochem. J., (1990) 272:333-337.
32 *Curling, et al., Biochem Journal, vol. 272 pp. 333-337 (1990).
33Dafler, F., In Vitro Cell Dev. Bio., vol. 26 pp 769-778 (1990).
34Darfler, In Vitro Cell. Dev. Bio., Vo 26 pp. 779-783 (1990).
35Decision of Appeal of the European Patent Office dated Jan. 15, 2004 T0962/98—3.3.1 Appl. No. 94931395.1.
36Decision of Boards of Appeal of the European Patent Office date Jan. 18, 2005 T0278/03—3.3.8 Appl. No. 91309595.6.
37Decision of Boards of Appeal of The European Patent Office dated Aug. 26, 2005 T0039/03—3.4.2 Appl. No. 99100131.4.
38Decision of Boards of Appeal of The European Patent Office dated Sep. 23, 2004 T0720/02—3.4.2 Appl. No. 97200957.5.
39Decision of Technical Boards of Appeal 3.4.2 dated Jul. 13, 2004 T1158/01—3.4.2 Appl. No. 99126075.3.
40Declaration of Dr. J Adair submitted in Opposition of EP0523949.
41DeCourcy et al., Experimental Cell Research, vol. 192 pp. 52-60 (1991).
42Deeds, et al., "Creating a New Medium to Meet the Variable Nutritional Requirements of Chinese Hamster Ovary (CHO) Cell Clones." Sigma Aldrich.com http://www.sigmaaldrich.com/sigma/general (Jul. 2003).
43DeWaele et al., European Journal of Biochemistry, vol. 176 (2-3) pp. 287-295 (1988).
44DiAugustine, et al. "Evidence of Isoaspartyl (Deamidated) Forms of Mouse Epidermal growth Factor." Anal. Biochem. (1987) 165(2):420-429.
45Dickman, Nature, vol. 329 p. 93 (1987).
46 *Dijkmans, et al., Journal of Biological Chemistry, vol. 262(6) pp. 2528-2535 (1987).
47Dulbecco and Freeman, Virology, vol 8 pp. 396-397 (1959) [composition of DMEM attached].
48Dyer et al., Blood, vol. 73 pp. 1431-1439 (1989).
49Dyer, et al., Leukemia and Lymophoma, (1989) 1:179-193.
50Eagle, Science, vol. 130 pp. 432-437 (1959) [composition of MEM attached].
51Ebert, Expression of Antibody C-DNA in CHO ("Chinese hamstet ovary") Cells, Dissertation Completed at the Institute for Applied Microbiology University for Soil Cultivation , Feb. 1991 (with Translation).
52Ehrlich et al., Human Antibod. Hybridomas, vol. 1(1) pp. 23-26 (1990).
53Ehrlich et al., Molecular Immunology, vol. 28(4-5) pp. 319-322 (1991).
54E-mail from Diane Fedyk in respect of GIBCO catalogue.
55English translation of Dissertation of Veronica Ebert Expression of Antibody c-DNA in CHO (Chinese Hamster Ovary) Cells University for Soil Conservation,Vienna Austria (1991).
56Enlarged version of figure from Hamilton et al., In Vitro, vol. 13 (9) pp. 537-547 (1977.
57EPO Communication in Opposition against European Patent No. 02 003 143.1, dated Jul. 21, 2010.
58Examination Report of Apr. 1, 2005.
59Expert declaration of Dr. Florian Ruker submitted in Opposition of EP0523949.
60Feldman, G., (Oct. 2001), "Amino Acid Production and the Associated Theoretical Risk of BSE Transmission from their Use in the Production of Biologicals, Drugs, and Medical Devices," FDA TSA Advisory Committee Meeting <www.fda.gov/Ohrms/dockets/ac/01/slides/.
61Felgenhauer, et al. "Nucleotide sequences of the cDNAs encoding the V-regions of H-and L-chains for a human monocalonal antibody specific to HIV-1-gp41," Nucleic Acids Research (1990) 18(16) 4927.
62 *Feys et al, Int J Cancer, 1988, Supplement 2, pp. 26-27.
63Feys et al., International Journal of Cancer, vol. 2 pp. 26-27 (1988).
