US20090258391A1 - Recombinant cell clones having increased stability and methods of making and using same - Google Patents

Recombinant cell clones having increased stability and methods of making and using same Download PDF

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US20090258391A1
US20090258391A1 US12/488,441 US48844109A US2009258391A1 US 20090258391 A1 US20090258391 A1 US 20090258391A1 US 48844109 A US48844109 A US 48844109A US 2009258391 A1 US2009258391 A1 US 2009258391A1
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protein
recombinant
serum
stable
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Manfred Reiter
Wolfgang Mundt
Friedrich Dorner
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Baxalta Innovations GmbH
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Priority to AT1073/97 priority Critical
Priority to AT0107397A priority patent/AT407255B/en
Priority to US09/100,253 priority patent/US6100061A/en
Priority to US09/324,612 priority patent/US6475725B1/en
Priority to US10/170,661 priority patent/US6936441B2/en
Priority to US11/123,362 priority patent/US7094574B2/en
Priority to US11/482,504 priority patent/US20070161079A1/en
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Priority to US12/488,441 priority patent/US20090258391A1/en
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    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract

Disclosed are a stable recombinant cell clones which are stable in serum- and protein-free medium for at least 40 generations, a biomass obtained by multiplying the stable cell clone under serum- and protein-free culturing conditions, and a method of preparing recombinant proteins by means of the biomass. Furthermore, the invention relates to a method of recovering stable recombinant cell clones.

Description

    CROSS-REFERENCES TO RELATED APPLICATIONS
  • This application is a continuation of U.S. application Ser. No. 11/482,504 filed Jul. 7, 2006, which is a divisional of U.S. Ser. No. 11/123,362 filed May 6, 2005, which is a continuation of U.S. Ser. No. 10/170,661 filed Jun. 12, 2002, now U.S. Pat. No. 6,936,441, which is a continuation of U.S. Ser. No. 09/324,612 filed Jun. 2, 1999, now U.S. Pat. No. 6,475,725, which is a continuation-in-part application of U.S. Ser. No. 09/100,253 filed Jun. 19, 1998, now U.S. Pat. No. 6,100,061, all of which are herein incorporated by reference in their entirety for all purposes.
  • BACKGROUND OF THE INVENTION
  • The present invention relates to a stable recombinant cell clone that is stable for at least 40 generations in serum- and protein-free medium, a biomass obtained by multiplying the stable cell clone under serum- and protein-free culturing conditions, and a method of preparing recombinant proteins by means of the biomass. Furthermore, the invention relates to a method of recovering stable recombinant cell clones. Furthermore, the invention relates to the production of a recombinant protein in a serum- and protein-free synthetic minimum medium.
  • Another aspect of the invention is a serum- and protein-free medium for culturing cells expressing a recombinant protein.
  • The preparation of recombinant proteins, in particular of biomedical products, such as blood factors, is gaining in importance. To allow for an optimum growth of recombinant cells, serum is added to the medium in most instances. Because of the high costs of serum and for avoiding possible contamination's in the culturing medium by viral or molecular pathogens from the serum, a number of serum-free media have been developed which, in particular, should not contain any additives of bovine or human origin. In addition to the low risk of contaminating the prepared products with viral and molecular pathogens, the use of such media in the preparation process also allows for a simpler purification of the expressed proteins.
  • In most instances, recombinant cells are first cultured in serum-containing medium up to a high cell density, e.g. for a working cell bank, and subsequently they are re-adapted to serum-free medium during the production phase.
  • Miyaji et al., Cytotechnology, 3:133-140 (1990) selected serum-independent cell clones in serum-free medium which contained insulin and transferrin. However, the living cell number and the expression rate proved to decrease continuously after 16 days. By co-amplification with a labeling gene, Mayaji et al., Cytotechnology, 4:173-180 (1990) tried to improve the expression rate and the productivity of the recombinant, cells.
  • Yamaguchi et al., Biosci. Biotechnol. Biochem., 56:600-604 (1992) established serum-independent recombinant CHO sub-clones by culturing serum-dependent cells on microtiter plates as monolayer for 3 to 4 weeks in serum-free medium that contained human serum albumin, insulin and transferrin. Approximately 0.1% of the cells were serum-independent. Part of the subclones also grew in suspension culture in serum-free medium, yet the cells aggregated and formed lumps. The duplicating time of the cells amounted to 1.5 days. Yet there are no data either on the stability of the serum-independent clones obtained, nor on the long time cultivation of these clones under serum-free conditions.
  • Media which allow for the maintenance of the metabolic activity and for a growth of cells during the serum-free phase frequently contain additional substances, e.g. growth factors, such as insulin or transferrin, or adherence factors which substitute the serum components.
  • To avoid the addition of polypeptide factors, such as insulin or transferrin, and to allow for protein free culturing conditions, various techniques have been developed. Thus, specifically defined, complete protein-free media have been developed which allow for a cell-growth also under protein-free conditions.
  • WO 97/05240 describes the preparation of recombinant proteins under protein-free conditions, the cells co-expressing a growth factor in addition to the desired protein.
  • JP 2696001 describes the use of a protein-free medium for the production of factor VIII in CHO cells by adding a non-ionic surface-active agent or cyclodextrin to increase the productivity of the host cells. To increase the effectiveness of these additives, the addition of, e.g., butyrate and lithium is recommended. As indicated in the specification, the addition of pluronic F-68 results in a marked increase in cell numbers.
  • WO 96/26266 describes the culturing of cells in a medium which contains a glutamine-containing protein hydrolysate whose content of free amino acids is less than 15 k of the total weight of the protein, and whose peptides have a molecular weight of less than 44 kD. As the culturing medium for the cell cultures, a synthetic minimum medium is used as the basic medium to which, inter alia, fetal calf serum, gentamycin and mercaptoethanol are added in addition to protein hydrolysate. The use of this serum-containing medium for the recombinant production of blood factors has not been mentioned.
  • U.S. Pat. No. 5,393,668 describes special synthetic surfaces which allow for a growth of adherent cells under protein-free conditions.
  • To stimulate cell proliferation, CHO cells which overexpress human insulin have been multiplied on an artificial substrate to which insulin is covalently bound (Ito et al., PNAS USA, 93:3598-3601 (1996)).
  • EP 0 872 487 describes the preparation of recombinant factor VIII in protein-free medium containing recombinant insulin to which polyols are added. According to the specification, the addition of pluronic F-68 results in an increased factor VIII productivity of BHK cells, and the addition of iron ions yet enhances this rise in productivity.
  • Reiter et al., Cytotechnology, 9:247-253 (1992) describe the immobilization of r-CHO cells first grown in serum-containing medium at a high density on carriers, and subsequent perfusion of the immobilized cells in protein-free medium during the production phase, wherein a continuous liberation of protein into the cell culture supernatant was found. There, the cells were perfused for less than 10 generations in protein-free medium.
  • Katinger et al, Adv. In Mol Cell Biol., 15a: 193-207 (1996) describe the preparation of stable cell cultures wherein the cells are immobilized on macroporous carriers. It is emphasized that perfusion cultures with porous carrier materials would be preferable to other methods. Stable clones expressing recombinant proteins, such as FVIII or von-Willebrand factor, are not described, the cells are invariably grown first in serum-containing medium and are only later transferred to serum- and protein-free medium.
  • Previous methods for the successful preparation of a large-scale cell culture under protein-free conditions have been described for continuous cell lines, in particular VERO cells (see, e.g., WO 96/15231). There, the cells are grown under serum- and protein-free conditions from the original ampule up to a large technical scale of 1200 1. However, these are not recombinant cells, but host cells which are used for the production of virus antigen in a lytic process.
