Title of Invention


Abstract A serum-free insect cell culture medium is described which provides improvements in the maximum cell density supported and replication of insect viruses within these cells, at a significantly lower cost than commercially-available media. An improved method for preparing a lipid emulsion which is easy to scale and which produces a more stable emulsion with a longer shelf life is also described.
Full Text FORM 2
THE PATENT ACT 1970 (39 of 1970)
The Patents Rules, 2003 COMPLETE SPECIFICATION (See Section 10, and rule 13]

a) Name
b) Nationality
c) Address

NEW JERSEY 07940-0874,

The following specification particularly describes the invention and the manner in which it is to be performed : -

WO 01/98517
/The present invention relates to fermentation and cell culture, 5 and more particularly, to cell culture media and concentrated li¬pid emulsions for use therein.
Many recent developments in biotechnology, .such as the industrial production of viral insecticides, recombinant products and the
10 like via insect cell lines, require in vitro cell culture an a large scale, which in turn demands considerable amounts of cell culture media. Unfortunately, the serum and serum albumin found in conventional cell culture media is problematic, and can be cost-prohibitive for large-scale tank fermentation. The Serum-
15 free alternatives described thus far in the prior art are also costly in large volumes.
The major problem with most commercially-available media is the need to supplement. the media with a large serum component (typi-
20 cally 5-20 %), which creates a significant limiting factor due to the high price and limited availability of the serum. Moreover, the use of animal serum and/or serum albumin in the media is also problematic from a production standpoint, since the presence of unidentified proteins in the sera can complicate downstream ef-
25 forts at product purification and contaminating animal viuuses can pose serious safety issues. Similarly, the unidentified pro¬teins found in these materials introduce an unwanted variable into smaller-scale experimental efforts as well. Further, the quality of the sera itself can vary from lot to lot, introducing
30 a contamination risk which must be investigated and resolved by the scientist or production engineer with each change in sera.
Another related problem with the use of serum and/or serum albu¬min in cell culture media involves their conventional role as the
35 carriSr for the lipid component, an essential requirement for most cell culture work. Since the direct addition of lipids to the media is impractical due to their low solubility, they are typically introduced along with the serum component in the form of water soluble lipoproteins. Alternatively, the lipids can be
40 bound to the albumin component and then added to media. Given the difficulties noted above with respect, to the use of these materials, however, it would be advantageous to provide a suitable lipid emulsion which does not require either serum or albumin.. Iscove, "Culture of Lymphocytes and Hematopoietic Cells
45 in Serum-Free Medium, " in Barnes et al., Methods for Serum-Free Culture of Neuronal and Lymphoid Cells, pp. 169-85 (1984). Although microemulsions have been described in the art to supply

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the necessary lipids for conventional insect serum-free media, Maoiella et al Bio/Technology, 6:1406 (1988), these prior art emulsions have proven unsatisfactory for a variety of reasons, inclnding high manufacturing costs" and short-term stability. 5
What is heeded, therefore, is a cell culture media, which provides the essential nutritional, biological and biophysical require¬ments needed for cell growth at substantially less cost. For large-scale tank fermentation, an ideal culture media would pro-
10 vide superior performance in cell growth and maximum cell
density, and still be easy to prepare from a relatively small number of low-cost ingredients. Preferably, the media should be serum-free. Also needed is a concentrated lipid emulsion which can supply critical lipids in a bioavailable form, but eliminates
15 the need for lipid carriers such as serum, serum albumin, or other proteins from culture media.
The present invention solves the problems in the prior art through the provision of a low-cost, serum-free cell culture 20 media, comprising a modified basal media having
a novel formulation of free amino acids" and vitamins, a peptone component which substitutes for the traditional serum component, and a lipid emulsion component.
25 "The starting-point for most conventional cell culture media is a basic basal medium, typically comprising an established mixture of amino acids, minerals, sugars and inorganic salts in an aqueous solution, together with vitamins, organic acids and/or appropriate buffers. For most conventional media a suitable
30 animal serum or serum albumin, together with a lipid component, is added to this basic basal medium to produce the complete media required to support cell life, growth, and reproduction, as well as for virus replication and the expression of recombinant or viral products.
Given the cost and the problems associated with the use of serum and/or serum albumin, several attempts have been made in the prior art to replace the sorum component with another protoin alternative. For example, U.S. Patent No. 5,024,947, the disclo-
40 sure of which is incorporated by reference herein, discloses a serum-free media consisting of 1) a conventional basal medium, 2) a lipid mixture, and 3) a peptone component, wherein the hydroly-zed protein products in the peptone component are intended to re¬place the proteins typically found in the serum or albumin. Alt-
45 hough capable of supporting insect cell growth to densities equivalent to serum-containing media, the "947 patent includes expensive ingredients (such as hydroxyproline and organic acids)

