JP2014223078A - Stable recombinant cell clones, production and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a system and a stable recombinant clone that enable continuous production of a recombinant protein for as long as possible in an industrial production of a recombinant protein under conditions containing neither serum nor proteins.SOLUTION: The method of the invention is characterized in that original recombinant cell clone obtained from cell culture is cultured in a culture medium containing serum, and then transferred to a serum- and protein-free culture medium to obtain a recombinant cell clone that is stable over multiple generations and expresses a recombinant protein under conditions free from proteins. The obtained cells are further cultured for at least 40 generations in a serum- and protein-free culture medium under the same conditions as the production conditions. An embodiment is a cell culture of the stable recombinant cell clone where the clone is derived from a mammalian cell, the mammalian cell is CHO cell, preferably CHO-dhfr cell, CHO-K1 cell or BHK cell, and the stable recombinant cell contains a sequence encoding a recombinant polypeptide or a recombinant protein.

Description

  The present invention provides a stable recombinant cell clone that is stable for at least 40 generations in serum-free protein-free medium, biomass obtained by growth of this stable cell clone under serum and protein free culture conditions, and It relates to a method for the preparation of recombinant proteins by means of biomass. Furthermore, the invention relates to a method for the preparation of stable recombinant cell clones. The present invention further relates to the preparation of recombinant proteins in a serum-free protein-free minimal medium.

  The preparation of recombinant proteins, especially biomedical products (eg blood factors), is becoming increasingly important. Serum is usually added to the medium to allow optimal growth of the recombinant cells. Many serum-free media have been developed due to the high cost of serum and to prevent contamination that can be caused by viral or molecular pathogens through the serum in the culture medium, especially these are bovine or human It must not contain any additive of origin. The use of such media in the preparation process not only allows for a low risk of contamination of the prepared product by viral and molecular pathogens, but also allows easier purification of the expressed protein.

  Recombinant cells are most often cultured in serum-containing medium until a high cell density (approximately equal to the cell density of the “working cell bank”) is achieved, and then during the production phase Recombinant cells are reapplied to serum-free medium.

  Miyaji et al. (Non-Patent Document 1) selected serum-independent cell clones in a serum-free medium containing insulin and transferrin. However, after 16 days, it was shown that the viable number and incidence decreased continuously. Miyaji et al. (Non-Patent Document 2) attempted to improve the expression rate and productivity of recombinant cells by co-proliferation with a marker gene.

  Yamauchi et al. (Non-Patent Document 3) is a serum-independent culture medium in which serum-dependent cells are cultured as a single layer on a microtiter plate in a serum-free medium containing human serum albumin, insulin and transferrin for 3 to 4 weeks. Recombinant CHO subclones were established. About 0.1% of the cells were serum independent. Some of the subclones also grew in suspension culture in serum-free medium, where the cells formed aggregates and clamps. The cell doubling time was 1.5 days. However, no suggestions are provided for the stability of the resulting serum-independent clones and for the long-term culture of these clones under serum-free conditions.

  Medium that allows maintenance of cellular metabolic activity and growth during the cell release phase often contained additional substances (eg, growth factors, insulin or transferrin, or an adhesion factor instead of a serum component).

  Various techniques have been developed to eliminate the addition of polypeptide factors such as insulin or transferrin and to allow protein-free media conditions. For example, specially defined, complete protein-free media have been developed that allow cell growth even under protein-free conditions.

  U.S. Patent No. 6,057,031 describes the preparation of recombinant proteins under protein-free conditions in which cells co-express growth factors in addition to the desired protein.

  U.S. Patent No. 6,057,031 describes the use of protein-free media for the preparation of Factor VIII in CHO cells by the addition of nonionic surfactants or cyclodextrins to improve host cell productivity. To increase the effectiveness of these additives, it has been recommended, for example, to add butyrate and lithium.