  • In contrast to adherent VERO cells, e.g. CHO cells are dependent on adhesion to a limited extent only. CHO cells grown by means of conventional methods under serum-containing conditions are capable of binding both to smooth and to porous micro-carriers (U.S. Pat. No. 4,978,616; Reiter et al., Cytotechnology, 9:247-253 (1992)). If CHO cells are grown under serum-free conditions, they lose this property and do not adhere to smooth carriers, such as, e.g., Cytodex 3, or they detach easily therefrom, unless adherence-promoting additives, such as, e.g., fibronectin, are put into the medium. Because of the slight adherence of CHO cells to carriers under serum-free conditions, the production of recombinant proteins thus mainly is effected in suspension culture. There, the production process may be effected as a continuous or as a batch-wise method. The recombinant cell culture at first is grown in a bioreactor up to an optimum cell density, optionally the protein expression is induced, and for harvesting, the medium containing the expressed proteins but also recombinant cells is withdrawn at certain intervals from the reaction tank and thus from the production process. By the continuous loss of biomass, the production efficiency in the bioreactor drops and increases again slowly only after the addition of fresh medium, since the cells must grow up to the desired cell density. Thus, despite the continuous process, repeatedly there is a phase of retardation, in which the production rate in this system drops. Furthermore, the growth and production capacity in such a system is limited by the maximum cell density attainable.
  • When adapting cells initially grown under serum containing conditions to protein-free medium, it has repeatedly been found that the yield of expressed protein and the productivity of recombinant CHO cells greatly drops after adaptation in protein-free medium as compared to serum-containing conditions (Paterson et al., Appl. Microbiol. Biotechnol., 40:691-658 (1994)). This is the consequence of an instability or reduced growth of the recombinant clones due to the changed culturing conditions. Despite the use of a stable original clone, on account of the altered fermentation conditions, repeatedly a large portion of the cells become cells with reduced expression or also non-producers, which overgrow product producers during the production process, whereby the fermented culture finally largely consists of non-producers or of such cells having a low expression.
  • As a consequence, the maximum production capacity of the fermentation culture drops continuously, and a maximum product production is restricted to a certain number of generations or cell passages.
  • Thus, there is a need for a system in which a continuous production is possible over as long a period of time as possible, in particular in the large-scale production of recombinant proteins under serum- and protein-free conditions.
  • It would furthermore be desirable to obtain a recombinant cell clone which is stable in the production phase for many generations under protein free conditions and which expresses recombinant protein.
  • BRIEF SUMMARY OF THE INVENTION
  • It is the object of the present invention to provide an efficient method of preparing recombinant proteins under serum- and protein-free cultivation and production conditions.
  • It is a further object to provide a stable recombinant cell clone.
  • It is another object of the present invention to achieve an increase in productivity of a recombinant cell clone by using a protein- and serum-free medium.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows the microscopy of a working cell bank of an original clone at the time of re-adaptation from serum-containing to serum- and protein-free medium (A), after 10 generations in serum- and protein-free medium (B), and after 60 generations in serum- and protein free medium (C).
  • FIG. 2 shows the microscopy of a cell culture starting with a stable recombinant cell clone under serum- and protein-free conditions at the working cell bank stage (A), after 10 generations (B) and after 60 generations (C).
  • FIG. 3 shows the results of culturing an rFVIII-CHO cell clone in a 10 l perfusion bioreactor.
      • a) F VIII-activity (mUnits/ml) and perfusion rate (1-5 day) over a period of 42 days.
      • b) Volumetric productivity (units factor VIII/1/day) in the perfusion bioreactor.
    DETAILED DESCRIPTION OF THE INVENTION
  • It is the object of the present invention to provide an efficient method of preparing recombinant proteins under serum- and protein-free cultivation and production conditions.
  • It is a further object to provide a stable recombinant cell clone.
  • It is another object of the present invention to achieve an increase in productivity of a recombinant cell clone by using a protein- and serum-free medium.
  • According to the invention, this object is achieved by providing a recombinant cell clone obtainable from a cell culture that is obtained after culturing a recombinant original cell clone on serum-containing medium and re-adapting the cells to serum- and protein free medium. The cells are continued to be cultured in serum- and protein-free medium under production equivalent conditions for at least 40 generations.
  • Preferably, culturing of the cells is effected without selection for the selection labeling and/or amplification gene, e.g. in the absence of MTX in case of CHO-dhfrcells.
  • By original cell clone within the scope of this invention a recombinant cell clone transfectant is understood which, upon transfection of host cells with a recombinant nucleotide sequence, expresses recombinant product in a stable manner under laboratory conditions. To optimize growth, the original clone is first grown in serum-containing medium. To increase the productivity, the original clone optionally is first grown in the presence of a selecting agent and selection for the selection marker and/or amplification marker. For the large-scale production, the original cell clone is first grown under serum-containing culturing conditions up to a high cell density, and shortly before the production phase it is re-adapted to serum- and/or protein-free medium. In this case, culturing is preferably effected without selection pressure.
  • In another preferred embodiment the recombinant original cell clone may be cultivated in serum- and protein-free medium already from the beginning, rendering re-adaptation unnecessary. Optionally, a selecting agent may also be used in this case, and selection may be for the selection and/or amplification marker. A respective method is, e.g., described in EP 0 711 835.
  • It has been found that under these conditions, a large part of more than 95% of the cells become nonproduct producers in such a cell culture which has been re-adapted to serum- and protein free medium. By means of immune fluorescence with product-specific antibodies it could be shown that in dependence on the generation time of the cells in serum- and protein-free medium, the number of the non-producers rises in a culture and overgrows the product-producers, whereby the production capacity of the culture decreases.
  • The cell culture obtained after re-adaptation to serum- and protein free medium is assayed for those cell clones of the cell population which are producers of the stable product under serum- and protein-free conditions, optionally in the absence of a selection pressure. This may be effected, e.g., by means of immunofluorescence with labeled antibodies specifically directed against the recombinant polypeptide or protein. Those cells which have been identified as product producers are isolated from the cell culture and again multiplied under serum- and protein-free, optionally under production-equivalent, conditions. Isolation of the cells may be effected by isolating the cells and assaying for product-producers. Optimally, the cell culture containing the stable cells is again assayed for stable recombinant clones, and the latter are isolated from the cell culture and cloned. Subsequently, the stable recombinant cell clones obtained under serum- and protein-free conditions are further multiplied under serum- and protein-free conditions.
  • The recombinant cell clone according to the invention is characterized in that it is stable in serum-free and protein-free medium for at least 40, preferably at least 50, in particular more than 60 generations and expresses recombinant product.
  • According to a particular aspect of the invention, the stable recombinant cell clone is present in isolated form. Departing from the stable cell clone, a cell culture is obtained under serum- and protein-free conditions by multiplying the stable cells.
  • The stable recombinant cell clone of the invention preferably is derived from a recombinant mammalian cell. The recombinant mammalian cells may be all cells that contain sequences which encode a recombinant polypeptide or protein. Included are all continuously growing cells which grow adherently and non-adherently. Particularly preferred are recombinant CHO cells or BHK cells. Recombinant polypeptides or proteins may be blood factors, growth factors or other biomedically relevant products.
  • According to the present invention, stable recombinant cell clones are preferred which contain the encoding sequence for a recombinant blood factor, such as factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, protein S, protein C, an activated form of any one of these factors, or VWF, and which are capable of stable expression of this factor over several generations. Particularly preferred are recombinant CHO cells, which express VWF or a polypeptide having VWF activity, factor VIII or a polypeptide having factor VIII activity, VWF and factor VIII, factor IX or factor II.
  • The cell clone of the invention selected under serum and protein-free conditions is particularly characterized in that it is stable in serum and protein-free medium for at least 40, preferably for at least 50 generations, particularly preferred for more than 60 generations.
  • To provide a master cell bank, 30 generations are required. To carry out an average batch culture on a 1,000 liter scale, at least approximately 40 generations are required. Thus it has become possible for the first time to prepare with one individual clone a master cell bank (MCB), a working cell bank (WCB) including approximately 8 to 10 generations and thus, a production-scale cell culture (production biomass) with up to 20 to 25 generations under these conditions, since so far cell clones have become unstable after having grown on serum- or protein-free medium for some generations, or have exhibited a reduced viability, whereby a) no uniform cell culture with product producers, and b) no stable product productivity has been possible over an extended period of time.
  • The cell clone according to the invention thus is stable and productive for at least 40 generations under production conditions in serum and protein-free medium. Previously described methods merely exhibited a product productivity for a generation number of less than 10 generations under protein-free conditions (Reiter et al., (1992) supra).
