US20030203448A1 - Medium for the protein-free and serum-free cultivation of cells - Google Patents

Medium for the protein-free and serum-free cultivation of cells Download PDF

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US20030203448A1
US20030203448A1 US10/405,794 US40579403A US2003203448A1 US 20030203448 A1 US20030203448 A1 US 20030203448A1 US 40579403 A US40579403 A US 40579403A US 2003203448 A1 US2003203448 A1 US 2003203448A1
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medium
cells
protein
free
accordance
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Manfred Reiter
Wolfgang Mundt
Friedrich Dorner
Leopold Grillberger
Artur Mitterer
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Baxalta Innovations GmbH
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Priority to US10/405,794 priority Critical patent/US20030203448A1/en
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Assigned to BAXTER AKTIENGESELLSCHAFT reassignment BAXTER AKTIENGESELLSCHAFT ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DORNER, FRIEDRICH, GRILLBERGER, LEOPOLD, MITTERER, ARTUR, MUNDT, WOLFGANG, REITER, MANFRED
Priority to US11/841,915 priority patent/US8021881B2/en
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Priority to US13/198,476 priority patent/US8722406B2/en
Priority to US14/231,221 priority patent/US9441203B2/en
Priority to US15/235,453 priority patent/US9982286B2/en
Priority to US15/244,302 priority patent/US20170002392A1/en
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
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    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C12N2500/92—Medium free of human- or animal-derived components

Definitions

  • the present invention pertains to a medium for the protein-free and serum-free cultivation of cells.
  • such complex preparations contain a plurality of proteins that can act in a disruptive manner, especially within the context of the purification process for the recombinant protein that is to be recovered from the cell culture. This applies particularly to those proteins that are homologous with or similar to the protein that is to be recovered.
  • these problems are especially acute in the case of the recombinant recovery of serum proteins because the biogenic pendant in the medium that is used (e.g., bovine protein) can be removed reliably within the context of purification only via quite specific differential purification (e.g., with antibodies that are directed specifically only against the recombinant protein but not against the bovine protein (Björck, L., J. Immunol., 1988, Vol. 140, pp. 1194-1197; Nilson et al., J. Immunol Meth., 1993, 164, pp. 33-40).
  • the cells that are used preeminently for a recombinant preparation are capable of adhering only to a limited extent.
  • CHO cells that have been bred by conventional methods bind to both smooth and porous microcarriers only under serum-containing conditions (see U.S. Pat. No. 4,973,616; Cytotechnology 9 (1992), 247-253).
  • other adhesion-promoting additions such as e.g., fibronectin, insulin or transferrin, have not been provided in the medium.
  • the cells can be bred using the suspension culture technique as well as e.g., using the batch process or using a continuous culture technique.
  • Cultivation preferably takes place using the chemostat process (Ozturk S. S. et al., 1996, Abstr. Pap. Am. Chem. Soc., BIOT 164, Payne G. F. et al., in “Large Scale Cell Culture Technology,” 1987, ed. Lydersen B. K., Hauser publishers; pp. 206-212).
  • Kattinger H. et al Advanced Mol. Cell.
  • the present invention has therefore an objective of improving the possibilities for the protein-free and serum-free cultivation of recombinant cells and of making agents and processes available with which recombinant cells can be cultivated efficiently in a serum-free or protein-free manner. Moreover, it should then be possible not only to culture surface-dependent cells, but also to use the suspension culture technique, whereby instability in the productivity of the cells is required to be repressed as much as possible.
  • a further objective of the present invention additionally is to efficiently increase the production of recombinant cells.
  • these tasks are accomplished by means of a medium for the protein-free and serum-free cultivation of cells, especially mammalian cells, characterized by the feature that the medium contains a proportion of soy hydrolysate.
  • FIG. 1 shows the results of the cultivation of a rFVIII-CHO cell clone in a 10-L perfusion bioreactor:
  • FIG. 2 shows a comparison of the Factor VIII productivity (mU/mL) in the case of cultivation, using the batch process, of CHO cells which express rFactor VIII, in various media.
  • Mix 1 consists of serum-free and protein-free medium without soy hydrolysate, but containing an amino acid mixture as listed in table 4;
  • Mix 2 consists of serum-free and protein-free medium containing soy hydrolysate;
  • Mix 3 consists of serum-free and protein-free medium containing soy hydrolysate and an amino acid mixture as listed in table 4 and
  • Mix 4 consists of serum-free and protein-free medium containing 2.5 g/l purified, ultrafiltrated soy hydrolysate and an amino acid mixture as listed in table 4.