64Feys, et al., Chemical Abstracts, vol. 108 (23) p. 514 (1988).
65Forthal et al., "Antibody from Patients with Acute Human Immunodefciancy Virus (HIV) Infection Inhibitis Primary Strains of HIV in the presence of Natural-Killer Effector Cell", Journal of Virology, 75(15):6953-6961 (2001).
66Fouser, et al., Biotechnology, vol. 10 pp. 1121-1127 (1992).
67Freshney, Culture of Animal Cells, a Manual of Basic Technique, pp. 71, 74. and 119-128, 1992.
68Freshney, Culture of Animal Cells, Second Edition, Wiley-Liss pp. 70-84 (1989).
69 *Freshney, R.I. (1988). Culture of Animal Cells. Alan R. Liss. New York USA, pp. 70-83.
70 *Furukawa and Kotaba, Molecular Immunology, vol. 28 (12) pp. 133-1340 (1991).
71Gaboriau, et al., Biochemical Pharmacology, vol. 67 pp. 1627-1637 (2004).
72Gardner-Lane et al, Decision on Preliminary Motions, Glaxo Welcome Inc v. Cabilly et al, 2002.
73Gasser et al., In Vitro Cellular Development Biology, vol. 21 (10) pp. 588-592 (1985).
74Gillies et al., Biotechnology, vol. 7 pp. 799-804 (1989).
75Gillies et al., J. Imminological Methods, vol. 125 pp. 191-202 (1989).
76Goeddel et al., Proceedings of the National Academy of Sciences USA, vol. 76 (1) pp. 106-110 (1979).
77Grady, et al., Journal of Biological Chemistry, vol. 284(34) pp. 20221-20220 (1989).
78Hale et al., Journal of Immunological Methods, vol. 103 pp. 59-67 (1987).
79Hale et al., Mol. Biol. Med., vol. 1 pp. 305-319 (1983).
80Hale et al., The Lancet, vol. 2 pp. 1394-1399 (1988).
81Hale et al., Tissue Antigens, vol. 35 pp. 118-127 (1990).
82Hale et al., Transplantation, vol. 45 pp. 753-759 (1988).
83Hale, et al., The Lancet, vol. 332(8625) pp. 1394-1399 (1988).
84Ham, Proceedings of the National Academy of Sciences, vol. 53 pp. 288-293 (1965) [composition of F12 attached].
85 *Hamilton et al, in Vitro, 1977, vol. 13, No. 9, pp. 537-547.
86Handa-Corrigan et al., Enzyme Microbial Technology, vol. 11 ppl. 230-235 (1989).
87Hata, et al., Cytotechnology (1992) vol. 10: pp. 9-14.
88Haynes, et al., Nucleic Acids Res., 11, 687-706 (1983).
89 *Hayter et al. "Recombinant Gamma-Interferon Production by CHO cells in Serum-free Medium." Advances in Cell Biology and Technology Bioprocessing. RE Spier et al (eds). Butterworths: Kent, England, 1989, pp. 280-282.
90Higuchi, K., Advances Applied Microbiology, vol. 16 pp. 111-136 (1973).
91Holtta et al., Biochemica et Biophysica Acta, vol. 721 pp. 321-327 (1982).
92Huang and Gorman, "The Simina Virus 40 Small-t Intron, Present in May Common Expression Vectors, Lead to Aberrant Splicing," Molecular and Cellular Biology (1990) 10(4):1805-1810.
93I5500, Biochemicals and Reagents for Life Science Research, p. 1147 (2002-2003) Sigma-Aldrich Company (Current website information also included).
94Isaacs et al., The Lancet, vol., 340 (8822) pp. 748-752 (1992).
95Iscove and Melchers, The Journal of Experimental Medicine, vol. 147 pp. 923-933 (1978).
96J. Cell. Biochem, 20th Annual Meeting (1991):122.
97Jefferies. "Structure Function relationship in Human Immunoglobulans" neth J Med (1991), vol. 39, No. 3-4, pp. 188-198. Abstract only.
98Jenkins, et al., Nature Biotechnology, (1996) 14:975-980.