  • The criterion for stability is held to be a minimum number of at least 40 generations, preferably more than 50 generations, particularly preferred more than 60 generations in the production process, during which a stable expression of the proteins takes place and the cells do not exhibit any tumorigenic properties.
  • Surprisingly it has been found that the cell clone according to the invention exhibits an increased product productivity under serum and protein-free conditions even in comparison to the original cell clone which had been cultured in serum containing medium.
  • In addition, it has surprisingly been found that the productivity of the cultivated cells may be increased by adding additional amino acids and/or purified, ultrafiltrated soybean peptone to the serum- and protein-free medium. In this case, the increase in productivity is not caused by the enhanced cell growth rate; rather, the culture conditions directly influence the productivity of the cells expressing a recombinant protein.
  • A particular aspect is the use of a protein- and serum-free medium to which a mixture of amino acids selected from the group of L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan and L-glutamine has been added.
  • The amino acids may be added to the medium individually or in combination.
  • Particularly preferred is the combined addition of all the amino acids listed in this group, i.e. L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan and L-glutamine.
  • The increase in productivity by the addition of the amino acid mixture, which may be thus achieved, was particularly surprising because the synthetic minimum media as described in the prior art, e.g. DMEM/HAM's F12, already contain low concentrations of amino acids.
  • According to a further aspect, the present invention provides a cell culture containing at least 90%, preferably more than 95%, particularly preferred more than 98%, stable recombinant cells which are stable under serum- and protein-free conditions for at least 40 generations, in particular for at least 50 generations, and express recombinant product.
  • Within the scope of the present invention, by cell culture a master cell bank (MCB), a working cell bank (WCB) or a production biomass in a large-technical production bioreactor is understood.
  • According to the invention, the cell culture is particularly obtained by culturing a stable recombinant cell clone of the above-defined kind under serum and protein-free conditions.
  • The cell culture of the invention is obtainable by multiplying the isolated stable cell clone from the individual clone, the seed cells up to the MCB, the WCB or a biomass on a production scale in the bioreactor under serum and protein-free conditions, preferably without selection pressure on the selection and/or marker gene. In particular, it has been shown that the recombinant cells in a cell culture which are obtained departing from the stable recombinant clone of the invention are stable under serum- and protein-free conditions for at least 40 generations.
  • The cell culture provided according to the present invention, which has been prepared from a serum and protein-independent stable cell clone, exhibits at least 90%, preferably at least 95%, particularly preferred at least 98%, stable recombinant cells under protein-free culturing and production conditions. By stable recombinant cells, in particular recombinant mammalian cells are understood which are derived from the stable cell clone. Preferred are recombinant CHO cells, preferably CHO-dhfr cells, CHO-K1 cells and BHK cells that express a blood factor, preferably recombinant vWF, factor VII, factor VIII and vWF, factor IX or factor II.
  • The cell culture according to the invention may contain the stable recombinant cells in the form of a suspension culture. The cells may also be immobilized on a carrier, in particular on a microcarrier, porous microcarriers being particularly preferred. Porous carriers, such as e.g. Cytoline® or Cytopore® have proved to be particularly suitable.
  • According to a further aspect, the present invention provides a method for the large-technical production of a recombinant product under serum and protein-free conditions, by using the stable cell clone according to the invention. The method comprises the steps of providing an isolated, stable recombinant cell clone of the above-defined kind for producing a cell culture. Multiplication of the isolated stable cell clone is effected from the stable individual cell clone up to the cell culture under serum and protein-free conditions. In particular, also sub-culturing of the stable cell clones is effected under protein-free conditions, in particular without the addition of a protease, such as, e.g., trypsin. Thus it is ensured that at no time during the production of a cell culture used in the production of a recombinant product, a contamination occurs which possibly could be caused by the addition of serum or protein-containing additives of human or animal origin to the cell culture. Thus, for the first time a method is described which allows for working under serum- and protein-free conditions, starting from the original clone, via the preparation of a working cell bank as far as to the production biomass and the subsequent production of recombinant protein.
  • The preparation of the recombinant products with the cell culture of the invention which contains more than 90%, preferably more than 95%, particularly preferred more than 98%, of stable product producer cells, may be effected as a suspension culture or with cells immobilized on carriers. The process may be effected as a batch-wise or a continuous method or by means of perfusion technique with serum and protein free medium.
  • Moreover, the culturing process may be effected by means of the chemostat method as extensively described in prior art (Wemer et al., J. Biotechnol., 22:51-68 (1992)). For example, a stirred bioreactor or an airlift reactor may be used.
  • The expressed recombinant proteins are then recovered from the cell culture supernatant, purified by means of methods known from the prior art, and further processed.
  • Any known synthetic medium can be used as the serum and protein-free medium. Conventional synthetic minimum media may contain inorganic salts, amino acids, vitamins and a carbohydrate source and water. It may, e.g., be DMEM/HAM's F-12 medium. The content of soybean or yeast extract may range between 0.1 and 100 g/l, particularly preferred between 1 and 5 g/l. As a particularly preferred embodiment, soybean extract, e.g. soybean peptone, may be used. The molecular weight of the soybean peptone can be less than 50 kD, preferably less than 10 kD.
  • The addition of ultrafiltrated soybean peptone having an average molecular weight of 350 Dalton has proven particularly advantageous for the productivity of the recombinant cell lines. It is a soybean isolate having a total nitrogen content of about 9.5% and a free amino acid content of about 13%.
  • Particularly preferred is the use of a purified, ultrafiltrated soybean peptone having a molecular weight of <1000 Dalton, preferably <500 Dalton, particularly preferably <350 Dalton.
  • Ultrafiltration may be effected by means of methods extensively described in prior art, e.g., using membrane filters with a defined cut-off.
  • The ultrafiltration soybean peptone may be purified by means of gel chromatography, for example using Sephadex chromatography, e.g., Sephadex G25 or Sephadex G10 or equivalent materials; ion exchange chromatography, or size exclusion chromatography or reversed phase chromatography. These are methods well known to a skilled artisan from prior art.
  • Particularly preferably a medium having the following composition is used: synthetic minimum medium (1 to 25 g/l) soybean peptone (0.5 to 50 g/l), L-glutamine (0.05 to 1 g/l), NaHC03 (0.1 to 10 g/l), ascorbic acid (0.0005 to 0.05 g/l), ethanol amine (0.0005 to 0.05), Na selenite (1 to 15 μg/l). Optionally, a non-ionic surface-active agent, such as, e.g., polypropylene glycol (PLURONIC F-61, PLURONIC F-68, SYNPERONIC F-68, PLURONIC F-71, or PLURONIC F-108) maybe added to the medium as a defoaming agent. This agent is generally applied to protect the cells from the negative effects of aeration, since without the addition of a surface-active agent, the rising and bursting air bubbles may damage those cells which are at the surface of these air bubbles (“sparging”). (See, e.g., Murhammer and Goochee, Biotechnol. Prog., 6:142148 (1990)).
  • The amount of non-ionic surface-active agent may range between 0.05 and 10 g/l, particularly preferred is as low an amount as possible, between 0.1 and 5 g/l. Furthermore, the medium may also contain cyclodextrine or a derivative thereof. The addition of non-ionic surface-active agent or of cyclodextrine is, however, not essential to the invention. Preferably, the serum and protein-free medium contains a protease inhibitor, such as, e.g., serine protease inhibitors, which are suitable for tissue culture and which are of synthetic or vegetable origin.
  • In another preferred embodiment the following amino acid mixture is additionally added to the above-mentioned medium: L-asparagine (0.001 to 1 g/l; preferably 0.01 to 0.05 g/l; particularly preferably 0.015 to 0.03 g/l), L-cysteine (0.001 to 1 g/l; preferably 0.005 to 0.05 g/l; particularly preferably 0.01 to 0.03 g/l), L-cystine (0.001 to 1 g/l; preferably 0.01 to 0.05 g/l; particularly preferably 0.015 to 0.03 g/l). L-proline (0.001 to 1.5 g/l; preferably 0.01 to 0.07 g/l; particularly preferably 0.02 to 0.05 g/l), L-tryptophan (0.001 to 1 g/l; preferably 0.01 to 0.05 g/l; particularly preferably 0.015 to 0.03 g/l) and L-glutamine (0.05 to 10 g/l; preferably 0.1 to 1 g/l). The above-mentioned amino acids may be added to the medium individually or in combination. Particularly preferred is the combined addition of the amino acid mixture containing all of the above-mentioned amino acids.
