IE84174B1 - Culture medium for CHO cells and adapted CHO cells - Google Patents

Description

PATENTS ACT ‘I964 COMPLETE SPECIFICATION "Culture Medium for CHO cells and adapted CHO cells" Patent Application by THE WELLCOME FOUNDATION LIMITED, a British Company of Unicorn House, 160 Euston Road, London NW1 2BP, England.

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 cells ovary (CHO) were first cultured by Puck 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 CHO-K1, is of which, proline-requiring and is diploid for the dihydrofolate reductase (dhfr) gene.

DUK B11) From this cell line a dhfr‘ cuo cell line (cuo (PNAS 11, 1930, 4216-4220) of dhfr function as a was developed which is characterised by the loss consequence of a mutation in one dhfr gene and the subsequent loss of the other gene.

These cells are functionally dhfr‘. Other CHO DUK sub-lines have been derived which are also phenotypically dhfr . dhfr- CHO cells which are cannot grow without nucleotide precursors such as thymidine, hypoxanthine, or the equivalent nucleosides.

Various proteins have been expressed in such CHO cells including the E,coli XGPRT gene (J.Mol.App.Gen. 1981, L, 165-175), human tissue-type & Cell Biol. 5, 1750-1759, 1985), interferon (PNAS 80 pp 4654-4658), plasminogen activator (Mol. human immune (7) human beta A dhfr- CHO cell line is transfected with a product gene and a dhfr gene which and interferon (Molecular and Cellular Biology 3, 166-172, l984). 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. inhibitor of the This drug is a direct 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 CHO cells which have system) which is described in W087/04462. 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 W087/04462.

Engineered CHO cells (those in which a CHO cell line is transfected with a product gene and a selectable marker gene) are routinely grown 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 This recent years with the emergence (BSE). but will represent an additional unknown factor. problem has become more acute in of Bovine Spongiform Encephalopathy 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 overall cost of production of the product. has been much research into finding a Consequently, there culture medium devoid of animal components which will support cellular growth, of CHO especially cells.

Unfortunately, the problems associated with the provision 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 Kl cells. of CHO The medium is a modification of the medium developed by Ham (Microbiology 53 l965 288-293) which is known as "Ham's F12". Other examples of media have been based on Ham's F12 medium for disclosed in EPA390327 and EP325190. example as 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 which range from 1 litre glass vessels to multi-thousand litre 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 C02 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 recombinant or source and a 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 the risk of highly pure conditions to minimise 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, and lipid carbohydrate isolated from an animal source, can be achieved. preferred culture medium of the invention contains no protein, lipid and carbohydrate isolated from an animal source.

It is advantageous to maintain osmolality in —350mOsm. the range 200—350mOsm preferably in the range Osmolality regulators are generally salts.

KCl, KN03.

Those which may be used in the medium include NaCl, 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 NaHC03; also chlorides, sulphates and phosphates such as CaCl 2H 0, MgSO 7H 0, 4 2 NaH2P0a2H2O, or sodium pyruvate, such buffers are generally present in an amount 50-500 mg/litre. 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 -10,000 mg/litre.

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

The non-ferous 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. 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 — .5 mg/litre; Vitamin C (ascorbic acid) present in an amount 10 ~ 30 mg/litre, Vitamin B2 (riboflavin) present in an amount 0.1 - 1.0 mg/litre and Vitamin B1 (thiamine), nicotinamide, Vitamin B5 (D calcium pentothenate), folic acid, i-inositol generally present in an amount 0.2 - 8.0 mg/litre.

It is preferable to include in the basal medium a lipid factor such as choline chloride, lipoic acid, oleic acid, phosphatidylcholine or methyl lineoleate, 0.05 — generally in an amount l0 mg/litre.