99Jungbauger, et al., Pilot scale production of a human monoclonal antibody against human immunodeficiency virus HIV-1, Journal of Biological and Biophysical Methods. (1989) 19:223-240.
100K. Loren, Vibrant Life, vol. 2 (1) 13 pgs (1999).
101Kalwy, S., et al., "Toward More Efficient Protein Expression," Molecular Biotechnology (2006) vol. 34 p. 151.
102Kaqawa et al., Journal of Biochemistry, vol. 68 pp. 133-136 (1970).
103Katsua & Takaoka, Methods of Cell Biology, vol. 6 pp. 1-42 (1973).
104Katsuta and Takaoka, Journal of Experimental Medicine, vol. 30 pp. 235-259 (1960).
105Katsuta and Takaoka, Methods of Cell Biology, vol. 6 pp. 1-42 (1973).
106Kaufman et al., Molec. Cell Biol., vol. 5(7) pp. 1750-1759 (1985).
107Kaufman, J. Mol. Biol., 159, 601-621 (1982).
108 *Kaufman, R.J. et al. (1985), "Coamplification and coexpression of human tissue-type plasminogen . . . " Molec. Cell Biol. 5(7):1750-59.
109Keay, Biotechnology and Bioengineering, vol. XVIII pp. 363-382 (1976).
110Kilburn, et al. Biotechnology and Bioengineering (1968) vol. X:801-814.
111Kim, et al., In Vitro Cell Dev. Biology, vol. 38 pp. 314-319 (2002).
112King, et al., Biochem Journal, vol. 281 pp. 317-323 (1992).
113Knight et al., Human Antibody Hybridomas, vol. 3 pp. 129-136 (1992).
114Köhrle, J., Biochimie, vol. 81 pp. 527-533 (1999).
115Kunert, et al. "Molecular Characteristics of Five Neutralizinf Anti-HIV Type I Antibodies: Indentification of Nonconventional D. Segments in the Human Monoclonal Antibodies 2G12 and 2F5" Aids Research 14(3):1115-1128 (1998).
116Kurano, et al., Journal of Biotechnology, vol. 15 pp. 101-112 (1990).
117Kyle et al., Journal of Rheumatology, vol. 18 (11) pp. 1737-1738 (1991).
118Large Scale Mammalian Cell Culturetechnology, published by Marcel Dekker, Inc., and edited by A.S. Lubiniecki (pp. 161-175, 417-449 and 515-541; Sep. 14, 1990).
119Larrick, et al., Biotechnology, vol. 7 pp. 934-938 (1989).
120Leatherbarrow, et al., Imuunology (1985) 22(4):401-413.
121Letter form head librarian of Boku submitted in Opposition of EP0523949.
122Letter form head librarian to "whom it may concern" submitted in Opposition of EP0523949.
123Levy et al., Gene, vol. 54 pp. 167-173 (1987).
124Lewis et al., Human Antibody Hybridomas, vol. 3 pp. 146-152 (1992).
125Lipoldova, "T-cell receptor V beta 5 usage defines reactivity to9 a human T-call receptor monoclonal antobody," Immunogenetics (1989) 30(3):162-8—abstract.
126Liu et al., J. Immunology, vol. 13(10) pp. 3521-3528 (1987).
127Liu, et al., Gene, vol. 54(1) pp. 33-40 (1987).
128Liu, et al., PNAS, vol 84(10) pp. 3439-3433 (1987).
129Luff, cited in "The BSE Inquiry," established fot the British Government.
130Madisen, et al., Growth Factors (1990) 3:129-138.
131Marquis et al., Cytotechnology vol. 2 pp. 163-170 (1989).
132Mather, et al., Methods of Enzymology, vol. 185 pp. 567-577 (1995).
133McCormick et al., Molec. Cell Biol., vol. 4 (1) pp. 166-172 (1984).
134 *McCormick, F., et al. (1984), "Inducible expression of amplified human beta . . . " Molec. Cell Biol. (4(1): 166-172.
135McKeehan et al., Proceedings of the National Academy of Sciences USA, vol. 73 pp. 2023-2027 (1976).
136Mendiaz et al., In Vitro Cell. Dev. Biol., vol. 22 pp. 66-74 (1986).