  • In a particular embodiment a serum- and protein-free medium is used additionally containing a combination of the above-mentioned amino acid mixtures and purified, ultrafiltrated soybean peptone.
  • Surprisingly, it has proven possible to heat the medium to 70 to 95° C., preferably 85 to 95° C., for about 5 to 20 minutes, preferably 15 minutes, without causing negative effects, e.g., in order to inactivate viruses or other pathogens.
  • The parameters for culturing the cells, such as O2 concentration, perfusion rate or medium exchange, pH, temperature and culturing technique will depend on the individual cell types used and may be determined by the skilled artisan in a simple manner. For instance, culturing of CHO cells may be effected in a stirring tank and under perfusion with protein-free medium at a perfusion rate of from 2 to 10 volume exchanges/day, at a pH of between 7.0 and 7.8, preferably at 7.4, and an O2 concentration of between 40% up to 60%, preferably at 50%, and at a temperature of between 340 and 38°, preferably of 37°.
  • Moreover, the cells may also be cultured by means of the chemostat method, using a pH of between 6.9 and 7.8, preferably 7.1, an O2 concentration of between 10% and 60%, preferably 20%, and a dilution rate D of 0.25 to 1.0, preferably 0.5.
  • According to a further aspect, the present invention provides a method of recovering a stable recombinant cell clone, comprising the steps of
      • multiplying a recombinant original clone up to the cell culture in serum-containing medium, preferably without selection pressure,
      • culturing the cells under serum and protein-free, preferably under production equivalent, conditions,
      • assaying the cell culture under serum and protein free conditions for product producers,
      • cloning the stable recombinant cell clones under serum- and protein-free conditions, wherein cloning may be effected by generally known techniques, such as diluting out and growing the individual cell clones,
      • multiplying the isolated cell clones under serum and protein-free conditions,
      • and optionally assaying the cell culture for product producers.
  • Only those recombinant cell clones are considered stable which express recombinant protein in a stable manner in protein-free medium for at least 10, preferably at least 20, and in particular at least 50 generations.
  • According to a further aspect, the invention relates to a method of recovering a stable recombinant cell clone, comprising the steps of
      • multiplying a non-recombinant starting cell or cell line under serum and protein-free conditions, and cloning a stable non-recombinant cell-clone under serum- and protein-free conditions,
      • transfecting the stable cell clone with a recombinant nucleic acid and isolating stable recombinant cell clones,
      • Culturing the stable cell clone transfectants in serum- and protein-free medium, optionally under production-equivalent conditions,
      • assaying the stable recombinant cells for production and product stability.
  • A particularly preferred aspect of the present invention provides the preparation of a stable cell clone comprising the steps of
      • multiplying a recombinant original clone up to the cell culture in serum- and protein-free medium, preferably without selection pressure;
      • culturing the cells under serum- and protein-free, preferably under production-equivalent conditions,
      • assaying the cell culture under serum- and protein-free conditions for product producers,
      • cloning the stable recombinant cell clones under serum- and protein-free conditions, wherein cloning may be effected by generally known techniques, such as diluting out and growing the individual cell clones,
      • multiplying the isolated cell clones under serum- and protein-free conditions, and
      • optionally assaying the cell culture for product producers.
  • Only those recombinant cell clones are considered stable which express recombinant protein in a stable manner in protein-free medium for at least 10, preferably at least 20, and in particular at least 50 generations.
  • Also preferred is the use of a serum- and protein-free medium to increase the productivity of a cell clone expressing a recombinant protein to which additionally a defined amino acid mixture and/or purified, ultrafiltrated soybean peptone have been added.
  • The invention will be described in more detail by way of the following examples, as well as drawings to which, however, it shall not be restricted.
  • EXAMPLES Example 1 Stability of rvWF-CHO Cells After Re-Adaptation from Serum-Containing to Serum- and Protein-Free Medium
  • CHO-dhfr cells were co-transfected with plasmids phAct-rvWF and pSV-dhfr, and vWF-expressing clones, as described in Fischer et al., FEBS Letters, 351:345-348 (1994)) were sub-cloned. From those sub-clones which expressed rvWF in a stable manner, a working cell bank (WCB) was set up under serum-containing conditions, yet in the absence of MTX, and the cells were immobilized on a porous microcarrier (Cytopore®) under serum-containing conditions. When a cell density of 2×107 cells/ml carrier matrix had been reached, readaptation of the cells to serum- and protein-free medium was effected. The cells were continued to be cultured for several generations under serum- and protein-free conditions. By means of immunofluorescence with labeled anti-vWF antibodies, the cells were assayed in serum- and protein-free medium at different points of time. The evaluation of the stability of the cells of the working cell bank was effected prior to medium readaptation, after 10 and after 60 generations in serum- and protein-free medium. Whereas the working cell bank still had 100% rvWF producers (FIG. 1A), the portion of rvWF producers after 10 generations in serum- and protein-free medium had decreased to approximately 50% (FIG. 1B). After 60 generations, more than 95% of the cells were identified as non-producers (FIG. 1C).
  • Example 2 Cloning of Stable Recombinant CHO Clones
  • From the cell culture containing rvWF-CHO cells according to Example 1, (the stable cell clone designated r-vWF-CHO F7 was deposited on Jan. 22, 1998, at the European Collection of Cell Cultures (ECACC), Salisbury, Wilshire, SP4 0JG, UK, according to the Budapest Treaty, and received the deposit number 98012206) which had been cultured for 60 generations in serum- and protein-free medium (FIG. 1C), a dilution was made, and 0.1 cells/well were each seeded in a micro-titer plate. The cells were cultured for approximately 3 weeks in DMEM/HAM's F12 without serum or protein additions and without selection pressure, and the cells were assayed by means of immunofluorescence with labeled anti-vWF antibody. A cell clone that had been identified as positive was used as the starting clone for the preparation of a seed cell bank. From the seed cell bank, a master cell bank (MCB) was prepared in serum- and protein-free medium, and individual ampoules were frozen off for the further preparation of a working cell bank. Departing from an individual ampoule, a working cell bank was prepared in serum- and protein-free medium. The cells were immobilized on porous microcarriers and continued to be cultured for several generations under serum- and protein-free conditions. At different points of time the cells were assayed for their productivity in serum- and protein-free medium by means of immunofluorescence with labeled anti-vWF antibodies. Evaluation of the stability of the cells was effected at the working cell bank stage and after 10 and 60 generations in serum- and protein-free medium. At the working cell bank stage (FIG. 2A) and also after 10 (FIG. 2B) and 60 generations (FIG. 2C), approximately 100% of the cells were identified as positive stable recombinant clones that express rvWF.
  • Example 3 Cell-Specific Productivity of the Recombinant Cell Clones
  • From defined stages during the culturing of recombinant cells, a defined number of cells was taken and incubated with fresh medium for 24 h. The rvWF:Risto-CoF activity was determined in the cell culture supernatants. Table 1 shows that the cell specific productivity in the inventive stable recombinant cell clones was still stable even after 60 generations in serum- and protein-free medium and was even higher in comparison to the original clone that had been cultured in serum-containing medium.
  • TABLE 1
    Cell specific Cell specific Cell specific
    productivity of the productivity after 10 productivity after 60
    working cells mU generations mU generations mU
    Cell Clone rvWF/106 cells/day rvWF/106 cells/day rvWF/106 cells/day
    rvWF-CHO #808.68 55 30 <10
    Original cell clone
    r-vWF-CHO F7 62 65 60
    Stable clone
  • Example 4 Composition of a Synthetic Serum- and Protein-Free Medium
  • Preferred amount
    (according to the
    knowledge at the time
    Component g/l of application) in g/l
    Synthetic minimum medium   1-100 11.00-12.00
    (DMEM/HAM's 12)
    Soybean peptone 0.5-50 2.5
    L-Glutamine 0.5-1  0.36
    NaHC03 0.1-10 2.00
    Ascorbic acid 0.0005-0.05   0.0035
    Ethanol amine 0.0005-0.05   0.0015
    Na-selenite 1-15 μg/l 8.6 μg/l
    optional: 0.01-10  0.25
    Synperonic F 68
  • Example 5 Culturing of rFVIII-CHO Cells in Protein- and Serum-Free Minimum Medium
  • A cell culture containing rFVIII-CHO cells was cultured in a 10 l stirring tank and with perfusion. A medium according to Example 4 was used. The cells were immobilized on a porous microcarrier (Cytopore©, Pharmacia) and cultured for at least 6 weeks. The perfusion rate was 4 volume exchanges/day, the pH was at 6.9-7.2, the O2 concentration was approximately 20-50%, 15 the temperature was 37° C.