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and does not support cell growth and virus infection cell densi¬ties at high enough levels to be economical for manufacturing. Thus, improvement is still needed, both in terms of the perfor¬mance and the overall cost of the media. 5
Surprisingly, the present inventors have discovered that another expensive ingredient of the basic basal medium, the free amino acids, can also be substantially reduced or even eliminated when replaced with correspondingly larger amounts of less expensive
10 vitamins, sugars and peptones. This modification can be made without compromising the performance of the media. In fact, in¬sect cells grown in the serumfree media of the present invention attain maximum cell densities twice that reported in the "947 patent and support productive virus infections at much higher
15 cell densities. Thus, the complete serum-free media of the pre¬sent invention provides additional cost savings in comparison with the serum-free compositions described in the Prior art, while providing superior results.
20 The present invention also provides a novel method for producing the lipid component of cell culture media and the improved lipid emulsions produced thereby, for use in large scale preparations of cell culture media such as the serum-free media described above. Prior art techniques, such as that described in U.S. Pa-
25 tent No. 5,372,943, the disclosure of which is incorporated by reference herein, require vigorous agitation such as sonication or vortexing to produce the emulsion. In contrast, the present invention employs a selective heating step which can sponta¬neously produce a concentrated lipid emulsion, thus eliminating
30 the significant manufacturing costs associated with vigorous agi¬tation of large volumes of liquid. Following the methods of the present invention, the emulsion forms spontaneously when the li¬pid component alone is heated , and a small volume of water at ambient temperature is added. Constant stirring can be advanta-
35 geously employed to reduce the droplet size of the resulting emulsion.
Thus, the lipid emulsions of the present invention provide consi¬derable cost savings and greatly simplify large-scale media pro-
40 duction, since the sonication or vortexing of large volumes of liquid is not necessary. The improved emulsions obtained follo¬wing the claimed method demonstrate superior stability in compa¬rison wich the emulsions produced in the prior art. As disclosed herein, the improved emulsions have a shelf life at least as long
45 as, six months and"retain biological activity during this period.

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■ 4 These lipid emulsions may be advantageously used in conjunction with the serum-free media of the present invention.
The preferred embodiment of the improved, serum-free media of the 5 present invention comprises a modified basal medium, to which is added a peptone component, a lipid component and a protective component. The modified basal medium together with the additional components provide the basic nutrients necessary to support cell life, growth and reproduction.

The basal medium provides a nutrient mixture of inorganic salts, sugars, amino acids, vitamins, organic acids and/or buffers. The prior art teaches the use of commercially-available basal media based an established recipes [see, e.g., U.S. Patent Wo.
15 5,024,947 at columns 7-8J; which typically incorporate mixtures of free amino acids, free organic acids and vitamins. The phrases ".free amino acid (s)" , "free organic acids" and "free vitamin (s)" are.used herein to refer to the purified preparations of indivi-dual amino acids, organic acids and vitamins which are commer-
20 cially available from a number of companies, such as Sigma
Chemical Company of St. Louis, Missouri. These free amino acid, organic acids and vitamin preparations have either been purified from natural sources or are mass produced in a substantially purified form, and are conventionally added .to the basic basal
25 medium independent of any amino acids and/or vitamins derived
from the peptone component of the complete medium. The free amino acids in particular represent a significant cost element of the basic basal media described in the prior art.
30 Unlike the prior art, the basal medium of the present invention has been modified so as to significantly reduce the amount of the costly free amino acids" such as L-arginine HCl L-aspartic acid, L-asparagine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-
35 phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-valine in comparison with conventional media preparations. Sur¬prisingly, the present inventors have discovered that one of the most expensive of the free organic acids, such as fumaric, malic, succinic and a-ketoglutamic acid and hydroxyproline, the amino
40 acid hydroxyproline, can be eliminated entirely from the media with no ill effect. Since commercial preparations of this amino acid typically cost five to six times more than the-next most ex-pensive purified amino acid, the elimination of hydroxyproline imparts considerable costs savings for a large scale production,
45 as does the overall reduction in free amino acids contemplated by the present invention.