  • a Sephadex® column was used for the purification of the ultrafiltrated soy hydrolysate.
  • FIG. 3 shows the Factor VIII productivity (U/L) in the case of the continuous growth of CHO cells, which express rFactor VIII, in a serum-free and protein-free medium after the start of the addition of purified, ultrafiltered soy peptone, namely on the 6th day of cultivation;
  • FIG. 4 shows BHK cells expressing recombinant Factor II that have been bred in a protein-free and serum-free medium that contains soy hydrolysate.
  • the medium in accordance with the invention preferably contains soy hydrolysate in a quantity of more than 10 wt % based on the total dry weight of the medium.
  • the soy hydrolysate in the medium is provided in a quantity of 4-40%.
  • soy hydrolysate is not critical in accordance with the invention.
  • a plurality of soy preparations which are to be found on the market, can be used in accordance with the invention, e.g., peptones from soy flour, digested enzymatically (e.g., by papain), with a pH value between 6.5 and 7.5 and a total nitrogen content between 9% and 9.7% and an ash content between 8 and 15%.
  • Ultrafiltration can take place in accordance with the process as described comprehensively in the prior art, e.g., with use being made of membrane filters with a defined cut-off limit.
  • the purification of the ultrafiltered soy peptone can take place by means of gel chromatography, e.g., by means of Sephadex chromatography, for example, with Sephadex G25 or Sephadex G10 or equivalent materials, ion-exchange chromatography, affinity chromatography, size exclusion chromatography or “reversed-phase” chromatography.
  • gel chromatography e.g., by means of Sephadex chromatography, for example, with Sephadex G25 or Sephadex G10 or equivalent materials, ion-exchange chromatography, affinity chromatography, size exclusion chromatography or “reversed-phase” chromatography.
  • those fractions can be selected which contain soy hydrolysate of defined molecular weight, i.e. ⁇ 1000 Dalton, preferably ⁇ 500 Dalton, more preferably ⁇ 350 Dalton.
  • the invention also comprises a process for producing a serum-free and protein-free cell culture medium, comprising obtaining a soy hydrolysate, ultrafiltering said soy hydrolysate using an ultrafiltration process, purifying said soy hydrolysate fraction using size exclusion chromatography and selecting the soy hydrolysate fractions consisting of soy hydrolysate having a molecular weight ⁇ 1000 Dalton, preferably ⁇ 500 Dalton, more preferably ⁇ 350 Dalton.
  • An especially advantageous soy hydrolysate is characterized by the feature that it has a free amino acids content of between 10.3 and 15.6% or, preferably, between 12 and 13.5%, a total nitrogen content of between 7.6 and 11.4% or, preferably, between 8.7 and 9.5% and an endotoxin content of ⁇ 500 U/g and whereby at least 40% or, preferably, at least 50% or, especially preferably, at least 55% thereof has a molecular weight of 200-500 daltons and at least 10% or, preferably, 15% thereof has a molecular weight of 500-1000 daltons. Most preferably, at least 90% of the soy hydrolysate is of a molecular weight of ⁇ 500 Daltons.
  • a soy hydrolysate is especially well suited to the industrial production of recombinant proteins since, because of its special features, it can be standardized especially easily and it is usable in routine processes.
  • the medium in accordance with the invention can also contain synthetic media in a way that is known as such, such as e.g., DMEM/HAM's F12, Medium 199 or RPMI, that are adequately known from the literature.
  • the medium in accordance with the invention also preferably contains amino acids, preferably those selected from the group comprising L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan, L-glutamine, or mixtures thereof.
  • L-asparagine in a quantity of 0.001-1 g/L of medium, preferably 0.1-0.05 g/L, especially preferably 0.015-0.03 g/L
  • L-cysteine (0.001-1 g/L, preferably 0.005-0.05 g/L, especially preferably 0.01-0.03 g/L)
  • L-cystine (0.001-1 g/L, preferably 0.01-0.05 g/L, especially preferably 0.015-0.03 g/L)
  • L-proline 0.001-1.5 g/L, preferably 0.01-0.07 g/L, especially preferably 0.02-0.05 g/L
  • L-tryptophan (0.001-1 g/L, preferably 0.01-0.05 g/L, especially preferably 0.015-0.03 g/L)
  • L-glutamine (0.05-1 g/L, preferably 0.1-1 g/L).