  • FIG. 3 shows the results of culturing an rFVIII-CHO cell clone in a 10 l perfusion bioreactor.
      • a) F VIII-activity (mUnits/ml) and perfusion rate (1-5/day) over a period of 42 days.
      • b) Volumetric productivity (units factor VIII/l/day) in the perfusion bioreactor.
  • Days of Cell-specific productivity Immunofluorescence (%
    culturing mU/106 cells/day) FVIII-positive cells)
    15 702 not indicated
    21 1125 not indicated
    28 951 >95%
    35 691 >95%
    42 970 not indicated
  • Table 2 shows the stability and specific productivity of the rFVIII-expressing cells. For these results, samples were taken after 15, 21, 28, 35 and 42 days, centrifuged at 300 g and re-suspended in fresh serum- and protein-free medium. After further 24 h, the factor VIII concentration in the cell culture supernatants and the cell number were determined. Based on these data, the specific FVIII productivity was calculated.
  • A stable average productivity of 888 mUnits/106 cells/day was attained. This stable productivity was also confirmed by immunofluorescence with labeled anti-FVIII antibodies after 15, 21, 28, 35 and 42 days in serum- and protein-free medium.
  • Example 6 Comparison of the Productivity of Recombinant FVIII-CHO Cells in Protein- and Serum-Free Medium Containing Additional Medium Components
  • A cell culture containing rFVIII-CHO cells was cultured using a batch method. A medium according to example 4 was used to which the following amino acids were added:
  • Preferred amount (according to
    the knowledge at the time of
    Amino acid mg/l application) in mg/l
    L-asparagine 1-100 20
    L-cysteine•HCl•H20 1-100 15
    L-cystine 1-100 20
    L-proline 1-150 35
    L-tryptophan 1-100 20
    L-glutamine 50-1000 240
  • The cells were cultured at 37° C., pH 6.9-7.2. The cells were cultivated over a period of 24-72 hours in a batch process.
  • The productivity of the recombinant FVIII-CHO cells was measured in the following media compositions:
  • Mix 1 consisting of serum- and protein-free medium without soybean peptone and additionally containing a mixture of amino acids as listed in the above table
  • Mix 2 consisting of serum- and protein-free medium containing soybean peptone
  • Mix 3 consisting of serum- and protein-free medium containing soybean peptone and a mixture of amino acids as listed in the above table
  • Mix 4 consisting of serum- and protein-free medium containing, in addition, a mixture of amino acids as listed in the above table and 2.5 g/l of purified, ultrafiltrated soybean peptone. The ultrafiltrated soybean peptone was purified by means of chromatography using a Sephadex© column.
  • Example 7 Culturing of Recombinant FVIII-CHO Cells in Protein- and Serum-Free Medium Using Chemostat Culture
  • A cell culture containing rFVIII-CHO cells was cultured in a 10 l stirred bioreactor tank. A medium according to example 4 not containing soybean peptone but containing an amino acid mixture according to example 6 was used.
  • The cells were cultivated at 37° C., pH 6.9-7.2; the oxygen concentration was in the range of 20-50% air saturation. In order to determine the titer of factor VIII and the cell concentration in culture supernatant, samples were taken every 24 hours. The total cell concentration was constant from day 2 to day 14. From day 6, ultrafiltrated soybean peptone was added to the medium. The factor VIII productivity is measured by means of a CHROGENIX COA FVIII:c/4 system. The lack of soybean peptone in the continuous culture lead to a marked decrease in factor VIII productivity after a few days, whereas the addition of the soybean peptone resulted in an almost 10-fold increase in productivity. Because said addition did not increase the cell number, this clearly indicates that ultrafiltrated soybean peptone causes a marked increase in productivity, which, however, is independent of cell growth.

Claims (14)

1. A method for producing a stable recombinant mammalian cell clone, which is stable under production conditions in serum- and protein-free medium for at least 40 generations, the method comprising:
providing a recombinant original mammalian cell clone,
cultivating the recombinant original cell clone on serum-containing medium,
adapting the cells to serum- and protein-free medium in the absence of a selection pressure,
testing the cell culture after adaptation for stable product-producers in the absence of a selection pressure and
cloning a stable product-producer-cell clone in serum- and protein-free conditions in the absence of a selection pressure.
2. The method according to claim 1, wherein the stable cell clone obtained is present in isolated form after the step of cloning.
3. The method according to claim 1, wherein the recombinant cell clone comprises a nucleic acid encoding a recombinant polypeptide or protein.
4. A stable recombinant mammalian cell clone, obtainable by a method according to of claim 1, wherein the cell clone is stable for at least 40 generations and expresses a recombinant product under production conditions in serum- and protein-free medium, in the absence of a selection pressure.
5. The cell clone according to claim 4, wherein the clone is present in isolated form after the step of cloning.
6. The cell clone according to claim 4, wherein the cell comprises a nucleic acid encoding a recombinant polypeptide or protein.
7. A cell culture obtainable by cultivating a stable recombinant cell clone according to claim 1.
8. The cell culture of claim 7, wherein the cell culture contains at least 90% stable recombinant cell clones.
9. The cell culture according to claim 7, wherein the stable recombinant cells comprise a nucleic acid encoding a recombinant polypeptide or protein.
10. The cell culture according to claim 7, wherein the stable recombinant cells are immobilised on a microsupport.
11. A method for industrial production of a recombinant product under serum- and protein-free conditions, the method comprising:
providing an isolated, stable recombinant cell clone according to claim 4,
propagating the stable cell clone in serum- and protein-free medium from the initial clone up to the cell culture in the absence of a selection pressure,
producing the cell culture containing stable cells in the bioreactor in the absence of a selection pressure, and
harvesting the recombinant product from the culture supernatant.
12. The method according to claim 11, wherein the serum- and protein-free medium is a synthetic minimal medium comprising an extract selected from the group consisting of: a yeast extract and a soy extract.