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6 ximately 0.02 and 0.1 g/1, more preferably between approximately 0.04 and 0.08 g/1, and most preferably between approximately 0.05 and 0.07 g/1. In comparison, the total concentration of free vi¬tamins found in conventional formulations of basal media such as 5 Weiss et al."s the IPL-41 medium of an the order of 0.02 g/1.
Thus, in the basal media of the present invention the ratio of free"amino acids to free vitamins will range from about 1:0.01 to l:0;1, usually from about 1:0.02 to 1:0.08, more usually from ab-
10 out 1:0.03 to 1:0.07, and most preferably from about 1:0.03 to
1:0.05. As demonstrated by the growth data provided in the experi¬mental section below, however, this significant alteration to the basic composition of the basal media does not adversely affect the performance of the complete media.
15 Of course, the relative concentrations of each individual amino acid and vitamin can be adjusted according the needs of the par¬ticular cell line. For example, although glutamine is essential for many insect cell lines, it is known in the art that some cell
20 lines can grow without glutamine and may be able to synthesize it from precursors. See Mitsuhashi, Appl. Entomol. 2ool. 22:533-36 (1987). Thus, depending an the intended use of the media one skil¬led in the art can easily adjust the concentrations of the indi¬vidual free amino acids and vitamins to accommodate the known
25 characteristics of the various insect cell lines.
The complete serum-free media of the present invention further comprises a peptone component, which also compensates for the reduction in free amino acids in the basic basal media, as well
30 as providing a replacement for the serum or albumin. The term "peptone" is intended to refer to hydrolyzed protein products, typically a mixture of protein cleavage products produced by par¬tial hydrolysis of a native protein using acid or enzyme and ha¬ving average molecular weights between about 5 and 30 kD. Optio-
35 nally, the individual peptone fractions combined to produce the peptone component will be prepurified by ultrafiltration or the like, to remove any residual proteases, endotoxins or other hig¬her molecular weight products which could potentially interfere with the purification and use of recombinant products expressed
40 by insect cells grown in the media.
The peptones contemplated for use in the present invention are therefore readily distinguishable from the higher molecular weight proteins supplied by serum, serum albumin and the like, 45 which are eliminated from the media of the present invention. The total peptpne concentration in the media will depend on a number of factors, such as the particular peptone fractions employed,

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7 the nature of the cell line to be cultured, the level at which a given peptone becomes toxic or inhibitory to cell growth, and the like, with the optimal concentration of each peptcne fraction being determined empirically. Generally, the total amount of pep-5 tone in the complete serum-free media of the present invention will range from about 8 to about 30 g/1, more usually from about 12 to. about 24 g/l.
Peptones suitable for use in the present invention are commer-
10 cially available and include"lactalbumin hydrolysate (e.g., Eda-min S, available from Sigma Chemical Co., St. Louis, Missouri, U.S.A.}, yeast extracts such as Yeastolate (e.g., TC Yeastolate (Difco, U.S.A ), ox liver digests such as Panmede (Paines & Byr¬nes, Ltd., Great Britain), caseine digests such as Bactocasitone
15 (Difco, U.S.A.), tryptose phosphate broth and any of a number of plant product hydrolysates (for example the Hy Pep products from rice, wheat or soybean, Quest International) . A preferred embodi¬ment of the peptone component comprises either TC Yeastolate, lactalbumin hydrolysate, or a combination of the two. In a parti-
20 cularly preferred and exemplary embodiment, the peptone component comprises a mixture of yeastolate and lactalbumin hydrolysate, each at a concentration of between about 6 and 14 g/l, more usually"between about 7 and 13 g/l, and preferably between about " 8 and 12 g/11
The serum-free media of the present invention may further include a lipid emulsion component, dissolved in an organic solvent. The prior art teaches the combination of these ingredients using a high energy input, vortexing method to emulsify the lipid
30 solution in a 10 % aqueous solution of -Pluronic F-68 protectant. [See, e.g., "943 patent, col. 6, 11. 21-31]. Unfortunately, howe¬ver, this method is difficult to perform and does not lend itself to scaling up for making large volumes of emulsion for use in ma¬nufacturing at the 1.000L, to 10,000L level. The present inven-
35 tion soives these problems through the provision of a low-cost, readily-scalable formula and method for forming the lipid micro-emulsion. The emulsion thus formed.is more stable and has a lon¬ger aholf lifs them prior art qmvilnionn.
40 According to the present invention the lipid emulsions of the present invention include the mixture of fatty acids, steroids, lipid-soluble vitamins and organic solvent as described in the prior art, wich certain modifications. In the lipid emulsions of the present invention, the fatty acidg comprise fatty acid alkyl
45 esters, and more preferably, polyunsaturated fatty acid alkyl esters such as polyunsaturated fatty acid methyl esters. Fatty acid esters have a chain legth of Ci2 to C22, preferred of C13 to