  • amino acids designated above can be added to the medium in accordance with the invention either individually or in combination.
  • the combined addition of an amino acid mixture, which contains all of the above-mentioned amino acids, is especially preferred.
  • a serum-free and protein-free medium is used in a special form of embodiment, whereby this medium additionally contains a combination of the above-mentioned amino acid mixture and purified ultrafiltered soy peptone.
  • the medium in order to inactivate viruses or other pathogens, can be heated, without negative effects, for approximately 5-20 min or, preferably, 15 min at 70-95° C. or, preferably, 85-95° C.
  • a known synthetic medium can be used in combination with the soy hydrolysate.
  • Conventional synthetic media can contain inorganic salts, amino acids, vitamins, a source of carbohydrates and water.
  • concentration of soy extract in the medium can preferably be between 0.1 and 100 g/L, especially preferably, 1 and 5 g/L.
  • soy peptone can be used which has been standardized in regard to its molecular weight.
  • the molecular weight of the soy peptone preferably is less than 50 kD, especially preferably less than 10 kD, most preferably, less than 1 kD.
  • ultrafiltered soy peptone has proven to be especially advantageous for the productivity of the recombinant cell lines, whereby the average molecular weight of the soy peptone is 350 daltons (Quest Company). This is a soy isolate with a total nitrogen content of approximately 9.5% and a free amino acid content of approximately 13%.
  • the medium in accordance with the invention also preferably contains auxiliary substances, such as e.g., buffer substances, oxidation stabilizers, stabilizers to counteract mechanical stress, or protease inhibitors.
  • auxiliary substances such as e.g., buffer substances, oxidation stabilizers, stabilizers to counteract mechanical stress, or protease inhibitors.
  • Use is especially made of a medium with the following composition: synthetic minimal medium (1-25 g/L), soy peptone (0.5-50 g/L), L-glutamine (0.05-1 g/L), NaHCO 3 (0.1-10 g/L), ascorbic acid (0.0005-0.05 g/L), ethanolamine (0.0005-0.05 g/L) and Na selenite (1-15 ⁇ g/L).
  • a nonionic surfactant such as, e.g., polypropylene glycol (PLURONIC F-61, PLURONIC F-68, SYNPERONIC F-68, PLURONIC F-71 or PLURONIC F-108) can be added to the medium as a defoaming agent in accordance with the invention.
  • a nonionic surfactant such as, e.g., polypropylene glycol (PLURONIC F-61, PLURONIC F-68, SYNPERONIC F-68, PLURONIC F-71 or PLURONIC F-108) can be added to the medium as a defoaming agent in accordance with the invention.
  • This agent is generally used in order to protect the cells from the negative effects of aeration since, without an addition of a surfactant, ascending and bursting air bubbles can lead to damage of those cells that are located on the surface of these air bubbles (“sparging”) (Murhammer and Goochee, 1990, Biotechnol. Prog. 6:142-148).
  • the quantity of nonionic surfactant can be between 0.05 and 10 g/L. Preferably, the smallest possible amount is between 0.1 and 5 g/L.
  • the medium in accordance with the invention can also contain cyclodextrin or a derivative thereof.
  • the serum-free and protein-free medium of the present invention preferably contains a protease inhibitor, such as a serine protease inhibitor, which is suitable for tissue culture and is of synthetic or plant origin.
  • a protease inhibitor such as a serine protease inhibitor
  • Cells that have already been adapted are preferably used as the cells for cultivation in the medium in accordance with the invention, i.e., cells that have already adapted to growth in the protein-free and serum-free media. It has been found that not only can increased yields be achieved with such preadapted cells, but their stability for serum-free and protein-free cultivation is also clearly improved by the use of the medium in accordance with the invention.
  • recombinant cell clones have proven to be especially valuable in accordance with the invention, whereby these are stable from the outset for at least 40 generations and, preferably, at least 50 generations in serum-free and protein-free media, and express recombinant products.
  • Such cell clones are obtainable from a cell culture that is obtained following the cultivation of a recombinant original cell clone on a serum-containing medium and readaptation of the cells to a serum-free and protein-free medium.