  U.S. Patent No. 6,057,031 describes culturing cells in a medium containing a glutamine-containing protein hydrolyzate, the free amino acid content of the hydrolyzate being less than 15% of the total protein weight, and the hydrolyzate peptide Has a molecular weight of less than 44 kd. Synthetic minimal medium is used as the basal medium in the culture medium for cell culture, in addition to protein hydrolysates, other additives including fetal calf serum, gentamicin and mercaptoethanol are also added. It is done. The use of this serum-containing medium for recombinant preparation of blood factors is not mentioned.

  U.S. Patent No. 6,057,031 describes a specific synthetic surface that allows the growth of adherent cells under protein-free conditions.

  In order to stimulate cell proliferation, CHO cells overexpressing human insulin were grown on an artificial substrate to which covalently bound insulin was bound (Non-patent Document 4).

  Reiter et al (5) fixed r-CHO cells cultured at high density in serum-containing medium to the support and subsequent immobilization in protein-free medium during the growth period. Cell perfusion was described, where continuous release of protein into the cell supernatant was observed. However, the cells were perfused for less than 10 generations in protein-free medium.

  For successful preparation of industrial “large-scale” cell cultures under protein-free conditions, methods available to date have been serially passaged cell lines (especially VERO cells). Has been described. The cells were here cultured from the original ampule to an industrial scale of 1200 L under serum and protein free conditions. However, the cells used are not recombinant cells but host cells used for the production of viral antigens in the lysis process.

  In contrast to adherent VERO cells, CHO cells, for example, are only dependent on adhesion to a limited extent. CHO cells cultured by conventional methods under serum-containing conditions can bind to both a smooth microporous support and a porous microsupport (Patent Document 6, Non-Patent Document 5). When CHO cells are grown under serum-free conditions, the CHO cells lose this property and do not adhere to a smooth support (eg Cytodex 3) or adhesion promoting additives (eg fibronectin, insulin or To the extent that no transferrin) is added to the medium, CHO cells are easily detached from the smooth support. Recombinant protein production is therefore usually carried out in suspension culture because of the low adherence of CHO cells to the support under serum-free conditions. Herein, this production process can be carried out by a continuous or batch process. Herein, recombinant cell culture is cultured in a bioreactor until optimal cell density is achieved; protein expression is induced as needed and not only expressed protein but also recombinant cells The medium containing also is flushed from the reaction tank at regular intervals for collection and is therefore removed from the production process. As a result of the continuous loss of biomass, the production efficiency in the bioreactor decreases and the production efficiency increases only after slow addition of fresh medium. This is because the cells must grow until the desired cell density is reached. Thus, and despite the continuous process, there is always a delay period in this system where the production rate decreases. Furthermore, the capacity for growth and production is limited in such systems by the maximum achievable cell density.

  Upon application of cells cultured under serum-containing conditions to protein-free medium, the yield of expressed protein and the productivity of recombinant CHO cells are severely reduced after application in protein-free medium compared to serum-containing conditions This was consistently observed (Non-Patent Document 6).

  This is explained by the instability or reduced growth of the recombinant clones as a result of changes in culture conditions. Due to altered fermentation conditions--and despite the use of stable original clones--the majority of cells always change to cells with reduced expression or to non-producing cells, which cells In the meantime, the product-producing cells are over-proliferated, resulting in a fermentation culture consisting mostly of non-producing cells or low-expressing cells.

  The result of this situation is that the maximum production capacity of the fermentation culture decreases continuously and the maximum product production is limited to a certain number of generations or cell passages.

WO97 / 05240 Patent No. 2696001 WO96 / 26266 U.S. Pat.No. 5,393,668 WO96 / 15231 U.S. Pat.No. 4,978,616

Miyaji et al., 1990, Cytotechonology 3: 133-140 Miyaji et al., 1990, Cytotechnology 4: 173-180 Yamauchi et al., 1992, Biosci. Biotechonol. Biochem, 56: 600-604 Ito et al., 1996, PNAS USA 93: 3598-3601 Reiter et al., 1992, Cytotechnology 9: 247-253 Paterson et al., 1994, Appl. Microbiol. Biotechnol. 40: 691-658

  Thus, there is a need for a system that allows continuous production for as long a period as possible, particularly in the industrial production of recombinant proteins under serum and protein free conditions.