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8 C19. The alkylcomponent is C1-C4 alkyl (methyl, ethyl, n-proplyl, i-propyl, n-butyl, i-butyl, tert-butyl) wherein methyl is most preferred, A particularly preferred mixture of polyunsaturated fatty acid methyl esters is provided by fish liyer oil, and more 5 preferably, by cod liver oil, which also contains vitamin A. The formulation can also be simplified by using pure oleic acid fatty acid methyl ester instead of cod liver oil. The steroids are pre¬ferably sterols such as lanosterol, stigmasterol, sitosterol and cholesterol, and in a particularly preferred embodiment, chole-
10 sterol, while the lipid soluble vitamin is selected from the
group consisting of phyllochxnon (vitamin K1), menachinon (vita-min K2) , menadion (vitamin K3), calciferol (vitamin D) cholecal-ciferol (vitamin D2), ergocalciferol (vitamin D3), retinol (vita¬min A), alpha-tocopherol (vitamin E) and the non-acetylated form
15 of alpha-tocopherol, wherein the non-acetylated form of alpha-to-copherol (vitamin E) is most preferred. Additionally, although a variety of alcohols (C1-C6 aklohols) are described for use in the prior art, the preferred organic solvent of the lipid emulsions of the present invention is n-propanol, i-propanol or a mixture
20 of both, wherein n-propanol is most preferred.
A mixture of polyunsaturated fatty acid alkyl esters preferably methyl alkyl esters such as cod liver oil is preferably present at a final concentration in the complete media of from 1 mg/1 to
25 50 mg/1; the steroid, preferably a sterol, most preferably chole¬sterol, is preferably at a concentration of 2 mg/1 to 7 mg/1, the fat soluble vitamin, preferrably non-acetylated alpha tocopherol is preferably at a concentration of 0.5 mg/1 to 4 mg/1, and the alkhol, preferably n-propanol, is preferably at a concentration
30 of 0.001 % to 0.01 %, more preferably of 0.002 % to 0.005 %, and most preferably between of 0.0025 % to 0.0035 %. A final concen¬tration of organic solvent which is non-toxic and non-inhibitory to cell growth is selected, depending an the chosen solvent, the type cells to be grown, and the like.
A surfactant is generally included in the lipid phase created by combining the above ingredients, prior to mixing with the aqueous phase and the consequent production of the lipid microemulsion. Preferably, the surfactant, will be an anionic surfactant, usually
40 a phospholipid or a non-ionic polymeric surfactant, usually a po¬lysorbate, and preferably polysorbate 20 or polysorbate 80 (com¬mercially available as Tween 80, ICI Americas Inc., Wilmington, Delaware, U;S.A.).
45 According to the method of the present invention, the above li¬pids are added to propanol and heated to a temperature of 40°C to 70°C; and preferably of 50 to 60°C, with constant stirring until