  • original cell clone can be understood to mean a recombinant cell clone transfectant that, after transfection of the host cells with a recombinant nucleotide sequence, expresses a recombinant product in a stable manner under laboratory conditions.
  • the original clone is bred in a serum-containing medium in order to optimize its growth.
  • the original clone is bred, optionally in the presence of a selection agent, with selection on the selection marker and/or amplification marker.
  • the original cell clone is bred, under serum-containing conditions of cultivation, to a high cell density and then it is readapted to a serum-free or protein-free medium just prior to the production phase. Cultivation preferably takes place without selection pressure in this case.
  • the cultivation of the recombinant original cell clone can take place from the beginning in a serum-free and protein-free medium; as a result, readaptation is no longer necessary. If required, use can also be made of a selection agent in this case and selection can take place on the selection marker and/or the amplification marker. A process for this is described in EP 0 711 835, for example.
  • the cell culture that is obtained after readaptation to a serum-free and protein-free medium is tested for those cell clones of the cell population which produce products in a stable manner under serum-free and protein-free conditions, optionally in the absence of selection pressure. This can take place, for example, by means of immunofluorescence with marked specific antibodies which are directed against the recombinant polypeptide or protein.
  • the cells that are identified as product producers are isolated from the cell culture and are re-bred under serum-free and protein-free conditions that are preferably equivalent to production conditions. The isolation of the cells can thereby take place by isolating the cells and testing them for product producers.
  • the cell culture, containing the stable cells can be tested again for stable recombinant clones, and these are isolated from the cell culture and subcloned.
  • the stable recombinant cell clones that are obtained under serum-free and protein-free conditions can then be bred further under serum-free and protein-free conditions.
  • the cell culture which is to be cultivated in accordance with the invention, is preferably derived from a recombinant mammalian cell.
  • Recombinant mammalian cells can hereby be all those cells that contain sequences which code for a recombinant polypeptide or protein. All continuously growing cells, which grow either adherently or nonadherently, are encompassed in this regard.
  • Recombinant CHO cells or BHK cells are especially preferred.
  • Recombinant polypeptides or proteins can be blood factors, growth factors or other biomedically relevant products.
  • cell clones which contain the coding 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 one of these factors, or vWF, and that are capable of expressing these in a stable manner over several generations.
  • 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 one of these factors, or vWF
  • Recombinant CHO cells that express vWF or a polypeptide with vWF activity, Factor VIII or a polypeptide with VIII activity, vWF and Factor VIII, Factor IX or Factor II, are especially preferred in this regard.
  • ⁇ 30 generations are required in order to start a master cell bank. At least approximately 40 generations are required in order to carry out an average batch culture on the 1000-L scale.
  • MCB master cell bank
  • WB working cell bank
  • a cell culture with up to 20-25 generations under protein-free and serum-free conditions on the production scale (production biomass) whereas, by contrast, some generations become unstable after growth on a serum-free or protein-free medium with previous cell clones and media and, as a result, a) a uniform cell culture with product producers is not possible and b) stable product productivity over an extended period of time is not possible.
  • the present invention also pertains to a process for the cultivation of cells, especially mammalian cells, that is characterized by the feature that these cells are introduced into a medium in accordance with the invention and then are cultured in this medium.
  • the present invention also pertains to the use of the medium in accordance with the invention for the cultivation of recombinant cells, preferably eukaryotic cells and, especially, mammalian cells.
  • the subject of the present invention accordingly, is also a cell culture that comprises the medium in accordance with the invention and cells, preferably eukaryotic cells, and especially mammalian cells.
  • the present invention further includes a process for the production of a desired protein (especially a recombinant protein) from cell culture comprising introducing cells which express such desired protein into a medium of the present invention; growing said cells in said medium and expressing said protein, thereby producing a mixture of said cells and said protein in the medium; and purifiying said protein from said mixture.
  • a desired protein especially a recombinant protein
  • recombinant proteins such as Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Protein S, Protein C, activated forms of these factors, and vWF can be produced.
  • CHO-dhfr cells were plasmid phAct-rvWF and pSV-dhfr co-transfected, and vWF-expressing clones were subcloned as described by Fischer et al. (1994, FEBS Letters 351:345-348).