  Furthermore, it is desirable to obtain a recombinant cell clone that is stable for many generations at the time of production under protein-free conditions and expresses the recombinant protein. Accordingly, it is an object of the present invention to provide an efficient method for the preparation of recombinant proteins under culture and production conditions, free of serum and protein.

  A further object is to provide stable recombinant cell clones.

  In accordance with the present invention, this problem is solved by making available recombinant cell clones that can be obtained from cell culture, where the cell clones are cultured in serum-containing medium and the original recombinant cell clones are cultured. Obtained after reapplying the cells to serum-free medium. Herein, the cells are further cultured for at least 40 generations in serum-free protein-free medium under the same conditions as the production conditions.

  Thus, the cell clones according to the invention form in a predominant part a cell population that can be cultured for at least 40 generations in a stable manner in serum-free protein-free medium. Herein, it is preferred that more than 80%, in particular more than 99%, of the cell population according to the invention or the cell clone according to the invention is stable for at least 40 generations.

FIG. 1 shows the time point when reapplied from serum-containing medium to serum-free protein-free medium (A), after 10 generations in serum-free protein-free medium (B), and after 60 generations in serum-free protein-free medium (C). A photomicrograph of the working cell bank of the original clone is shown. FIG. 2 shows working cell bank stage (A), 10 generation (B), and 60 generation (C) microscopes of cell cultures starting from recombinant cell clones stable under serum and protein free conditions. Show photos. FIG. 3 shows the results of culturing rFVIII CHO cell clones in a 10 L perfusion bioreactor. a) FVIII activity (mU / mL) and perfusion rate (1-5 / day) over a period of 42 days. b) Volumetric productivity in the perfusion reactor (unit VIII / l / day).

Herein, culturing cells is preferably performed without selection for a selectable marker and / or growth gene (eg, in the absence of MTX in the case of CHO-dhfr ⁻ cells).

  In the context of the present invention, the term original cell clone (Ursprungszellklon) means a recombinant cell clone transfectant, which is in a manner that is stable under laboratory conditions after transduction of host cells by recombinant nucleotide sequences. To express the recombinant product. Original clones are cultured in serum-containing medium for growth optimization. In order to increase productivity, original clones are cultured in the presence of selection reagents and with selection for selection markers and / or proliferation markers, if necessary. For industrial production, the original cell clone is cultured under serum-containing culture conditions until high cell density is achieved, and the original cell clone is serum-free and / or protein-free immediately before the production phase. Applied to the medium. As used herein, culturing is preferably performed without selective pressure.

  Under these conditions, it was found that the majority (greater than 95%) of cells in such cell cultures reapplied to serum-free protein-free media turned into non-product producing cells. The method of immunofluorescence with product-specific antibodies increases the number of non-producing cells in culture as a function of the generation time of the cells in serum-free protein-free medium, causing the product-producing cells to overgrow and consequently the culture It can be shown that the productivity of is reduced.

  Cell cultures obtained after reapplication to serum-free and protein-free media are free of selective pressure as needed for cell clones of cell populations that produce stable products under serum and protein free conditions. And tested. This can be achieved, for example, by immunofluorescence with a specifically labeled antigen made against a recombinant polypeptide or protein. Cells identified as product producing cells are isolated from the cell culture and grown again under serum and protein free conditions, which are preferably equal to production conditions. Cell isolation can be done herein by cell isolation and testing for product producing cells. If necessary, cell cultures containing stable cells are tested again for stable recombinant clones, which are then isolated and cloned from the cell culture. The stable recombinant cell clone obtained under serum and protein free conditions is then further expanded under serum and protein free conditions.