137Meri & Bonsdorff, ency. Of Imm., Delves & Roidd (eds), (1998) pp. 617-619.
138Merten et al., Production of biologicals from animal cells in culture research, development, and achievements, 10th Mfg., Avignon, France (1990).
139Mesa, et al., J. Interferon and Cytokine Res. (1995) 15:309-315.
140Michel, et al., Proceedings of the National Academy of Sciences, vol. 81 (24) pp. 7708-7712 (1984).
141Miyaji, et al., Expression of Human beta-interferon in Namalwa KJM-1 which was adapted to a serum-free medium., Cytotechnology vol. 3, pp. 133-140, 1990.
142Miyazaki, et al. "Production of Monoclonal antibodies against human erythropoietin and their use in purification of human urinary erythropoietin" J. Immunol Methods. (Oct. 1988) 26:113(2)261-7 Abstract.
143 *Mizouchi, et al., Biochem Journal, vol. 254 pp. 599-603 (1998).
144Moellering, et al., "Electrophoretic Differences in Mab Expressed in Three Media." BioPharm. (Feb. 1990) 30-38.
145Morrison et al., PNAS, vol. 81 pp. 6851-6855 (1984).
146Morrison, "Transfection Provide Novel Chimeric Antibodies," Science (1985) 229:1202-1205.
147Morrison, "Trasfection Provide Novel Chimeric Antibodies," Science (1985) 229:1202-1205.
148Mountain and Adair, Biotechnology and Genetic Engineering Reviews, vol. 10 pp. 1-142 (1992).
149Murphy, Science, vol. 273 (5276 pp. 746-747 (1996).
150Nakamori et al., Applied and Environmental Microbiology, vol. 64 (5) pp. 1607-1611 (1998).
151Neidhardt, et al., J. Bacteriol., 119, No. 3, 3, 736-747 (1947).
152Neuberger et al., Nucleic Acids Research, vol. 16(14) pp. 6713-6724 (1988).
153Newman, et al., Biotechnology, vol. 10 pp. 1455-1460 (1992).
154Nippon Zenyaku Industries, Reply of Applicant concerning EP92306420.8, May 28, 1998.
155Nishimura, et al., Cancer research (1987) 47:999-1005.
156Noda et al., Chem. Abs., vol. 110 (19) Abstract p. 652 (1988).
157Ogata et al., Applied Mcrobiology Biotechnology, vol. 38 (4) pp. 520-525 (1993).
158Oka et al., Bioprogress Technol., vol. 10 pp. 72-92 (1990).
159Opposition Division's Decision of revocation in respect of parent case (EP-B-0481791).
160Opposition Division's Preliminary Opinion in respect of parent case (EP-B-0481791).
161Organic Chemistry, John Wiley & Sons Inc, vol. II Second Edition pp. 1129-1130 and 1136-1138 (1943).
162Page and Sydenham, Biotechnology, vol. 21 (10) pp. 64-68 (1991).
163Page et al., Biotechnology, vol. 9 pp. 64-68 (1991).
164Painter, Encyclopedia Of Immunology, Second Ed. Delves et al., (1998):1208-1211.
165Parekh, et al., Nature 316:452-457 (1985).
166Patel, et al. "Different culture methods lead to differences in mrine IgG monoclonal antibody" Biochem J. 285, 839-845 (1992).
167Patterson, et al., 1994: Appl Microbial Biotechnologiy 40: p. 691-698.
168 *Paulson and Colley, Journal of Biological Chemistry, vol. 264(30) pp. 17615-17618 (1989).
169 *Paulson, TIBS Jul. 14, 1989.
170Pearson et al., The 19th Meeting of the European Society for Animal Cell Technology, pp. 1-11 (2005).
171Persson et al. PNAS, vol. 88 pp. 2432-2436 (1991).
172Phillips, et al., Cytotherapy (2001) 3 (3):233-242.
173Phillpotts, Cytotechnology, vol. 2 pp. 161-162 (1989).
174Prokop, et al., Editors, Annals of the New York Academic of Sciences, 646:Table of Contents and Prefaces, Dec. 27, 1991.