WO 01/98517


an anhydrous lipid phase is achieved. One or more small aliquots of water are then added to the lipid phase, again with constant stirring, and the mixture is cooled to room temperature. Although the emulsion generally forms spontaneously, stirring is usually 5 required to reduce the droplet size such that it can be filter sterilized. The ratio of lipid to aqueous phases ranges from ap-proximately 3:0.4 to 3:0.6 an a volume basis. A ratio of approxi-mately 3:0.5 (v/v) is preferred. Additional organic solvent can be added as needed to maintain a liquid phase. A larger volume of 10 water or 1 % Pluronic F-68 can then be slowly added to the con-centrated emulsion with constant stirring to facilitate long-term storage.
Importantly, and contrary to the teachings in the prior art (see
15 ,943 patent, cols. 6-8), the present inventors have determined that the presence of higher concentrations of protectants or other emulsifiers in the aqueous phase actually destabilizes the lipid microemulsion and greatly reduces the time the emulsion may be stored before addition to the basal medium. The method and
20 formula of the present invention therefore solves a significant problem with prior art commercial lipid emulsions, which typi¬cally have a shelf life only days long and must be ordered or prepared immediately prior to use. In accordance with.the prefer-red embodiment of the present invention, the concentration of
25 emulsifier added to the aqueous component will be less than about 2%, more preferably less than about 1 %, and most preferably no additional emulsifier will be added to the aqueous component of the lipid emulsion. The beneficial effects of these compounds in the overall media can ,be obtained by adding them separately to
30 the complete medium, to avoid the stability problems" they create in the emulsion component.
The microemulsion can be added directly to basal medium at this point and filter sterilized wich the complete medium, or filter
35 sterilized alone and stored refrigerated and in the dark for at least one month and usually up to six months. This method offers the advantages that it can be scaled up to any needed volume wi¬thout-special equipment, is easy to perform, and results in an emulsion wich a long storage life, all characteristics needed to
40 reduce media formulating costs for manufacturing purposes.
Additional ingredients may also be added to the complete serum- free media of the present invention for a variety of purposes, such as, for example, a protective component to help prevent cell 45 damage in agitated and/or sparged cell cultures. The protective component preferably comprises block copolymers of propylene oxide and ethylene oxide (polyoxypropylene polyoxyethylene con-

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10 densates), and more preferably Pluronic polyols such as Pluronic F68 and F88 available from BASF Wyandotte Corp. (Parsippany, New Jersey, U.S.A.). Other suitable materials for use as protective components include hydroxyethyl starch, methyl cellulose, carbo-5 xymethyl cellulose, dextran sulfate, polyvinylpyrrolidone, fi-coll, alginic acid, and polypropyleneglycol. As noted above, in accordance with the methods of the present invention the protec¬tive component is preferably added separately to the complete me¬dium rather than combined wich the lipid component as suggested 10 in the prior art.
The media of this invention can also include other water-soluble ingredients, for example, insulin to enhance glucose uptake, transferrin for iron transport, trace elements as selenium, cata-
15 lase as a peroxidation protectant, ethanolamine as a lipid pre¬cursor, steroid hormones such as testosterone, thyroid hormones such as triiodothyronine, nucleic acid precursors such as hypo-xanthine, thymidine, deoxyadenosine, and deoxycytidine, as well as other nutrients contained in conventional serum-supplemented
20 or serum free media for Gell culture.
The method of preparing a culture medium is not critical. The me¬dium may be prepared, for example, by dissolving all the ingre¬dients and additives.in water in their respective appropriate
25 concentrations first and then filtering the solution an a mem¬brane Filter under pressure to get a sterilized culture medium. As noted above, when growing insect cells for the expression of recombinant or viral products, the peptone component of the media will preferably be prepurified, such as by prefiltration followed
30 by ultrafiltration using a membrane having a molecular weight cu¬toff selected to be smaller than any recombinant or viral product that is -to be collected to facilitate later purification.
The method of culturing cells with the medium of this invention 35 is also not critical. Cells are cultured in the medium of this invention under approximately the same conditions as those for conventional culture media. In general, cells grown in the Serum-free medium of this invention are cultured in a temperature range and under conditions appropriate for the particular cell line se-40 lected. For example, Spodoptera frugiperda cells, preferably Sf9 cells, are cultured in a -temperature range of from about 20°C to about 28°C and wherein the pH of the culture medium is preferably maintained in a range from about 6 to about 7, more preferably about 6.2 to about 6.4. In some cases it may be advantageous to 45 grow the cells under well-aerated conditions, that is, in agita¬ted and/or, sparged cultures. See, e.g., International Application No. PCT/US90/03756, published as International Publication No. WO