  The recombinant cell clones according to the invention are stable and express recombinant proteins, in particular in serum-free protein-free medium for at least 40 generations, preferably at least 50 generations and particularly advantageously over 60 generations It is characterized by In this specification, this stability appears without assistance, such as, for example, as a support, a matrix or a solid surface. Furthermore, according to the present invention, it is not necessary to perform a culture utilizing a high cell density.

  In accordance with certain aspects of the present invention, stable recombinant cell clones exist in isolated form. Starting from stable cell clones, cell culture is achieved by growth of stable cells under serum and protein free conditions.

  Stable recombinant cell clones according to the invention are preferably derived from recombinant mammalian cells. As used herein, a recombinant mammalian cell can be any cell that contains a sequence encoding a recombinant polypeptide or recombinant protein. This definition includes all continuously growing cells (both adherent and non-adherent). It is particularly preferred to use recombinant CHO cells or recombinant BHK cells. Recombinant polypeptides or proteins can be blood factors, growth factors and other biomedical related products.

  In accordance with the present invention, recombinant blood factors (eg, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Protein S, Protein C, one of these factors It is preferred to use stable recombinant cell clones that contain coding sequences for one activated form, or vWF), and are capable of expressing these blood factors for several generations under stable conditions. As used herein, a polypeptide having vWF or vWF activity, a polypeptide having factor VIII or VIII activity, a CHO cell expressing vWF and factor VIII, factor IX or factor II may be used. ,preferable.

  Cell clones selected under serum- and protein-free conditions according to the present invention are in particular serum-free protein-free medium for at least 40 generations, preferably for at least 50 generations and particularly advantageously for more than 60 generations. It is characterized by being stable.

  Thirty generations are required to develop a master cell bank. In order to perform average batch culture on a 1000 L scale, at least 40 generations are required. Thus, first, from each clone, using about 8-10 generations, a “master cell bank (MCB)”, a “working cell bank (WCB)” is prepared and then under these conditions 20-25 generations Can be used to prepare cell cultures on a production scale (production biomass). Because cell clones available to date become unstable after several generations of growth in serum or protein-free medium, a) uniform cell culture with product-producing cells, and b) stable product productivity over time Because, it becomes powerless to get.

  Thus, cell clones according to the present invention are stable for at least 40 generations under production conditions in serum-free protein-free medium. The methods described to date provided only generations of less than 10 generations with product productivity under protein-free conditions (Reiter et al., 1992, supra).

  As a criterion for stability, a minimum number of at least 40 generations, preferably more than 50 and particularly preferably more than 60 generations are used in the production process, during which stable expression of the protein occurs, And cell morphology and phenotype do not change, and no oncogenetic features appear.

  Unexpectedly, cell clones according to the present invention were shown to show increased productivity even under serum-free and protein-free conditions, even compared to original cell clones cultured in serum-containing media. It was.

  According to another aspect, the present invention provides at least 90%, preferably more than 95% and particularly more advantageously more than 98% stable recombinant cells under serum and protein free conditions. A stable cell culture comprising, wherein the stable recombinant cell is stable for at least 40 generations, in particular at least 50 generations, and expresses the recombinant product. In the context of the present invention, cell culture means production biomass in a master cell bank (MCB), a working cell bank (WCB-Arberitszell-bank) or an industrial production bioreactor.

  In accordance with the present invention, cell culture is obtained by culturing stable recombinant cell clones of the type described above, in particular under conditions free of serum and protein.

  A cell culture according to the invention is preferably used herein on a production scale in a bioreactor under conditions free of serum and protein, from individual clones (ie seed cells) to MCB, WCB or biomass. It can be obtained by growth of isolated and stable cell clones without selection and / or selective pressure against the marker gene. In particular, recombinant cells in cell cultures obtained from stable recombinant clones according to the present invention have been shown to be stable for at least 40 generations under serum and protein free conditions.