  • a working cell bank (WCB) was started from the subclones, which expressed rvWF in a stable manner, under serum-containing conditions but in the absence of MTX, and the cells were immobilized on a porous microcarrier (Cytopore) under serum-containing conditions. Switching the cells to a serum-free and protein-free medium took place after a cell density of 2 ⁇ 10 7 cells/mL of the matrix had been reached.
  • the cells were cultured further for several generations under serum-free and protein-free conditions.
  • the cells were tested in a serum-free and protein-free medium at various points in time by means of immunofluorescence with labelled anti-vWF antibodies.
  • the evaluation of the stability of the cells took place using the working cell bank prior to switching the medium, after 10 generations and after 60 generations in the serum-free and protein-free medium. Whereas the working cell bank still exhibited 100% rvWF producers, the proportion of rvWF producers declined to approximately 50% after 10 generations in the serum-free and protein-free medium. After 60 generations, more than 95% of the cells were identified as nonproducers.
  • a dilution series was prepared from the cell culture containing rvWF-CHO cells in accordance with Example 1 (this stable cell clone that was designated r-vWF-CHO F7 was filed, in accordance with the Budapest convention, with the ECACC (European Collection of Cell Cultures), Salisbury, Wiltshire SP4 OJG, UK, on Jan. 22, 1998, and acquired the deposition number 98012206) which had been cultured for 60 generations in a serum-free and protein-free medium and 0.1 cells were seeded out in each well of a microtiter plate.
  • the cells were cultivated for approximately 3 weeks in DMEM/HAM's F12 without serum additions or protein additions and without selection pressure, and the cells were tested via immunofluorescence with labelled anti-vWF antibodies.
  • a cell clone which had been identified as positive, was used as the starting clone for the preparation of a seed cell bank.
  • a master cell bank (MCB) was started from the seed cell bank in a serum-free and protein-free medium and individual ampules were put away and frozen for the further preparation of a working cell bank.
  • a working cell bank was prepared in a serum-free and protein-free medium from an individual ampule.
  • the cells were immobilized on porous microcarriers and cultivated further for several generations under serum-free and protein-free conditions.
  • the cells were tested for productivity at various points in time in a serum-free and protein-free medium by means of immunofluorescence with labelled anti-vWF antibodies.
  • the evaluation of the stability of the cells took place at the working cell bank stage and after 10 and 60 generations in a serum-free and protein-free medium. Approximately 100% of the cells were identified as positive stable clones, which express rvWF, at the working cell bank stage, after 10 generations, and 60 generations.
  • a cell culture containing rFVIII-CHO cells was cultivated in a 10-L stirred tank with perfusion.
  • a medium in accordance with Example 4 was used in this case.
  • the cells were thereby immobilized on a porous microcarrier (Cytopore, Pharmacia) and then cultivated for at least 6 weeks.
  • the perfusion rate was 4 volume changes per day; the pH was 6.9-7.2; the O 2 concentration was approximately 20-50% and the temperature was 37° C.
  • FIG. 1 shows the results of the cultivation of a rFVIII-CHO cell clone in a 10 L perfusion bioreactor.
  • Table 3 shows the stability and specific productivity of the rFVIII-expressing cells.
  • samples were taken after 15, 21, 28, 35 and 42 days and then centrifuged at 300 g and resuspended in fresh serum-free and protein-free medium.
  • the Factor VIII concentration in the supernatant liquors of the cell cultures and the cell count was determined after a further 24 h.
  • the specific FVIII productivity was calculated from these data.
  • a stable average productivity of 888 milliunits/10 6 cells/day was achieved. This stable productivity was also confirmed by immunofluorescence with labelled anti-FVIII antibodies after 15, 21, 28, 35 and 42 days in a serum-free and protein-free medium.
  • a cell culture containing rFVIII-CHO cells was cultivated batchwise.
  • the cells were bred at 37° C. and pH 6.9-7.2. The cells were bred using the batch process over periods of 24-72 h.
  • Mix 1 comprising a serum-free and protein-free medium without soy peptone and additionally containing an amino acid mixture in accordance with the table designated above.
  • Mix 2 comprising a serum-free and protein-free medium containing soy peptone.
  • Mix 3 comprising a serum-free and protein-free medium containing soy peptone and additionally containing an amino acid mixture in accordance with the table designated above.