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91/00341, the disclosure of which is hereby incorporated in its entirety.
In general,, if insect, and especially Lepidopteran, cells can be 5 grown successfully in media wherein serum, albumin, or other pro-teinaceous lipid carriers or other non-protein lipid carriers are employed, then the cells can be grown in the media of this inven¬tion wherein the serum or proteinaceous lipid carriers are repla¬ced with peptones and a lipid microemulsion is provided, optio-
10 nally together with other required honuones - and growth factors. For" example, there are a wide variety of commercially available media for insect cell culture which include such commercially-available basal media as, for example, TC10 without tryptose broth [commercially available from Microbiological"Associates;
15 see Gardiner et al.., J. Invert. Pathol, 25:363 (1975)], Grace"s Antheraea medium [Vaughn et al., TCA Manual, 3(1) (1976); Yunker et al., Science, 155:1565-1566 (19 67.)]. Medium M20 of Marks [Vaughn et al., TCA Manual, 3(1) (1976); Marks, "In Kruse et al. (eds), Tissue Culture Methods and Applications, pp. 153-156
20 (1973)], Goodwin"s I PL-"5 2 Medium [Goodwin, In Vitro, 11:369-378 (1975)], Goodwin"s IPL Medium [Goodwin et al., In Kurstak et al. (eds.), Invertebrate Systems In Vitro (1980)], Goodwin"s IPL-76 Peptone Medium [Goodwin et al. , id; Goodwin et al., Jn vitro, 14:485-494 (1978)], Hink"a TNMFH Medium (Revised) [Hink, Nature
25 (London), 226:466-467 (1970)], Medium S-301 of Hansen [Hansen, In Maramorosch (ed.), Invertebrate Tissue Culture Research Applica¬tions, pp. 75-99 (1976)]; Vaughn et al., TCA Manual, 3(1) (1976)] , and the IPL-41 Medium discussed above [Weiss et al., In Vitro, 17(6) .-495-502 (1981)].
The media of this invention are employable not only for "the growth of cells, but also for the production of useful physiolo¬gically active substances such as interferons, lymphokines and antibodies. Heterologous proteins that have been expressed in in-
35 sect cells via a baculovirus expression vector system (BEVS) are outlined in Summers et al. , Banbury Report: Genetically Altered vi-ruses in the Environment, 22:319-329 (1985). However, those skilled in the art who have the benefit of this disclosure, will reco¬gnize that many other recombinant proteins can be produced by an-
40 imal, plant and/or microbial cells according to this invention. Exemplary recombinant proteins include, without limitation colony stimulating factors [for example, Jong and short form CSF-1 or M-CSF, 6-CSF, GM-CSF and interleukin-3 among others], modified pro-urokinase or urokinase, tissue plasminogen activator (TPA),
45 TPA-urokinase hybrids, toxic proteins "such as whole ricin toxin, ricin A chain, products containing ricin A, as well as, interfe¬rons a, and y and hybrids thereof), interleukins, tumor necrosis

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12 factor, erythropoietin and other hemotopoietic growth factors, human growth hormone, as well as porcine, bovine and other growth hormones, epidermal growth factor, insulin, hepatitis B vaccine, herpes simplex virus glycoprotein vaccines, superoxide dismutase, 5 Factor VIII, Factor VIII C, atrial natriuretic factor, feline leukemia virus vaccines, as, for example, gp70 polypeptides, the light and heavy chains of antibody molecules, lectins such as Ri cin communis agglutinin (RCA) , diphtheria toxin, gelonin, exotoxin from Pseudomonas aeruginosa, toxic proteins from Phytolacca ameri-10 cana (PAPI, PAPII and PAP-s), insecticidal proteins from Bacillus thuringiensis, many enzymes as for example, CAT, as well as innu¬merable other hybrid proteins.
Although the exemplary embodiments described herein are adapted
15 more specifically for use in insect cell culture, the present in¬vention contemplates that media having the claimed characteri¬stics can be advantageously used in the culture of virtually any cell type. The cells can be of animal, microbial or plant origin. If animal cells, they can be from vertebrates or invertebrates.
20 Preferably, the cells are those which can produce recombinant, viral and/or natural products. Exemplary vertebrate cells are mammalian cells, for example, lymphocytes, fibroblasts, epithe¬lial cells, ovarian cells, and their transformed cells, various neoplastic cells, and hybridomas derived therefrom. More specific
25 examples of mammalian cells include Chinese Hamster Ovarian
cells, Epstein-Barr Virus (EBV)-transformed human lymphoblastoid cell lintes such as UMCL and C51804, human Burkitt"s lymphoma-de-rived Namalwa cells, murine lymphoid cellderived myeloma SPI cells, human fibroblast cells such as HEL and IMR-90, human ru-
30 mor-derived epithelial cells such as HeLa-S3 Hep-2 and KB, human primary cultured cells, rat Yoshida sarcoma cells, human fibro-blast cells BHK-21, murine fibroblast cells 3T3, murine lymphoma cells YAC-l, human/mouse hybridomas such as stable cell line D-234-4-27-8 -which produces anti-LPS IgM (ATCC No. HB 8598) and
35 hybridomas which produce monoclonal antibodies to human fibro¬blast interferon as described in U.S. Ser. No. 325,969, filed No¬vember 30, 1981.
Exemplary invertebrate cells are insect cells, preferably cells 40 which can produce viral or recombinant products upon infection, respectively, with either wild-type viruses-or recombinant bacu-. loviruses and which have been shown to grow, reproduce and ex¬press recombinant and/or viral products in a medium containing serum, albumin, another protein and/or other lipid carriers. Such 45 insect cell lines include Bombyx mori, Lymantria dispar, Tricho-plusia ni,. Helicoverpa zea and Spodoptera frugiperda. [See gene¬rally, Granados et al. (eds.), The Biology of Baculoviruses (CRC