Under culture and production conditions almost free of protein, cell cultures made available according to the present invention (prepared from serum and protein-independent stable cell clones) are at least 90%, preferably at least 95%, particularly preferably at least 98% of stable recombinant cells. As used herein, the term “stable recombinant cell” specifically refers to a recombinant mammalian cell derived from a stable cell clone. Herein, it is preferred to use recombinant CHO cells, preferably CHO-dhfr ⁻ cells, CHO-K1 cells and BHK cells, which cells are blood factors, preferably recombinant vWF, Expresses Factor VIII, Factor VIII and vWF, Factor IX or Factor II.

  The cell culture according to the invention may comprise stable recombinant cells as a suspension culture. The cells can also be fixed to a support, in particular a fine support, where a porous fine support is particularly preferred. For example, porous supports such as Cytoline® or Cytopore® have been found to be particularly suitable.

  According to another aspect, the present invention provides a method for industrially producing recombinant products under serum and protein free conditions using stable cell clones made available according to the present invention. Show. The method herein comprises the preparation of an isolated stable recombinant cell clone of the type described above for the preparation of a cell culture. As used herein, the growth of isolated stable cell clones occurs under conditions free of serum and protein, from stable individual cell clones to cell cultures. In particular, subculture of stable cell clones also takes place under protein-free conditions, in particular without the addition of proteases such as trypsin. As a result, at any time during the preparation of cell cultures utilized in the production of recombinant products, this can occur in certain circumstances by the addition of additives containing serum and proteins of human or animal origin to the cell culture It is guaranteed that no contamination will occur. Thus, for the first time, a method has been described that allows for the preparation of working cell banks, production of biomass, and subsequent production of recombinant proteins under serum and protein free conditions, starting with the starting clone. The

  The preparation of recombinant products using cell cultures according to the invention, which comprises more than 90%, preferably more than 95% and particularly advantageously more than 98% stable product producing cells, It can be performed as a suspension culture or with the cells fixed to a support. Herein, this process can be carried out in batch or continuous format or by perfusion techniques with serum-free protein-free media.

  The expressed recombinant protein is then obtained from the cell culture supernatant and then purified and further processed by known state of the art methods.

  Any known synthetic medium can be used as the serum-free protein-free medium. Traditional synthetic minimal media can include inorganic salts, amino acids, vitamins, and carbohydrate sources and water. For example, it can be DMEM / HAM F12 medium. The content of soy extract and yeast extract may be 0.1-100 g / L, particularly preferably 1-5 g / L. In particularly preferred embodiments, soy extract (eg, soy peptone) may be used. The molecular weight of soy peptone is less than 50 kd, preferably less than 10 kd.

It is particularly preferred to use a medium containing 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), sodium selenite (0.0001) ~ 0.01 g / L). If necessary, a nonionic surfactant (eg, polypropylene glycol (PLURONIC F-61, PLURONIC F-68, SYNPERONIC F-68, PLURONIC F-71, or PLURONIC F108) can be added to the medium as an antifoaming agent. .

  This drug is usually used to protect cells from the negative effects of aeration. Because, without the addition of surfactant, rising aerated bubbles can cause damage to these cells located on the surface of these bubbles (“sparging”) (Murhammer and Goochee, 1990, Biotechonol. Prog 6: 142-148).

  In the present specification, the amount of the nonionic surfactant may be 0.05 to 10 g / L, but it is particularly preferable to use the nonionic surfactant as the smallest possible amount of 0.1 to 5 g / L. In addition, the medium can also contain cyclodextrin or a derivative thereof.

  However, the addition of a nonionic surfactant or cyclodextrin is not essential to the present invention. It is preferred that the serum-free protein-free medium comprises a protease inhibitor (eg, a serine protease inhibitor suitable for use in tissue culture and synthetic or plant derived).