13. The method according to claim 11, wherein the medium contains cyclodextrin or a derivative thereof.
14. The method according to claim 11, wherein the serum- and protein-free medium contains a protease inhibitor.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060148074A1 (en) * 1996-08-30 2006-07-06 Invitrogen Corporation Serum-free mammalian cell culture medium, and uses thereof

Families Citing this family (55)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6475725B1 (en) * 1997-06-20 2002-11-05 Baxter Aktiengesellschaft Recombinant cell clones having increased stability and methods of making and using the same
US6747003B1 (en) * 1997-10-23 2004-06-08 Regents Of The University Of Minnesota Modified vitamin K-dependent polypeptides
US7220837B1 (en) 2000-04-28 2007-05-22 Regents Of The University Of Minnesota Modified vitamin K-dependent polypeptides
US7812132B2 (en) * 2000-04-28 2010-10-12 Regents Of The University Of Minnesota Modified vitamin K-dependent polypeptides
AT409379B (en) 1999-06-02 2002-07-25 Baxter Ag Medium for the protein and serum-free cultivation of cells
PL204285B1 (en) * 2000-02-11 2009-12-31 Bayer Healthcare Llc FACTOR VII OR VIIa−LIKE MOLECULES
US20030211094A1 (en) 2001-06-26 2003-11-13 Nelsestuen Gary L. High molecular weight derivatives of vitamin k-dependent polypeptides
CN102766668B (en) * 2000-10-02 2016-03-16 诺沃挪第克健康护理股份公司 Clotting factor type vii glycosyl
KR20040039472A (en) * 2001-10-02 2004-05-10 노보 노르디스크 헬스 케어 악티엔게젤샤프트 Method for production of recombinant proteins in eukaryote cells
US20040185534A1 (en) * 2000-10-02 2004-09-23 Knudsen Ida Molgaard Industrial-scale serum-free production of recombinant proteins in mammalian cells
AU2003221469A1 (en) * 2002-04-30 2003-11-17 Bayer Healthcare Llc FACTOR VII OR VIIa POLYPEPTIDE VARIANTS
JP3916516B2 (en) * 2002-06-10 2007-05-16 独立行政法人物質・材料研究機構 Hard tissue - soft tissue interface for reproduction scaffolding material
NZ538094A (en) * 2002-07-09 2007-01-26 Baxter Int Serum free and animal protein free culture medium for cultivation of cells
US20060166874A1 (en) * 2002-09-30 2006-07-27 Haaning Jesper M Fvii or fviia variants having increased clotting activity
DK2085470T3 (en) * 2003-03-20 2012-08-06 Bayer Healthcare Llc FVII- or FVIIa variants
US20040185535A1 (en) * 2003-03-21 2004-09-23 Giles Wilson Industrial-scale serum-free production of recombinant FVII in mammalian cells
CN1806040A (en) * 2003-06-10 2006-07-19 株式会社岛津制作所 Extract from cultured mammalian cell, process for preparation thereof and method of cell-free protein synthesis using the extract
NZ573412A (en) * 2003-06-19 2010-09-30 Maxygen Holdings Ltd Factor VII or VIIa Gla domain variants
US7255288B2 (en) * 2004-03-08 2007-08-14 Wan Shan Chan Aroma therapy for fountain
BRPI0514984A (en) 2004-09-07 2008-07-01 Archemix Corp aptamers for von Willebrand factor and its use as a therapeutic for thrombotic disease
WO2006050050A2 (en) * 2004-10-29 2006-05-11 Centocor, Inc. Chemically defined media compositions
US20060094104A1 (en) * 2004-10-29 2006-05-04 Leopold Grillberger Animal protein-free media for cultivation of cells
US8273553B2 (en) * 2004-11-02 2012-09-25 Ares Trading S.A. Production of growth hormone in serum-free cell culture medium for mammalian cells
KR100679112B1 (en) 2004-11-17 2007-02-07 삼성정밀화학 주식회사 Method for Animal Cell Culture
JP2008520250A (en) * 2004-11-19 2008-06-19 バイオジェン・アイデック・エムエイ・インコーポレイテッド The method for producing a mammalian cell
US20070231895A1 (en) * 2005-11-02 2007-10-04 Lee Gene W Methods for adapting mammalian cells
BRPI0618178A2 (en) * 2005-11-02 2011-08-23 Wyeth Corp methods for adapting mammalian cells
ES2474573T3 (en) * 2006-01-04 2014-07-09 Baxter International Inc Cell culture medium without oligopptidos
WO2007149406A2 (en) * 2006-06-19 2007-12-27 Nautilus Technology Llc Modified coagulation factor ix polypeptides and use thereof for treatment
EP2592148B1 (en) * 2007-10-12 2018-09-12 F. Hoffmann-La Roche AG Protein expression from multiple nucleic acids
KR101798813B1 (en) 2007-12-27 2017-11-16 박스알타 인코퍼레이티드 Cell culture processes
JP5451884B2 (en) 2009-07-31 2014-03-26 バクスター・インターナショナル・インコーポレイテッドBaxter International Incorp0Rated Cell culture medium for Adamts protein expression
GB0915481D0 (en) 2009-09-04 2009-10-07 Arecor Ltd Stable manufacture of factor V111
MX362382B (en) 2009-09-21 2019-01-14 Baxter Int Stabilized liquid and lyophilized adamts13 formulations.
WO2011065940A1 (en) 2009-11-24 2011-06-03 Biogen Idec Ma Inc. Method of supplementing culture media to prevent undesirable amino acid substitutions
US9458222B2 (en) 2010-07-08 2016-10-04 Baxalta Incorporated Method of producing recombinant ADAMTS13 in cell culture
TWI621625B (en) 2010-09-17 2018-04-21 Baxalta Inc Stabilization of immunoglobulins through aqueous formulation with histidine at weak acidic to neutral ph
TWI595004B (en) 2010-11-03 2017-08-11 Catalyst Biosciences Inc Modified factor ix polypeptides and uses thereof
EP2702077A2 (en) 2011-04-27 2014-03-05 AbbVie Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US9181572B2 (en) 2012-04-20 2015-11-10 Abbvie, Inc. Methods to modulate lysine variant distribution
US9334319B2 (en) 2012-04-20 2016-05-10 Abbvie Inc. Low acidic species compositions
US9512214B2 (en) 2012-09-02 2016-12-06 Abbvie, Inc. Methods to control protein heterogeneity
ES2633960T3 (en) 2012-10-15 2017-09-26 Bristol-Myers Squibb Company Culture processes mammalian cells for protein production
SG11201507230PA (en) 2013-03-12 2015-10-29 Abbvie Inc Human antibodies that bind human tnf-alpha and methods of preparing the same
US9499614B2 (en) 2013-03-14 2016-11-22 Abbvie Inc. Methods for modulating protein glycosylation profiles of recombinant protein therapeutics using monosaccharides and oligosaccharides
US9067990B2 (en) 2013-03-14 2015-06-30 Abbvie, Inc. Protein purification using displacement chromatography
EP2970843A1 (en) 2013-03-15 2016-01-20 Genzyme Corporation High-density cell banking methods
US9700486B2 (en) 2013-04-24 2017-07-11 Corning Incorporated Delamination resistant pharmaceutical glass containers containing active pharmaceutical ingredients
US9598667B2 (en) 2013-10-04 2017-03-21 Abbvie Inc. Use of metal ions for modulation of protein glycosylation profiles of recombinant proteins
US9017687B1 (en) 2013-10-18 2015-04-28 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same using displacement chromatography
US9181337B2 (en) 2013-10-18 2015-11-10 Abbvie, Inc. Modulated lysine variant species compositions and methods for producing and using the same
US9085618B2 (en) 2013-10-18 2015-07-21 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
WO2015073884A2 (en) 2013-11-15 2015-05-21 Abbvie, Inc. Glycoengineered binding protein compositions
KR20170054507A (en) 2014-09-18 2017-05-17 젠자임 코포레이션 Ultra-high density cell banking methods
WO2018162517A1 (en) 2017-03-10 2018-09-13 F. Hoffmann-La Roche Ag Method for producing multispecific antibodies

Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4431629A (en) * 1980-05-13 1984-02-14 Novo Industri A/S Method of producing an egg white substitute material
US4443540A (en) * 1980-05-09 1984-04-17 University Of Illinois Foundation Protein hydrolysis
US4767704A (en) * 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
US4978616A (en) * 1985-02-28 1990-12-18 Verax Corporation Fluidized cell cultivation process
US5122469A (en) * 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
US5316938A (en) * 1990-10-17 1994-05-31 Burroughs Wellcome Co. Defined media for serum-free tissue culture
US5378612A (en) * 1990-05-11 1995-01-03 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Culture medium for production of recombinant protein