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 mgglitre L-Alanine ' 20 - so L-Arginine (HCl) 50 — 100 L-Asparagine (H20) 20 — SO» L—Aspartic Acid 20 — SO L-Cystine (disodium salt) 50 - 100 L-Glutamic acid 50 - 100 L-Glutamine 400 - 600 Glycine 20 ~ 50 L-Histidine (HC1.H2O) ' 30 - so L-Isoleucine 50 — 150 L-Leucine . 50 - 150 L-Lysine (HCI) 100 - 200 L-Methionine 20 - 50 L—Pheny1alanine 40 — 80 L-Proline 30 - 60 L-Serine 30 - 60 L-Threonine 50 - 120 L-Tryptophan 10 - 20 L-Tyrosine (disodium salt) 50 - l20 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.

It may be advantageous to include in the medium a pH indicator for example Phenol red sodium salt for example at 5 -_50 mg/litre.

Medium A as set out in Table l, 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 ZYG. It is similar to a published culture medium (lscoves 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' and lecithin) DHEH lackin albumin transferrin Ingredient mg/litre L-Alanine 25.00 L-Arginine HCl 84.00 L-Asparagine H20 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.H20 42.00 L-lsoleucine 105.00 L-Leucine 105.00 L—Lysine HCI 146.00 L-Methionine 30.30 L-Phenylalanine L-Praline L-Serine L-Threonine L-Tryptophan L-Tyrosine disodium salt L—Va1ine Biotin D.Ca1cium Pantothenate Choline chloride Folic acid i~Inosito1 Nicotinamide Pyridoxal RC1 Riboflavin Thiamin RC1 Vitamin B 12 CaC122H20 KC1 MgSOh7H2O NaCl NaHCO3 NaH2P0h2H2O D-Glucose HEPES Phenol red sodium salt Sodium pyruvate Sodium selenite DMEM modification of Iscoves N and Melcher (1978), J.Exp.Med. 1+ . . 40. 42. 95. 16. 104. .00 .013 .00 .00 .00 .20 .00 .00 .40 .00 .013 219. 330. .076 200. 4505. 3024. 141. 4500. 5958. . 110. .017 #0 ¥-‘ <3 :~ c: -D s~ ~4 .b ¢~ :~ <3 O0 00 00 00 00 00 00 O0 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/litre or L-Ascorbic acid generally in an amount 20 - mg/litre to help minimise the potential toxic effects of ferrous or ferric ions, and oxygen. Further use of such as chelating citrate or agents Ethylenediaminetetraacetic acid (EDTA) or 'a free radical scavenger such as a—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 These contamination. are usually included in an amount 10,000 — 100,000 Iu/litre Growth factors which may be added to the basal medium are synthetic or recombinant and include insulin. Other factors such as p1atelet-deri- ved growth factor (PDGF), 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 (TM of 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 Spg - Smg/litre. 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 Smg/litre. and present in an amount 0.25 - Examples include ferric and ferrous salts such as ferric citrate or ferrous sulphate. 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. _ 10 _ The concentration of ferric should be controlled as these may help generate superoxides and free radicals in or ferrous ions carefully 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 0.01 — preferably added in an amount 1.0 mg/litre.

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 of the and the dhfr genes.

Therefore, in another aspect invention there is provided a culture medium for the CH0 growth of engineered dhfr‘ 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 O.l - 5.0 pH, (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 concentra- of the dhfr may be present in the medium, but the presence of these two nucleotide tions which are insufficient to bypass selection system precursors is not preferred for use with the present invention. scale In large fermenters, 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 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. polyol F68 is Of these, Pluronic (TM of BASF Wyandotte Corp) 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 Tryptone, 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, 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 x 105 1.5 x 106 mg/1. cells/ml up to or greater than cells/ml and product secretion of 30 mg/1 up to greater than 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. 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 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 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 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 particular a CHO cell invention. In 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.

Figure 1 shows growth of ClH 3Dll* AA in WCM5 (protein—free medium) in a 1 litre fermenter measured as cell count/ml over 90 days.

Figure 2 shows antibody production from ClH 3Dll* 44 cells in WCM5 in a 1 litre fermenter measured as micrograms of antibody/ml over 80 days.