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The following examples serve to illustrate certain preferred em¬bodiments and aspects of the present invention and are not to be 5 construed as limiting the scope thereof.
In the experimental disclosure which follows, the following ab¬breviations apply: eq (equivalents); M (Molar) ; pM (micromolar) ; N (Normal) ; mol (moles) ; mmol (millimoles) ; [xmol (micromoles) ; nmol 10 (nanomoles); g (grams); mg (milligrams); kg (kilograms); jig (mi¬crograms); L (liters); ml (milliliters); ph(microliters) ; cm (cen¬timeters) ; mm (millimeters); pm (micrometers); nm (nanometers); EC (degrees Centigrade); h (hours); min (minutes); sec (seconds); msec (milliseconds) .
The individual materials described below were purchased from Sigma Chemcial Company, St. Louis Missouri, with the exception of TC Yeastolate, DF5577-08, which was obtained from VWR Scientific, Brisbane, California.
Preparation of Serum Free Media
25 A specific emodiment of the serum-free media of the present in vention, labeled EM028, was prepared using the ingredients and
amount"s listed in Table 1:



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X=fold dilution)


The lipid emulsion was prepared as described in Example 2 below. The liquid concentrates were prepared with the components and 5 amounts listed in Tables 2 through 4 below. The concentrates and powdered components were added in the order listed in Table 1 above to 800 ml of water. The pH of the final mixture was then adjusted to 6.3 and the volume brought up to 1 liter.
10 TABLE 2


Thiamin 32.64
Riboflavin 89.6
Niacin 576
Vitamin B6 92
Folic acid 11.88
Vitamin B12 15
Biotin 11.896
Pantothenic acid 361.6
Choline 1908
para-Aminobenzoic acid 16
Inositol 20



KC1 65.65
MgS04(anhydrous) 46.17
NaCl 150.31
NaH2P04 6H20 58.00



CoCl2 • 6H20 76.68
CuCl2 • 2H20 108.58
MnCl2 • 4H20 38.82
FeSO* 7H20 " 1469.85

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The amino acids, wich the exception of glutamine, were prepared
according to Table .5 below: TABLE 5
5.00 2.50
L-tyrosine 2Na-2H20

35 The above amino acids, wich the exception of tyrosine, are added to 900 ml cf water in the order given above in Table 5.If an in dividual amino acid fails to dissolve in a reasonable amount of

time, 6N HCl can be added until the pH IS 1.7. After all of the
40 other amino acids have been added, the tyrosine is dissolved in
50 ml of water and added slowly to the previous mixture. The nal volume is then adjusted to 1 liter.

iVO 01/98517 PCT/EP01/07121
Preparation of Lipid Emulsion 5 TABLE 6

Cod Liver Oil Fatty Acid Methyl Esters 1.0
Cholesterol 0.45
Alpha-Tocopherol 0.2
Tween 8 0 2.5
15 The surfactant and lipid ingredients were added to a large glass beaker and heated gently to about 50°C to 60°C. The mixture was stirred continuously with a magnetic stir bar until the solids melted and a liquid phase was achieved. While continuing to heat and mix, 0.5 ml of water was slowly added, followed by 1.3 ml
20 propanol. The mixture was cooled to room temperature, then an¬other 1.7 ml of propanol was added with mixing to again achieve a liquid phase. Then, 8.3 ml of water or 1% Pluronic F-68 in water was added dropwise with mixing. The stirring rate was increased and 0.8 ml of water or 1% pluronic was added. The mixture was
25 heated gently for 15 minutes wich stirring (the temperature was kept below 40°C) and water or 1% Pluronic was added to bring the volume up to l.OL. In order to store the emulsion for later use, the emulsion was filter sterilized and refrigerated.