Herein, parameters for cell culture such as O ₂ concentration, rate of perfusion or change in the medium, pH, temperature and culture technique depend on the individual cell type used and these are Can be determined in a simple manner by those skilled in the art. For example, culture of CHO cells can be performed in a stirred vessel, and perfusion with protein-free medium is perfusion rate 1-10 volume changes / day, pH 7.0-7.8 (preferably pH 7.4). O ₂ concentration 40-60% (preferably 50%) and temperature 34-38 ° C. (preferably 37 ° C.).

In a further aspect, the present invention makes available a method for obtaining stable recombinant cell clones comprising the following steps;
The original recombinant clone is grown in a serum-containing medium, preferably without selective pressure, to a cell culture,
-Culturing the cells under serum and protein free conditions, preferably equal to production conditions;
Testing the cell culture for product-producing cells under serum and protein free conditions;
The step of cloning stable recombinant cell clones under serum and protein free conditions, where this cloning is usually performed by known techniques (eg isolation of cells by dilution and propagation of individual clones) Can be performed by a process,
-Proliferating isolated cell clones under serum and protein free conditions; and-optionally testing cell cultures for product regenerative cells.

  As used herein, these recombinant cells that express a stable recombinant protein in protein-free medium for at least 10 generations, preferably for at least 20 generations, and particularly advantageously for at least 50 generations. Only clones should be considered stable.

According to another aspect, the present invention makes available a method for obtaining stable recombinant cell clones comprising the following steps;
-Proliferating 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 stable cell clones with recombinant nucleic acids and isolating stable recombinant cell clones;
-Optionally culturing a transfectant of a stable cell clone in a serum-free protein-free medium under the same conditions as the production conditions;
Testing stable recombinant cells for production and product stability.

  The present invention is incorporated by reference for the following examples, but is not limited to these examples.

(Example)
(Example 1: Stability of rvWF CHO cells after reapplication from serum-containing medium to serum-free protein-free medium)
CHO-dhfr ⁻ cells cotransfected with plasmids phAct-rvWF and pSV-dhfr, and vWF expressing clones were subcloned as described in Fischer et al. (1994, FEBS Letters 351: 345-348). Working cell banks (WCBs) were prepared from subclones that expressed rvWF in a stable manner under conditions that contained serum but no MTX, and porous ultrafine supports under conditions that contained serum Cells were fixed on (Cytopore®). After the support matrix has reached a cell density 2 × 10 ⁷ cells / mL, it was converted in cells to serum and protein free medium. In addition, for several generations, cells were cultured under serum and protein free conditions. Cells were tested at various times in serum-free protein-free medium by immunofluorescence using labeled anti-vWF antibodies. Assessment of cell stability was performed in the working cell bank after 10 and 60 generations in serum-free protein-free medium prior to medium change. The working cell bank still showed 100% rvWF producing cells (FIG. 1A), but some of the rvWF producing cells were reduced to about 50% after 10 generations in serum-free protein-free medium (FIG. 1B). After 60 generations, more than 95% of the cells were identified as non-producing cells (FIG. 1C).

Example 2: Cloning of stable recombinant CHO clones
Dilutions were prepared from cell cultures containing rvWF CHO cells according to Example 1, which were cultured for 60 generations in serum-free protein-free medium (FIG. 1C), and in each case 0 .1 cells / well were seeded in microtiter plates. The cells were cultured for about 3 weeks in DMEM / HAM12 medium without serum or protein additives and without selective pressure, and the cells were tested by an immunofluorescence method using labeled anti-vWF antibody. Cell clones identified as positive were used as starting clones for seed cell bank preparation. From this seed cell bank, a master cell bank (MCB) was prepared in serum-free protein-free medium, and individual ampoules were frozen and stored for later preparation of the working cell bank. Starting from individual ampoules, working cell banks were prepared in serum-free protein-free medium. The cells were fixed on a porous ultrafine support and continued to be cultured under serum and protein free conditions for several generations. The cells were tested for productivity in serum-free protein-free medium at different times by immunofluorescence using a labeled anti-vWF antibody. Cell stability was assessed at the working cell bank stage and after 10 and 60 generations in serum-free protein-free medium. Approximately 100% of cells were identified as positive stable recombinant clones expressing rvWF after working cell bank stage (FIG. 2A) and after 10 generations (FIG. 2B) and 60 generations (FIG. 2C). .