Example 1.

Formulation for medium WCM4. _ 13 - Medium A: (Iscoves modification of DMEK without BSA, transferrin and lecithin as set out in Table 1). + + + + + + + + + ml/litre mg/litre S0 mg/litre mg/litre S0 mg/litre mg/litre mg.litre 0.062 mg.litre 1.36 mg.1itre 0.2 mg/litre 0.088 mg/litre lpM mg/litre l mg/litre 0.0025 mg/litre mg/litre 50,000 Iu/litre ,000 Iu/litre 0.16 mg/litre which can bypass methotrexate glycine which cannot by selection. itself bypass selection. mM L glutamine L proline L threonine Lrmethionine L cysteine L tyrosine ascorbic acid vitamin B6 vitamin B12 lipoic acid methyl linoleate methotrexate FeSO ZnSOQ CuSO4 recombinant insulin (Nucellin) polymyxin neomycin putrescine-2 HCL.

This medium does not contain hypoxanthine, thymidine or folinic acid The medium does contain Therefore, this medium maintains full selection for methotrexate resistance.

Example 2.

Formulation for Medium WCMS - 14 _ Medmium A: (Iscoves modification of DMEM without BSA, transferrin or lecithin).

+ + + + + + + + + + + + + + Example 3 ml/litre mg/litre mg/litre mg/litre mg/litre mg/litre mg/litre 0.062 mg.litre 1.36 mg.litre mg/litre mg/litre 0.0025 mg.1it 50,000 IU/litre ,000 IU/litre 3 pl/litre .16 mg/litre S mg/litre mM L glutamine L proline L threonine L methionine L cysteine L tyrosine L ascorbic acid Vitamin B6 Vitamin B12 Ferric citrate Zinc sulphate Copper sulphate Polymyxin Neomycin Ethanolamine Putrescine Recombinant Insulin (Nucellin) Growth of and Production from ClH 3Dll* an in WCMA ClH 3Dll* cells are genetically engineered CHO DUK Bll cells (Urlaub and Chasin (1980)) PNAS 77, 7 pp 4216-4220). CHO DUK Bll cells cannot produce dihydrofolate reductase (dhfr). to produce a humanised IgG antibody, Campath lH These cells were engineered (Winter et al" Nature, 1988, Q22, 323-327), using plasmid constructs to express heavy _ 15 - and light antibody chains and the mouse dhfr. Expression is amplified Clh 3Dll* cells growing as a monolayer in Iscoves + lO% FBS Flow, non-essential and maintained using the folate antagonist methotrate. amino acids, l0‘6M Methotrexate and antibiotics were approximately 90% confluent. These cells were removed from the plastic with trypsin/versene, Iscoves medium without washed in supplements, centrifuged and resuspended at S x l04/ml in WCM4 medium + 0.25% peptone + 0.1% polyethylene glycol (PEG) 10,000 + 0.5% fetal bovine serum (FBS) without methotrexate (MTX). up with 10ml of cell Three 25cm2 flasks were set suspension + hypoxanthine (H), thymidine (T) or 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 PEG, WCM4 + MTX without peptone or 75cm2 flask. and were transferred to a These cells were used to seed a 500ml Techner spinner, incubated at .5oC 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 pg/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, these cells produced antibody in excess of 70pg/ml and regularly achieved levels of l00ug/ml or more.

CIH 3011* 44.

The cells are denoted _ 16 - Example 4 Growth and Production of CIH 3Dll* 44 in WCM5 in a 1 litre fermenter.

ClH 3D1l*44 cells from Example 3 which had been growing serum—free for over 2 months were a SGi 1 litre transferred to fermenter with a stainless steel angled paddle turning at 70rpm. The temperature was set at 37°C, dO2 at 10% and pH control to 7-7.2. The fermenter was seeded on day 0 with 0.22 x 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 02. The cells were routinely passaged using fresh medium and a split rate typically between l to 2 and l 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.l% pluronic F68. growth and antibody levels achieved are shown the the The resulting attached graphs (Figs l and 2), and demonstrate the capacity of the invention to allow protein-free production of antibody in excess of l00pg/ml in fermenters.