175R.H. Kimberlin, Symposium of Virological Aspects of the Safety of Biological Products London, England 1990, Develop. Biol. Standard, vol. 75 pp. 75-82 (1991).
176Rabbi S. Emanuel, MK Vaad Hair, vol. IV (7) 9 pgs (2003) .
177Rabbi S. Emanuel, MK Vaad Hair, vol. IV (7) 9 pgs (2003) <www.mk.ca/page6_11.php>.
178Ray, et al., ann. NY Aca Sci., (1990) 589:443-457.
179Regenstein et al., E-Journal , Kosher Issues for Today's Dairy Industry, 2002.
180Regenstein et al., E-Journal <www.Kashrut.com>, Kosher Issues for Today's Dairy Industry, 2002.
181Response to Examination Report of Apr. 1, 2005.
182Riechmann et al., Nature, vol. 322, pp. 323-327 (1988).
183Ripka, et al., Archives of Biochemistry and Biophysics (Sep. 1986) 249(2):533-545.
184Roberts, et al. J.Cell Biochem 20th Annual Meeting p. 122 (2001).
185Robinson et al., Human Antibody Hybridomas, vol. 2 pp. 84-92 (1991).
186Roitt Brostoff and Male, Immunology 6th edition, p. 74 (2001).
187Rose et al., Molecular Immunology, vol. 29(1) pp. 131-144 (1992).
188 *Rudd, et al., Molecular Immunology, vol. 28(12) pp. 1369-1378 (1991).
189Rüker, et al., Annals New York Acad. Sci., pp. 212-219 (1991).
190Saban, T., (2004) "Food Additives From Islamic Perspective," Version 1.3 .
191Saban, T., (2004) "Food Additives From Islamic Perspective," Version 1.3 <wwwsrvl.mycity.at/privat.9704236/Im/LM-en.html>.
192Sakar, et al., Proceedings of the National Academy of Science, vol. 92 pp. 3323-3327 (1995).
193Salahuddin et al., Journal of Experimental Medicine, vol. 155 pp. 1842-1857 (1982).
194Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition pp. 8.8-8.9 (1989).
195Sano et al., Cell Struct. Funct., vol. 13 (2) pp. 142-159 (1988).
196Sano et al., Cell Structure and Function, vol. 13 (2) pp. 143-159 (1988).
197Scanhill et al., Proceedings of the National Academy of Sciences, vol. 80 pp. 4654-4658 (1983).
198Schneider & Laviox, Journal of Immunological Methods, vol. 129 pp. 251-268 (1990).
199Schnider, Journal of Immunological Methods, vol. 116 pp. 65-77 (1989).
200Sertich, et al., Journal of Cellular Physiology, vol. 127 pp. 114-120 (1986).
201Sims, et al. "A Humanised CD18 Antibody can Block Fucntion without Cell Destruction" J. Immumol. 151:2296-2309 (1993).
202 *Spellman, et al., Journal of Biological Chemistry, vol. 264(24) pp. 14100-14111 (1989).
203Spira et al., Trends in Animal Cell Culture Technology, Proc. Ann. Meeting Jpn. Tech., pp. 67-73 (1990) [Reporting conference meeting in 1989].
204Takagi, et al., Journal of Bioscience and Bioengineering, vol. 90(5) pp. 509-514 (2001).
205Taylor et al., Mutation Research, vol. 67 pp. 65-80 (1979).
206Thilly, et al., Mammalian Cell Technology, pp. 26-29 (1986).
207Titeux et al., Journal of Cellular Physiology, vol. 121 pp. 251-256 (1984).
208Tsujimoto et al., Journal of Biochemistry, vol. 106 pp. 23-28 (1989).
209U.S. Appl. No. 10/145,712, filed May 16, 2002, Page, et al.
210U.S. Appl. No. 10/145,992, filed May 16, 2002, Page, et al.
211U.S. Appl. No. 10/765,067, filed Jan. 28, 2004, Page, et al.
212U.S. Appl. No. 11/289,714, filed Nov. 29, 2005, Crowe, et al.
213U.S. Appl. No. 11/804,729, filed May 18, 2007, Shadle, et al.