(Example 3: Cell-specific productivity of recombinant cell clone)
From the defined stage during the cultivation of the recombinant cells, the defined number of cells was removed and cultured in fresh medium for 24 hours. rvWF: Risto-CoF [ristocetin cofactor] activity was determined in cell culture supernatants. Table 1 shows that cell-specific productivity in stable recombinant cell clones according to the present invention was stable even after 60 generations in serum-free protein-free medium, and this productivity was expressed in serum-containing medium. It shows improvement compared with the original clone cultured in

（実施例４：タンパク質および血清を含まない最小培地でのｒＦＶＩＩＩ ＣＨＯ細胞の培養）
ｒＦＶＩＩＩ ＣＨＯ細胞を含む細胞培養物を、１０Ｌの攪拌タンク内で培養し、そして灌流させた。ここでは、無血清無タンパク質培地を使用した。本明細書中で、この細胞を、多孔性超超微細支持体（Cytopore（登録商標）、Pharmacia）に固定し、そして少なくとも６週間培養した。灌流速度は、４容積変化／日で、ｐＨは６．９〜７．２で、Ｏ_２濃度は約２０〜５０％で、そして温度は３７℃であった。

Example 4: Incubation of rFVIII CHO cells in minimal medium without protein and serum
Cell cultures containing rFVIII CHO cells were cultured in a 10 L stirred tank and perfused. Here, a serum-free protein-free medium was used. Herein, the cells were fixed to a porous ultra-fine support (Cytopore®, Pharmacia) and cultured for at least 6 weeks. The perfusion rate was 4 volume changes / day, the pH was 6.9-7.2, the O ₂ concentration was about 20-50%, and the temperature was 37 ° C.

  FIG. 3 shows the results of culturing rFVIII CHO cell clones in a 10 L perfusion bioreactor.

  a) FVIII activity (mU / mL) and perfusion rate (1-5 / day) over a period of 42 days.

  b) Volumetric productivity in the perfusion reactor (unit factor VIII / L / day).

表２は、ｒＦＶＩＩＩ発現細胞の安定性および特異的生産性を示す。これらの結果のために、１５、２１、２８、３５および４２日後にサンプルを取り出し、３００Ｇで遠心分離し、そして新鮮な無血清無タンパク質培地に再懸濁した。さらに２４時間後に、細胞培養上清中の第ＶＩＩＩ因子濃度および細胞数を決定した。これらのデータから、特異的ＦＶＩＩＩ生産性を計算した。

Table 2 shows the stability and specific productivity of rFVIII expressing cells. For these results, samples were removed after 15, 21, 28, 35 and 42 days, centrifuged at 300 G, and resuspended in fresh serum-free protein-free medium. After an additional 24 hours, factor VIII concentration and cell number in the cell culture supernatant was determined. From these data, specific FVIII productivity was calculated.

A stable average productivity of 888 mU / 10 ⁶ cells / day was achieved. This stable productivity was also confirmed by immunofluorescence with labeled anti-FVIII antigen after 15, 21, 28, 35 and 42 days in serum-free protein-free medium.