Example 5 Growth of CHO AJl9 MCBl in WCM4 and compared to CH0 AJl9 MCBl grown in serum containing medium Chinese hamster ovary cells, (Urlaub & Chasin CHO AJl9 MCBI, 77, 7, derived from CHO DUK cells, 1980), PNAS, pph2l6—4220, were genetically engineered to produce tPA under methotrexate selection.

This cell line had been routinely grown in a fermenter as a suspension culture using normal growth medium consisting of RPMI 1640 medium _]_7_ (GIBCO), 2.5% acid hydrolysed adult bovine serum (Imperial), 0.5% Tryptone, SOIU/ml polymycin, 500nM methotrexate (MTX).

IU/ml neomycin, Medium WCM4 was formulated to which was added - B 0.25%w/V N-Z Soy Peptone (Sigma P1265), 0.l%w/v Polyethylene glycol (PEG) 20,000 (Serva, Carbowax 20M), luM MTX.

C 0.25%w/V Yeast extract (Sigma Y0500), 0.1%w/v PEG 20,000 luM MTX.

In this medium the Iscoves' in CM4 was replaced by RPMI 1640 medium (ICN FLOW). 46D O.25%w/v Yeast extract, 0.l%w/v PEG 20,000, luM HTX.

E 0.25%w/v Yeast extract, 0.l%w/v PEG 20,000, 0.25% Foetal bovine serum (Imperial), luM 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 0.2um disposable filter (Gelman, Supor Vac), then diluted for use.

The cells were incubated at 37°C in a humidified incubator containing % CO2.

Cells growing in normal growth medium were pelleted by centrifugation at l200g +400 for 5 minutes, were washed in RPMI l640 without The cells l0S cell/ml in normal growth medium (46A) C, 46D or 46E). supplements and pelleted again. were then resuspended at and the other media (46B, well plates (costar mm wells) were seeded with lml/well and incubated, at 37°C in an incubator containing 5% CO2. On days 3, 4, 5 haemcytometer and trypan blue exclusion. Two and 6 one well of each was counted using a further wells of each were harvested, pooled and pelleted at 1200g +400 5 minutes. The supernatant was separated and stored at -20°C. These samples were - 13 _ 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 pg/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 EXPERIMENT DAYS CELLCOUNT X10-5 MEAN tPA MEAN tPA "SPECIFIC IN VIABLE NONVIABLE ACTIVITY CONTENT ACTIVITY CULTURE IU/ml ug/ml MegIU/mg (n=3) (n=3) 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 0.355 468 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 0.2 lOl80 9080 .70 3h.25 .3h3 0.26i E 4 7.3 0.5 46E 5 6.1 l.3 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 AJl9 MCBI in WCM4 CHO AJl9 MCBI in WCMQ cells growing in normal growth medium were pelleted and washed as in Example 5 and were resuspended at 7x10"/ml cells were transferred to a Techne in 500ml of medium 463. These spinner flask and incubated, as above, stirring at 40rpm. 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. t of tPA in various cell subcultures The specific activi y 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 activizies of different cell passages are summarised in Table 3.

Table 3 tPA present in supernatant was PASS CELLCOUNT x1o'5 SPLIT conc. tPA SPECIFIC VIABLE NONVIABLE RATE ug/ml ACTIVITY ACTIVITY (n-3) IU/ml Heg.U/mg (n=3) 7 l 9.75 0.65 1-10 ND ND ND 2 4.95 0.01 l-5 ND ND ND 13 3 6.35 0.0 1-10 22.2 8865 0.3 _ 20 _ .8 0.0 -10 7.25 1914 0.264 21 S 7.2 0.8 1-10 15.08 4331 0.287 24 6 4.1 0.3 1-10 8.28 2040 0.246 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 Experiment terminated ND - not done.