US5393668A (en) * 1991-09-11 1995-02-28 Hans-Wilhelm Doerr Cultivation of mammalian cells in a protein-free medium on a polyvinylformal and/or polyvinyl butyral surface
US5441868A (en) * 1983-12-13 1995-08-15 Kirin-Amgen, Inc. Production of recombinant erythropoietin
US5573937A (en) * 1989-12-07 1996-11-12 Snow Brand Milk Products Co., Ltd. Serum free culture medium
US5719050A (en) * 1993-12-24 1998-02-17 Eiken Chemical Co., Ltd. Animal cell culturing media containing N-acetyl-L-glutamic acid
US5741705A (en) * 1995-02-23 1998-04-21 Quest International Flavors & Food Ingredients Company, Division Of Indopco, Inc. Method for in vitro cell growth of eucaryotic cells using low molecular weight peptides
US5789247A (en) * 1994-04-01 1998-08-04 Ballay; Annick Expression in non-tumoral human lymphoblastoid lines with an integrative vector
US5811299A (en) * 1994-02-08 1998-09-22 Renner; Wolfgang A. Methods for the activation of proliferation of animal cells
US5851800A (en) * 1996-05-14 1998-12-22 Pharmacia & Upjohn Ab Process for producing a protein
US6048728A (en) * 1988-09-23 2000-04-11 Chiron Corporation Cell culture medium for enhanced cell growth, culture longevity, and product expression
US6100061A (en) * 1997-06-20 2000-08-08 Immuno Aktiengesellschaft Recombinant cell clone having increased stability in serum- and protein-free medium and a method of recovering the stable cell clone and the production of recombinant proteins by using a stable cell clone
US20020182679A1 (en) * 1997-06-20 2002-12-05 Baxter Healthcare Corporation Recombinant cell clones having increased stability and methods of making and using the same
US20030203448A1 (en) * 1999-09-28 2003-10-30 Manfred Reiter Medium for the protein-free and serum-free cultivation of cells
US20060148074A1 (en) * 1996-08-30 2006-07-06 Invitrogen Corporation Serum-free mammalian cell culture medium, and uses thereof

Family Cites Families (80)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT165999B (en) 1947-06-26 1950-05-25 Delle Atel Const Electr Einirchtung for the protection of three-phase motors against overcurrent
FR2196386A1 (en) 1972-08-17 1974-03-15 Cudennec Alain Culture media selection - for identification of unknown bacteria
US6455275B1 (en) 1980-02-25 2002-09-24 The Trustees Of Columbia University In The City Of New York DNA construct for producing proteinaceous materials in eucaryotic cells
AU546577B2 (en) 1980-02-25 1985-09-05 Trustees Of Columbia University, The Use of eucaryotic promoter sequences in the production of proteinaceous materials
DE3126388A1 (en) * 1981-07-03 1983-01-27 Bayer Ag A process for preparing hydrazidines
JPS59501533A (en) 1982-08-10 1984-08-30
US4637510A (en) 1982-10-02 1987-01-20 Anderson Strathclyde Plc Line pans
NZ206699A (en) 1982-12-30 1989-08-29 Bio Response Inc Process for the production of serum independent cell lines
IL74909A (en) 1984-04-20 1992-01-15 Genentech Inc Preparation of functional human factor viii and dna sequences,expression vectors,transformed microorganisms and cell lines used therein
US4965199A (en) 1984-04-20 1990-10-23 Genentech, Inc. Preparation of functional human factor VIII in mammalian cells using methotrexate based selection
GB8415186D0 (en) 1984-06-14 1984-07-18 Ciba Geigy Ag Polypeptides
US4672007A (en) * 1984-08-16 1987-06-09 Kollmorgen Technologies Corporation Electrodeposition composition and process for providing a Zn/Si/P coating on metal substrates
EP0217822B1 (en) 1985-02-13 1993-05-12 Scios Nova Inc. Human metallothionein-ii promoter in mammalian expression system
WO1986006408A1 (en) 1985-04-22 1986-11-06 Genetics Institute, Inc. HIGH YIElD PRODUCTION OF ACTIVE FACTOR IX
US4822560A (en) * 1985-10-10 1989-04-18 The Furukawa Electric Co., Ltd. Copper alloy and method of manufacturing the same
WO1988000967A1 (en) 1986-08-04 1988-02-11 The University Of New South Wales Serum free tissue culture medium containing polymeric cell-protective agent
US5104796A (en) * 1987-04-06 1992-04-14 International Minerals & Chemical Corp. High titer production of human somatomedin c
CA1331157C (en) 1987-04-06 1994-08-02 Randal J. Kaufman Method for producing factor viii:c-type proteins
WO1989000192A1 (en) 1987-06-30 1989-01-12 Amgen Inc. Production of kallikrein
JP2507882B2 (en) 1988-02-17 1996-06-19 工業技術院長 External growth factor-independent growth preparation of good cell lines
US5284756A (en) 1988-10-11 1994-02-08 Lynn Grinna Heterodimeric osteogenic factor
US5202246A (en) * 1989-08-16 1993-04-13 Armour Pharmaceutical Company Treatment of immobilized matrices for preparation of pharmaceutical and biological products with anti-microbial agents to remove pyrogen-producing organisms and pyrogens
SE465222C5 (en) 1989-12-15 1998-02-10 Pharmacia & Upjohn Ab A recombinant human factor VIII derivative and method for its preparation
CA2074363C (en) 1990-01-22 2004-11-09 David Thomas Vistica Co2-independent growth medium for maintenance and propagation of cells
JP2844484B2 (en) 1990-02-22 1999-01-06 味の素株式会社 The method of producing a recombinant protein
JP2859679B2 (en) 1990-03-01 1999-02-17 協和醗酵工業株式会社 New cell lines
JPH049498A (en) * 1990-04-26 1992-01-14 Nkk Corp Metallic plate plated with nickel-phosphorus alloy which has excellent peeling property and high hardness and production thereof
GB9022543D0 (en) 1990-10-17 1990-11-28 Wellcome Found Antibody production
AU643077B2 (en) 1990-10-19 1993-11-04 Unilever Plc Detergent compositions
JPH04228066A (en) 1990-10-23 1992-08-18 Rikagaku Kenkyusho Culture cell for expressing exogenote
GB9027917D0 (en) 1990-12-21 1991-02-13 Ici Plc Expression systems
JP2696001B2 (en) 1991-04-15 1998-01-14 帝人株式会社 Medium for recombinant protein production
US5353721A (en) 1991-07-15 1994-10-11 Manufacturing And Technology Conversion International Pulse combusted acoustic agglomeration apparatus and process
JPH05123178A (en) 1991-11-01 1993-05-21 Ajinomoto Co Inc Production of l-phenylalanine
NZ256921A (en) 1992-10-02 1996-05-28 Pharmacia Ab Formulations of coagulation factor viii and a non-ionic surfactant and their preparation
DE4311872C2 (en) * 1993-04-10 1998-07-02 Heraeus Gmbh W C Leadframe for integrated circuits
AU7895898A (en) 1993-04-26 1998-10-08 Hans Wolf Mammal cell lines and method of obtaining glycoproteins
DE4313620A1 (en) 1993-04-26 1994-10-27 Biotechnolog Forschung Gmbh Hamster cell lines and methods for Glykoproteingewinnung
EP0625574A1 (en) 1993-05-19 1994-11-23 Takeda Chemical Industries, Ltd. Production for biologically active polypeptide
DK0874057T3 (en) 1993-06-10 2004-11-22 Bayer Ag Vector and mammalian cell line with enhanced production capacity
US5405637A (en) 1993-06-30 1995-04-11 Bristol-Myers Squibb Company Milk protein partial hydrolysate and infant formula containing same
JP2766165B2 (en) 1993-08-02 1998-06-18 株式会社バイオポリマー・リサーチ Method for producing a bacterial cellulose
US5733782A (en) 1993-10-25 1998-03-31 Creative Biomolecules, Inc. Methods and compositions for high protein production from non-native DNA
EP0733100A1 (en) 1994-09-09 1996-09-25 James E. Bailey Chemical process for promoting the proliferation of animal cells
AT542891T (en) 1994-11-10 2012-02-15 Baxter Healthcare Sa A process for the production of biological products in protein-free culture
AT403167B (en) 1994-11-14 1997-11-25 Immuno Ag Selection and expression of foreign proteins by means of a selection amplification systems
WO1996018734A1 (en) 1994-12-16 1996-06-20 Novartis Ag Production of recombinant secretory component
CA2211630A1 (en) 1995-02-23 1996-08-29 Quest International B.V. Peptides for tissue and cell culture media
WO1996040866A1 (en) 1995-06-07 1996-12-19 Novartis Ag Serum-free media for primitive hematopoietic cells and methods of use thereof
AUPN442295A0 (en) 1995-07-26 1995-08-17 Commonwealth Scientific And Industrial Research Organisation Regulated autocrine growth of mammalian cells