Claims (25)

A method for preparing a stable recombinant cell clone comprising the following steps:
Preparing an original recombinant cell clone,
Culturing the original recombinant cell clone in a serum-containing medium;
Reapplying the cells to serum-free protein-free medium;
Testing the cell culture for stable product-producing cells, and cloning the stable product-producing cell clone under serum and protein free conditions;
A method characterized by containing.   2. The method of claim 1 characterized by obtaining the stable cell clone in isolated form after cloning.   The method according to claim 1 or 2, characterized in that a recombinant mammalian cell is prepared as said original clone.   4. The method of claim 3, wherein the mammalian cell is characterized by being a recombinant CHO cell or a recombinant BHK cell.   5. The method according to any one of claims 1 to 4, wherein the recombinant cell clone is characterized by comprising a sequence encoding a recombinant polypeptide or recombinant protein.   6. The method of claim 5, wherein the recombinant protein is Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, protein. A method characterized by being a blood factor selected from the group consisting of S, protein C or one active form of these factors, or vWF.   7. The method according to any one of claims 1 to 6, characterized by preparing recombinant CHO cells expressing von Willebrand factor as the original cell clone.   7. A method according to any one of claims 1 to 6, characterized by preparing recombinant CHO cells expressing factor VIII as said original cell clone.   The method according to any one of claims 1 to 6, characterized by preparing recombinant CHO cells co-expressing factor VIII and vWF as said original cell clone.   7. The method according to any one of claims 1 to 6, characterized by preparing recombinant CHO cells expressing factor IX as the original cell clone.   7. A method according to any one of claims 1 to 6, characterized by preparing recombinant CHO cells expressing factor II as the original cell clone.   A recombinant cell clone characterized by being prepared by the method of any one of claims 1-11. A cell culture comprising the following steps:
Growing the original recombinant clone in a serum-containing medium;
Culturing the cells to a cell culture under serum and protein free conditions;
Testing the cell culture for product-producing cells under serum and protein free conditions;
Cloning a stable cell clone under serum and protein free conditions, and growing a stable cell clone under serum and protein free conditions;
A cell culture characterized by being obtainable by:   A cell culture characterized in that it can be obtained by culturing a stable recombinant cell clone according to claim 12.   15. A cell culture according to claim 13 or 14, characterized by being capable of being induced to express the recombinant product under serum and protein free conditions.   16. A cell culture according to any one of claims 13 to 15, characterized in that the stable recombinant cell clone is a mammalian cell. The mammalian cell is, CHO cells, preferably CHO-dhfr ^- characterized by cells, a CHO-K1 cells, or BHK cell, cell culture of claim 16.   18. A cell culture according to any one of claims 13 to 17, wherein the stable recombinant cell is characterized by comprising a sequence encoding a recombinant polypeptide or protein.   19. A cell culture according to any one of claims 13 to 18, characterized in that the cells are immobilized on a fine support. A method for industrial production of recombinant products under serum and protein free conditions comprising the following steps:
Preparing an isolated, stable recombinant cell clone according to claim 12;
Growing the stable cell clone in serum-free protein-free medium from the starting clone to the cell culture;
Preparing a cell culture containing the stable cells in a bioreactor, and collecting protein from the culture supernatant;
A method characterized by comprising.   21. The method of claim 20, wherein the serum-free protein-free medium is a synthetic minimal medium comprising yeast extract or soy extract.   The method according to claim 20 or 21, wherein the medium is characterized by containing cyclodextrin or a derivative thereof.   23. A method according to any one of claims 20 to 22, wherein the serum-free protein-free medium is characterized by comprising a protease inhibitor. A method for obtaining a stable recombinant cell clone comprising the following steps:
Proliferating the original recombinant clone to cell culture in serum-containing medium;
Culturing the cells under serum and protein free conditions equivalent to production conditions;
Testing the cell culture for product-producing cells under serum and protein free conditions;
Cloning the recombinant cell clone under serum and protein free conditions, and propagating the stable cell clone under serum and protein free conditions;
A method characterized by inclusion. A method for obtaining a stable recombinant cell clone comprising the following steps:
Growing non-recombinant starting cells under serum and protein free conditions;
Cloning a stable non-recombinant cell clone under serum and protein free conditions;
The stable cell clone is transfected with the recombinant nucleic acid and the transfectant is cultured in serum-free protein-free medium under conditions equivalent to the isolated production conditions of the stable transfectant and the product Testing the cells for stability;
A method characterized by comprising.

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