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Long Term Stability of Lipid Emulsion
5 Table 7 demonstrates the long term stability Of the lipid emul¬sion produced using the procedure described in this patent. The maximum cell density achieved and AcMNPV PIB production of Sf9 cells grown in three different lots of EM28 medium are presented. Lots 10 and 11 were made with 7 month old lipid,emulsions contai-
10 nihg cod liver oil fatty acid methyl esters or pure oleic acid fatty acid methyl ester, respectively. All other solutions were freshly prepared: Lot 12 was made using freshly prepared solu¬tions and lipid emulsion (the lipid emulsion contained the cod liver oil fatty acid methyl esters). For virus production, cultu-
15 res were infected at a density of 1.8 x 106 cells/ml.

Lot 10 10.5 x 10s 1.2 X 10a
Lot 11 10.3 x 106 1.0 X 108
Lot 12 10.3 x 108 1.0 x 108 .
Production of PIBs
Table 8 illustrates the production of AcMNPV baculovirus polyhe¬dral occlusion bodies (PIBs) by Sf9 cells growing in either a commercially-available serum-free insect medium (Excell 401, JRH Biosiences, Lanexa, Kansas) or EM028 medium with and without the lipid microemulsion. The cells were infected at a density of 2.4 x 10S cells/ml in shake flasks.

Commercial serum free 4.0 X 107
EM028 without lipid emulsion 5.0 x 105
EM028 with lipid emulsion 1.5 x 108
It is evident"from the above results that cells grown in the low-45 cost, serum-free medium of the present invention can achieve cell densities superior to those described for prior art serum-free media formulations (Figure.1). The present invention medium also

WO 01/98517


supports productive replication of Baculoviruaes in high density cell cultures at lower cost. Furthermore, Example 3 demonstrates the superior stability of the lipid emulsion produced according to the present invention. 5
All publications and patent applications mentioned in this speci¬fication are Indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the 10 same extent as if each individual publication or patent applica-don was specifically and individually indicated to be incorpora¬ted by reference.
Although the foregoing invention has been described in some de-
15 tail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and mo¬
difications may be practiced within the scope of the appended
claims. For example, the relative amounts of the individual com¬
ponents set forth in Tables 1-6 can be modified by one of skill
20 in the art in accordance with the specific needs of the particu-
lar cell line of interest, which needs are well known and readily
available to one of skill in the art. Generally, the specific
amounts recited in Tables 1-6 above can vary by up to approxima¬
tely 50 %, and more preferably, by up to approximately 20% .
Brief description of Figures:
Figure 1 is a graph illustrating the growth of S. frugiperda Sf-9 Cells in EM028 low-cost, serum-free media compared to growth in a 30 typical commercially available serum free insect medium.

We claim:
1. A method for producing a lipid emulsion at large scale for use in cell
culture media, said method comprising:
a) combining in an organic solvent a surfactant and a mix- hire of lipids to create a lipid phase, wherein said li- pids are selected from die group consisting essentially of fatty acids, sterols, and lipid-soluble vitamins;
b) heating said lipid phase to a temperature of 40°C to 70°C to create a liquid phase; and adding an aqueous phase to said lipid phase to produce a stable lipid micro-emul- sion, wherein said aqueous phase consists of water.

2. A method according to Claim 1, wherein said aqueous phase excludes an emulsifier or surfactant.
3. A method according to Claims 1 to 2, wherein said temperature is of 50°C to 60°C.
4. A method according to Claims 1 to 3, wherein said organic solvent is n-propanol, i-propanol or a mixture of both.
5. A method according to Claims 1 to 3, wherein said lipid solu- ble vitamin is non-acetylated alpha-tocopherol.
Dated 23rd day of December, 2002.




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Patent Number 214203
Indian Patent Application Number IN/PCT/2002/01870/MUM
PG Journal Number 13/2008
Publication Date 28-Mar-2008
Grant Date 07-Feb-2008
Date of Filing 23-Dec-2002
Name of Patentee WYETH
# Inventor's Name Inventor's Address
PCT International Classification Number C12N 5/06
PCT International Application Number PCT/EP01/07121
PCT International Filing date 2001-06-22
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/213 , 665 2000-06-23 U.S.A.