US6479279B2 (en) 1995-12-29 2002-11-12 Estonian Biocentre Episomal vectors and uses thereof
US6025155A (en) 1996-04-10 2000-02-15 Chromos Molecular Systems, Inc. Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes
AU4751697A (en) 1996-10-10 1998-05-05 Douglas Danner Animal cell culture media comprising plant-derived nutrients
US5804420A (en) * 1997-04-18 1998-09-08 Bayer Corporation Preparation of recombinant Factor VIII in a protein free medium
WO1998054296A1 (en) 1997-05-28 1998-12-03 Chiron S.P.A. Culture medium with yeast or soy bean extract as aminoacid source and no protein complexes of animal origin
US6099624A (en) * 1997-07-09 2000-08-08 Elf Atochem North America, Inc. Nickel-phosphorus alloy coatings
EP0986644B1 (en) 1997-07-23 2006-10-04 Boehringer Mannheim GmbH Production of erythropoietin by endogenous gene activation with viral promoters
WO1999057246A1 (en) 1998-05-01 1999-11-11 Life Technologies, Inc. Animal cell culture media comprising non-animal or plant-derived nutrients
US6406909B1 (en) 1998-07-10 2002-06-18 Chugai Seiyaku Kabushiki Kaisha Serum-free medium for culturing animal cells
US6252607B1 (en) * 1998-07-27 2001-06-26 Attachmate Corporation Method and system for character spacing in graphics terminal emulation
US6051401A (en) 1998-07-28 2000-04-18 Bayer Corporation Methods and constructs for protein expression
US6210922B1 (en) 1998-11-30 2001-04-03 National Research Council Of Canada Serum free production of recombinant proteins and adenoviral vectors
US6358703B1 (en) 1998-12-10 2002-03-19 Bayer Corporation Expression system for factor VIII
US6855544B1 (en) 1999-04-15 2005-02-15 Crucell Holland B.V. Recombinant protein production in a human cell
US6338964B1 (en) 1999-05-07 2002-01-15 Bayer Corporation Process and medium for mammalian cell culture under low dissolved carbon dioxide concentration
US6323128B1 (en) * 1999-05-26 2001-11-27 International Business Machines Corporation Method for forming Co-W-P-Au films
US6767509B1 (en) * 1999-06-16 2004-07-27 Kimberly-Clark Worldwide, Inc. Self-sterilizing packaging
PT1200561E (en) 1999-08-05 2006-09-29 Baxter Ag Clone cellular recombinant stable, its production and utilization
IL148060D0 (en) 1999-08-25 2002-09-12 Immunex Corp Compositions and methods for improved cell culture
US6406611B1 (en) * 1999-12-08 2002-06-18 University Of Alabama In Huntsville Nickel cobalt phosphorous low stress electroplating
AT434035T (en) 2000-09-25 2009-07-15 Polymun Scient Immunbio Forsch Live influenza vaccine and process for its manufacture
IT1318937B1 (en) * 2000-09-27 2003-09-19 Getters Spa Method for the production of porous getter devices with ridottaperdita of particles and thus' products devices
JP4270768B2 (en) * 2000-11-08 2009-06-03 Jfeスチール株式会社 Tin-plated steel sheet and the chemical conversion treatment solution
EP1208966A1 (en) 2000-11-27 2002-05-29 Cheng-Kun Liao Manufacturing process of patio tabletop glass with broken protection
US6518873B1 (en) * 2001-09-13 2003-02-11 Bourns, Inc. Variable resistive element
CA2365749A1 (en) * 2001-12-20 2003-06-20 The Governors Of The University Of Alberta An electrodeposition process and a layered composite material produced thereby
US6808614B2 (en) * 2002-01-17 2004-10-26 Lucent Technologies Inc. Electroplating solution for high speed plating of tin-copper solder
US6726827B2 (en) * 2002-01-17 2004-04-27 Lucent Technologies Inc. Electroplating solution for high speed plating of tin-bismuth solder
US6730209B2 (en) * 2002-02-22 2004-05-04 Lucent Technologies Inc. Solder electroplating bath including brighteners having reduced volatility
NZ538094A (en) 2002-07-09 2007-01-26 Baxter Int Serum free and animal protein free culture medium for cultivation of cells

Patent Citations (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4443540A (en) * 1980-05-09 1984-04-17 University Of Illinois Foundation Protein hydrolysis
US4431629A (en) * 1980-05-13 1984-02-14 Novo Industri A/S Method of producing an egg white substitute material
US4767704A (en) * 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
US5441868A (en) * 1983-12-13 1995-08-15 Kirin-Amgen, Inc. Production of recombinant erythropoietin
US4978616A (en) * 1985-02-28 1990-12-18 Verax Corporation Fluidized cell cultivation process
US6048728A (en) * 1988-09-23 2000-04-11 Chiron Corporation Cell culture medium for enhanced cell growth, culture longevity, and product expression
US5573937A (en) * 1989-12-07 1996-11-12 Snow Brand Milk Products Co., Ltd. Serum free culture medium
US5378612A (en) * 1990-05-11 1995-01-03 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Culture medium for production of recombinant protein
US5122469A (en) * 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
US5633162A (en) * 1990-10-17 1997-05-27 Glaxo Wellcome Inc. Method for culturing Chinese hamster ovary cells
US5316938A (en) * 1990-10-17 1994-05-31 Burroughs Wellcome Co. Defined media for serum-free tissue culture
US5393668A (en) * 1991-09-11 1995-02-28 Hans-Wilhelm Doerr Cultivation of mammalian cells in a protein-free medium on a polyvinylformal and/or polyvinyl butyral surface
US5719050A (en) * 1993-12-24 1998-02-17 Eiken Chemical Co., Ltd. Animal cell culturing media containing N-acetyl-L-glutamic acid
US5811299A (en) * 1994-02-08 1998-09-22 Renner; Wolfgang A. Methods for the activation of proliferation of animal cells
US5789247A (en) * 1994-04-01 1998-08-04 Ballay; Annick Expression in non-tumoral human lymphoblastoid lines with an integrative vector
US5885835A (en) * 1995-02-23 1999-03-23 Quest International Flavors & Food Ingredients Co., Division Of Indopco, Inc. Kit for in vitro cell growth of eucaryotes using low molecular weight peptides containing L-glutamine
US5741705A (en) * 1995-02-23 1998-04-21 Quest International Flavors & Food Ingredients Company, Division Of Indopco, Inc. Method for in vitro cell growth of eucaryotic cells using low molecular weight peptides
US5851800A (en) * 1996-05-14 1998-12-22 Pharmacia & Upjohn Ab Process for producing a protein
US20060148074A1 (en) * 1996-08-30 2006-07-06 Invitrogen Corporation Serum-free mammalian cell culture medium, and uses thereof
US6100061A (en) * 1997-06-20 2000-08-08 Immuno Aktiengesellschaft Recombinant cell clone having increased stability in serum- and protein-free medium and a method of recovering the stable cell clone and the production of recombinant proteins by using a stable cell clone
US20020182679A1 (en) * 1997-06-20 2002-12-05 Baxter Healthcare Corporation Recombinant cell clones having increased stability and methods of making and using the same
US20030203448A1 (en) * 1999-09-28 2003-10-30 Manfred Reiter Medium for the protein-free and serum-free cultivation of cells

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060148074A1 (en) * 1996-08-30 2006-07-06 Invitrogen Corporation Serum-free mammalian cell culture medium, and uses thereof
US20090280533A1 (en) * 1996-08-30 2009-11-12 Life Technologies Corporation Serum-free mammalian cell culture medium, and uses thereof
US8198084B2 (en) 1996-08-30 2012-06-12 Life Technologies Corporation Serum-free mammalian cell culture medium, and uses thereof
US8455246B2 (en) 1996-08-30 2013-06-04 Life Technologies Corporation Serum-free mammalian cell culture medium, and uses thereof
US8785194B2 (en) 1996-08-30 2014-07-22 Life Technologies Corporation Serum-free mammalian cell culture medium, and uses thereof
US8815573B2 (en) 1996-08-30 2014-08-26 Life Technologies Corporation Serum-free mammalian cell culture medium, and uses thereof
US9321996B2 (en) 1996-08-30 2016-04-26 Life Technologies Corporation Serum-free mammalian cell culture medium, and uses thereof

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