214U.S. Appl. No. 90/006,997, filed Apr. 5, 2004, Crowe, et al.
215Ungemach et al., The 50th Meeting of the joint FAO/WHO Expert Committee on Food Additives (JECFA), World Health Organization, 1998.
216Urban & Chasin, Proceedings of the National Academy of Sciences, vol. 77(7) pp. 4216-4220 (1980).
217 *Urblan, G and L.A. Chasin (1980). "Isolation of Chinese hamster cell mutants . . . " Proc. Natl. Acad. Sci. USA 77(7):4216-20.
218 *Urlaub et al, Proceedings of the National Academy of Sciences of The United States of America, 1980, vol. 77, No. 7, pp. 4216-3220.
219Waldman, et al. "A Clonal Derivative of Tunicamysin-resiistant Chinese Hamster Ovary Cells with Increased N-Acetylglucosamine-Phosphate Transferase Activity Has Altered Asparagine-Linked Glycosylation" J. Cell. Physio. 131 pp. 302-317 (1987).
220Weber et al., Journal of Neuroimmunology, vol. 22 pp. 1 to 9 (1989).
221Weidle et al., Gene, vol. 51 pp. 21-29 (1987).
222Weidle et al., Gene, vol. 60 pp. 205-216 (1987).
223Whitaker et al., Biopharm, vol. 3 (8) p. 5 (Sep. 1990).
224Whittle et al., Protein Eng., vol. 1 pp. 499-505 (1987).
225Wiebe et al., ESACT 9th Meeting, Editors Spier R.E. et al., pp. 68-71 (1989).
226Wood, et al, Journal of Immunology, vol. 145 pp. 3011-3016 (1990).
227 *Yan, et al., TIBS Jul. 14, 1989.
228Yang et al., Proceedings of the National Academy of Sciences USA, vol. 81 pp. 2752-2756 (1984).
229Zekauskas et al., J. Okla. State Med. Assoc. vol. 83 pp. 447-448 (1990).
230Zettlemeissl et al., Biotechnology, vol. 5 pp. 720-725 (1987).
231Zettlemeissl, et al., Journal of Biological Chemistry, vol. 264 (35) pp. 21153-21159 (1989).
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US8343974Jul 9, 2010Jan 1, 2013Scott Iii Linzy OMethods and compositions for treating thyroid-related medical conditions with reduced folates
US8575171Nov 28, 2012Nov 5, 2013Linzy O. Scott, IIIMethods and compositions for treating thyroid-related medical conditions with reduced folates
US9248130Oct 1, 2013Feb 2, 2016Linzy O. Scott, IIIMethods and compositions for treating thyroid-related medical conditions with reduced folates
US9481861Jul 10, 2012Nov 1, 2016Foodchek Systems, Inc.Culture medium, method for culturing Salmonella and E. coli and method for detecting Salmonella and E. coli
US20110008464 *Jul 9, 2010Jan 13, 2011Scott Iii Linzy OMethods and compositions for treating thyroid-related medical conditions with reduced folates
WO2013006960A1 *Jul 11, 2012Jan 17, 2013Foodchek Systems, Inc.Culture medium, method for culturing listeria, and method for detecting listeria
Classifications
U.S. Classification435/383, 435/387, 435/386, 435/384
International ClassificationC12N5/00, C12R1/91, C12N5/02, C07K16/28, C12N5/06, C12N9/72
Cooperative ClassificationC12N5/0043, C12Y304/21069, C07K16/2893, C12N2500/38, C12N2500/34, C12N2501/135, C12N2501/33, C12N2500/36, C12N2501/392, C12N2500/32, C12N9/6459, C12N2501/23, C12N2500/20, C12N2501/39, C12N2500/76, C12N2500/74, C12N2501/395
European ClassificationC12N5/00M2, C07K16/28W, C12N9/64F21Q68
Legal Events
DateCodeEventDescription
May 13, 2010ASAssignment
Owner name: GLAXOSMITHKLINE LLC, PENNSYLVANIA
Free format text: CHANGE OF NAME;ASSIGNOR:SMITHKLINE BEECHAM CORPORATION;REEL/FRAME:024377/0976
Effective date: 20091027