Method for culturing Chinese hamster ovary cells

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.

REEXAMINATION RESULTS

The questions raised in reexamination request no. 90/006656, filed Jun. 2, 2003 have been considered and the results thereof are reflected in this reissue patent which constitutes the reexamination certificate required by 35 U.S.C. 307 as provided in 37 CFR 1.570(e), for ex parte reexaminations, or the reexamination certificate required by 35 U.S.C. 316 as provided in 37 CFR 1.997(e) for inter partes reexaminations.

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.

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 dhfr⁻ CHO 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 dhfr⁻ cannot 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 dhfr⁻ CHO 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 insolation 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 transformats, there is normally concomitant amplification resulting in increased expression of the desired produce 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. 5, 1750-1759, 1985; PNAS 80 pp 4654-4658; Molecular and Cellular Biology 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 fermenter5 ml/liter +5 ml/liter 200 mM L glutamine +50 mg/liter L proline +50 mg/liter L theonine +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 +1μM methotrexate +1 mg/liter FeSO₄ +1 mg/liter ZnSO₄ +0.0025 mg/liter CuSO₄ +5 mg/liter recombinant insulin (Nucellin) +50,000 lu/liter polymyxin +20,000 lu/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

Medmium Medium 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 (1980) PNAS 77, 7 pp 4216-4220). CHO DUK B11 cells cannot produce dihydrofolate reductase (dhfr). These cells were engineered to produce a humanised 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 monolayer 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 cm² 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 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 or 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 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, 77, 7, pp4216-4220, 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 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 μM MTX.
  • 46C 0.25% w/v Yeast extract (Sigma Y0500), 0.1% w/v PEG 20,000 1 μM 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 μM MTX.
  • 46E 0.25% w/v Yeast extract, 0.1% w/v PEG 20,000, 0.25% Fetal bovine serum (Imperial), 1 μM 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 ×10−6	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 differing 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	0.258
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.

Claims

1. A method for growing CHO cells which comprises culturing CHO cells under cell growing conditions in the absence of serum in 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 method for culturing CHO cells in accordance with claim 1 wherein the medium further comprises non-ferrous metals, vitamins or cofactors.

3. A method for culturing CHO cells in accordance with claim 1, wherein the osmolality regulator maintains the medium at 200-350 mOsm.

4. A method for culturing 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 method for culturing 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 method for culturing CHO cells in accordance with claim 5, wherein the energy source is a monosaccharide.

7. A method for culturing 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, 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 method for culturing CHO cells in accordance with claim 1, wherein the concentration of L-glutamine is within the range of 400-600 mg/liter.

9. A method for culturing CHO cells in accordance with claim 2, wherein the medium comprises a lipid factor in an amount of 0.05-10 mg/liter.

10. A method for culturing 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 method for culturing CHO cells in accordance with claim 1, wherein the growth factor comprises recombinant or synthetic insulin, platelet derived growth factor, thyroxine T₃, thrombin, interleukin, progesterone, hydrocortisone or vitamin E.

12. A method for culturing CHO cells in accordance with claim 11, wherein the growth factor is recombinant or synthetic insulin.

13. A method for culturing cells in accordance with claim 1, wherein the medium further comprises a peptide digest, hydrolysate or extract.

14. A method for culturing cells in accordance with claim 1, wherein the medium is essentially free of hypoxanthine and thymidine.

15. A method for culturing cells in accordance with claim 14, wherein the medium further comprises methotrexate.

16. A method for culturing CHO cells which comprises culturing and growing Chinese hamster ovary cells in the absence of serum in 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, 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 genetically engineered CHO cells in suspension 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, NaH₂PO₄.2H₂O, 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, or about 0.01-0.2 mg of sodium selenite;

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 insulinand 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. ;

a base medium containing

	L-Alanine	20-50	mg/L
	L-Arginine (HCl)	50-100	mg/L
	L-Asparagine (H2O)	20-50	mg/L
	L-Aspartic Acid	20-50	mg/L;
	L-Cystine (disodium salt)	50-100	mg/L;
	L-Glutamic acid	50-100	mg/L;
	L-Glutamine	400-600	mg/L;
	Glycine	20-50	mg/L;
	L-Histidine (HCl•H2O)	30-60	mg/L;
	L-Isoleucine	50-150	mg/L;
	L-Leucine	50-150	mg/L;
	L-Lysine (HCl)	100-200	mg/L;
	L-Methionine	20-50	mg/L;
	L-Phenylalanine	40-80	mg/L;
	L-Proline	30-60	mg/L;
	L-Serine	30-60	mg/L;
	L-Threonine	50-120	mg/L;
	L-Tryptophan	10-20	mg/L;
	L-Tyrosine (disodium salt)	50-120	mg/L;
	L-Valine	80-120	mg/L;
	Biotin	0.01-0.5	mg/L;
	D Calcium Pantothenate	0.2-8	mg/L;
	Folic acid	0.2-8	mg/L;
	i-Inositol	0.2-8	mg/L;
	Nicotinamide	0.2-8	mg/L;
	Pyridoxal HCL	about 4	mg/L;
	Riboflavin	0.1-1	mg/L;
	Thiamin HCl	0.2-8	mg/L;
	Vitamin B12	0.1-0.5	mg/L;

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 CaCl₂2H₂O, MgSO₄7H₂O, NaH₂PO₄2H₂O, sodium pyruvate, N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulphonic acid] (HEPES), 3-[N-morpholino]-propanesulfonic acid (MOPS), and NaHCO₃ in an amount sufficient to maintain the medium within the pH range of about 6.5-7.5; 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 method in accordance with claim 1, wherein each component of the medium is selected from an inorganic, synthetic, recombinant, plant or bacterial source.