  • Mix 4 comprising a serum-free and protein-free medium, and additionally containing an amino acid mixture in accordance with the table designated above and 2.5 g/L of purified, ultrafiltered soy peptone. The purification of the ultrafiltered soy peptone took place chromatographically over a Sephadex column.
  • a cell culture containing rFVIII-CHO cells was cultivated in a 10-L stirred bioreactor tank.
  • the cells were bred at 37° C. and pH 6.9-7.2; the oxygen concentration was 20-50% air saturation. Samples were taken every 24 h in order to determine the Factor VIII titer and the cell concentration in the supernatant liquor of the culture. The total cell concentration was constant from the 2nd day to the 14th day. Ultrafiltered soy peptone was added to the medium starting from the 6th day.
  • the Factor VIII productivity is illustrated in 3; the measurements took place by means of a CHROMOGENIX CoA FVIII:C/4 system. Immunofluorescence was carried out with labelled anti-FVIII antibodies. It can be seen from the data that a distinct increase in Factor VIII productivity and hence an increase in the volumetric productivity of the bioreactor system, occurred as a result of the addition of soy peptone, whereby this did not lead to a distinct increase in cell growth.
  • the absence of soy peptone in the continuous culture leads to a distinct decline in Factor VIII productivity after a few days, whereas the addition of soy peptone leads, as a consequence, to an almost 10-fold increase in productivity.
  • this addition does not increase the cell count, it is hereby clearly shown that ultrafiltered soy peptone leads, as a consequence, to a distinct increase in productivity which is independent of cell growth.
  • a rFVIII-CHO cell culture was cultivated batchwise. In this case, use was made of a serum-free and protein-free medium as described in Example 4 to which different hydrolysates (from soy, yeast, rice and wheat) had been added. A serum-free and protein-free medium, to which no hydrolysate had been added, was used as the control.
  • the initial cell density was 0.6 ⁇ 10 5 and 0.4 ⁇ 10 6 , respectively.
  • the cells were cultured at 37° C. using the batch process at pH 6.9-7.2.
  • Table 5 shows the results of the cultivation experiments with rFVIII-CHO cells in a serum-free and protein-free medium to which soy hydrolysate (ultrafiltered) and yeast hydrolysate had been added.
  • the initial cell density was 0.6 ⁇ 10 5 cells.
  • a serum-free and protein-free medium without hydrolysate additions was used as the control.
  • Table 6 shows the results of the cultivation experiments with rFVIII-CHO cells in a serum-free and protein-free medium to which soy hydrolysate (ultrafiltered), rice hydrolysate and wheat hydrolysate had been added.
  • the initial cell density was 0.6 ⁇ 10 5 cells.
  • a serum-free and protein-free medium without hydrolysate additions was used as the control.
  • TABLE 6 Final cell density FVIII titer vWF - Antigen Hydroysate ( ⁇ 10 6 /mL) (mU/mL) ( ⁇ g/L) Soy 3.1 1142 6.7 Rice 3.0 419 3.2 Wheat 3.4 522 3.9 Control 3.0 406 3.1
  • Table 7 shows the results of the cultivation experiments with rFVIII-CHO cells in a serum-free and protein-free medium to which soy hydrolysate (ultrafiltered) and wheat hydrolysate had been added.
  • the initial cell density amounted to 0.4 ⁇ 10 6 cells.
  • TABLE 7 Final cell FVIII FVIII VWF- density titer Antigen Antigen Hydrolysate ( ⁇ 10 6 /mL) (mU/mL) ( ⁇ g/mL) ( ⁇ g/mL) Soy 1.6 1427 166 17.2 Wheat 1.0 1120 92 1.9
  • BHK-21 (ATCC CCL 10) cells were co-transfected three times with the following plasmids by means of a CaPO 4 procedure: 25 ⁇ g of the plasmid pSV-FII (Fischer, B. et al., J. Biol. Chem., 1996, Vol. 271, pp. 23737-23742) which contains the human Factor II (prothrombin)-cDNA under the control of a SV40 promotor (SV40 early gene promoter); 4 ⁇ g of the plasmid pSV-DHFR for methotrexate resistance and 1 ⁇ g of the plasmid pUCSV-neo (Schlokat, U. et al., Biotech. Appl.
  • Stable cell clones were selected by means of cultivation in a medium, which contained 500 ⁇ g/mL of G418, by increasing the methotrexate concentration in a stepwise manner up to a concentration of 3 ⁇ M.

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