Over a 48 day period, based on the above split rate, one cell This is population doublings with a doubling time of 38 hours. could have divided to give 3.77xl08 cells. 31.8 equivalent to 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 (18)

1.Claims 1 .

2.A method for culturing recombinant CHO cells to obtain a product, which _comprises growing CHO cells in a medium which is free from protein, lipid and carbohydrate isolated from an animal source including transferrin and comprises water, an osmolality regulator to maintain the osmolality of the medium within the range of about 200 - 350mOsm, a buffer to maintain the pH of the medium within the range of about 6.5 — 7.5 and an energy source in the range 1000 —— 10,000mg/litre, and in addition, a supplement which comprises; amino acids, including L—G|utamine in the range 400 — 600mg/l, an inorganic or recombinant iron source, a lipid source in the range 0.05 — 10mg/litre, a recombinant insulin or insulin analogue, a cell growth enhancer which is involved in the folate pathway selected from the group consisting of folic acid, vitamin B6 and vitamin B12, wherein said medium is capable of supporting both the growth of the cells in suspension at a density greater than 1 X 105 cells/ml and the subsequent secretion of the product from said cells in the absence of serum. 22 .

3.A method as claimed in claim 1 wherein the media further comprises a peptide digest, hydrolysate or extract in the range 1% - 0.025% w/v. .

4.A method as claimed in claim 2 wherein the peptide digest, hydrolysate or extract is selected from the group consisting of Tryptone, casein hydrosylate, yeast extract or papain digested soya peptone. .

5.A method as claimed in any one of the preceding claims wherein the media -further comprises a factor selected from any one of the group consisting of PDGF, thyroxine T3, thrombin, interleukins such as lL2 and lL6, progesterone, hydrocortisone and Vitamin E. .

6.A method as claimed in any one of the preceding claims wherein the amino acids are one or more selected from: L—Alanine L-Arginine L-Asparagine L-Aspartic Acid L-Cysteine L-Giutamic Acid Glycine L-Histidine L-lsoleucine L-Leucine L—Lysine L-Methionine L—Phenytalanine L—Proline L-Serine L—Threonine L-Tryptophan L-Tyrosine ‘further L-Valine A method as claimed in any one of the preceding claims wherein the energy source is a monosaccharide.

7.A method as claimed in any one of the preceding claims wherein the inorganic or recombinant iron source is in the range 0.25 — 5mg/I.

8.A method as claimed in any one of the preceding claims wherein the media further comprises selenium or L-Ascorbic acid to help minimise the potential toxic effects of ferrous or ferric ions, and oxygen.

9.A method as claimed in any one of the preceding claims wherein the media chelating citrate or Ethylenediaminetetraacetic acid (EDTA) or a free radical scavenger such as or comprises agents such as - tocopherol (Vitamin E) to reduce free radical damage.

10.A method as claimed in any one of the preceding claims wherein the media further comprises antibiotics such as polymyxin, neomycin, penicillin or streptomycin n the range 10,000 — 100,000 lU/litre to prevent bacterial contamination.

11.A method as claimed in any one of the preceding claims wherein the media further comprises putrescine or a salt thereof in a range 0.01 — 1.0mg/litre.

12.A method as claimed in any one of the preceding claims wherein the media further comprises methotrexate.

13.A method as claimed in any one of the preceding claims wherein the media further comprises a cell protectant selected from the group consisting of polyethylene glycol, polyvinyl alcohols or pluronic polyols.

14.A method as claimed in claim 13 wherein the cell protectant is Pluronic polyol F68. (ft 24

15. A method as claimed in any one of the preceding claims wherein the iron source of the culture medium is added last to the culture medium to avoid precipitation.

16. A method according to any of claims 1 to 15, wherein the product is an antibody.

17. A method according to claim 16, wherein the product is an anti-CDw52 antibody.

18.A method for culturing recombinant CHO cells to obtain a product substantially as hereinbefore described with reference to the examples 1 1 462/Temp