Patent Application
20100197011
The present invention relates to biotechnology, specifically to a method of recovering stable cell clones adapted to serum- and protein-free medium by a two-stage adaptation process.
Since the development of the in vitro cultivation of mammalian cells the demand for large scale production of these cells has increased due to diagnostic and therapeutic potential of many of the products they produce. These useful agents include monoclonal antibodies, human growth hormone, lymphokines, erythropoietin, blood clotting factors and tissue plasminogen activators.
The use of recombinant monoclonal antibodies (rMab) for therapy and in vivo diagnosis of different diseases imply in many cases the use of high dose treatments. This fact makes necessary the production of large amount of the rMab of interest with a very high purity.
Several recombinant monoclonal antibodies with potential use in cancer and autoimmune diseases therapy and diagnostic have been expressed in NSO myeloma cells at Center of Molecular Immunology. U.S. Pat. No. 5,891,996, describe the obtainment of the chimeric and humanized antibodies against Epidermal Growth Factor receptor (EGF-R), useful in diagnosis and therapy of tumors expressing said receptor. WO 97/19111 describes anti-CD6 monoclonal antibodies useful in diagnosis and therapy in patients suffering psoriasis. Gavilondo et al. in Hybridoma 9 No. 5, 1999 reported an anti-CD3 monoclonal antibody called IOR-T3a
For protein-free culturing conditions, various techniques have been developed. Thus, specifically defined, complete protein-free media have been developed which allow the cell growth under protein-free conditions.
WO 97/05240 describes the expression of recombinant proteins under protein-free conditions.
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.
WO 96/26266 describes the culturing of cells in a medium which contains a glutamin-containing protein hydrolysate whose content of free amino acids is less than 15% 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, gentamycine and mercapto-ethanol 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 A describes special synthetic surfaces which allow the growth of adherent cells under protein-free conditions.
To stimulate cell proliferation, CHO cells which over express human insulin have been multiplied on an artificial substrate to which insulin is covalently bound (Ito et al. 1996 PNAS U.S.A. 93:3598-3601).
Reiter et al. (1992. Cytotechnology 9:247-253) describe the immobilisation 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 maintained for less than 10 generations in 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 (WO 96/15231). There, the cells are grown under serum- and protein-free conditions from the original ampoule up to a large technical scale of 1200 liters.
To adapt cells initially grown under serum-containing conditions to protein free medium is a rather troublesome process which usually takes long time; in addition, 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. 1994. Appl. Microbiol. Biotechnol. 40:691-658). 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 culture of the fermenter finally largely consists of non-producers or of such cells having a low expression.
In the present invention we have established an approach to develop stable cell lines adapted to serum- and protein-free media. Following this approach several clones were isolated in protein-free medium.
This procedure comprises mammalian cell lines, for which it's not possible to carry out a direct procedure of adaptation from serum-supplemented or serum-free medium to protein-free medium.
The method of the present invention consists of a two stages process during adaptation of cell lines to protein-free medium (PFM).
The first step which is consider as a Non critical stage: the reduction of protein contents occurs without the lost of cell viability and there is not an important decrease of population doubling time in each step of protein concentration. The non-critical stage is observed usually between 5 and 0.5 mg/mL of total protein concentration in the culture medium and the culture shows almost the same growth rate that in the initial culture medium.
This first stage starts with cell line viability between 80 and 100% and cells are grown in culture media with consecutive protein concentration reduction up to a critical protein concentration at which cell viability drop to 0%. This protein concentration is the start point for the next stage.
The second step which is consider as a Critical stage: At this stage it occurs a decrease in cell viability and population-doubling time of the cells and it will take more time to adapt from one step of protein concentration to another. There exist critical protein concentrations in the culture medium, which is not possible to bypass during the adaptation process. These critical protein concentrations are specific for each recombinant cell line, but usually are below 0.6 mg/mL. Once the cells have recovered the initial viability and growth rate at these critical protein concentrations there is possible to subculture to the follow condition with lower protein concentration.
Once the critical protein concentration is fixed, its closed higher protein concentration which support cells growth is consider the pre-critical protein concentration. Starting from the pre-critical protein concentration it is reduced slowly up to cells recover the initial viability and growth rate.
The selected combination of steps to reduce the protein concentration at the critical stage will determine the total adaptation time in this stage and the rate of adaptation (Vadapt.), calculated as the relationship:
However this step combination will not have influence upon the time needed for adaptation to each protein concentration, including critical concentrations. In order to determine the end of non-critical stage and the critical protein concentrations it's necessary to carry out an stepwise adaptation, by serial dilutions of the cell culture in the desired protein free medium reducing two fold the protein concentration each time (Table 1). This reduction can be done by the decrease of serum concentration or supplementing the basal medium with different levels of some rich in proteins serum substitute.
Before to start the adaptation procedure the cells should be maintained with more than 80% of viability in T-flasks in the standard medium usually employed to culture the cells.
The adaptation process is carry out step by step following stages described below.
In the procedure of the present invention the initial culture medium contains a range between 5 to 10% of fetal bovine serum.
The mammalian cell line adapted to growth in protein-free medium is a myeloma, particularly NSO cells.
The present invention also could be useful when using NSO cell line transfected with a polypeptide or a recombinant protein, particularly when they are transfected with a sequence encoding a recombinant antibody or fragments thereof. The mammalian cell lines modified by procedure of the present invention growing in protein-free medium are also disclosed.
In a different embodiment of the present invention it is disclosed any mammalian cell line expressing a humanized or chimeric antibody selected from the group consisting of the anti-EGF receptor hR3, anti-CD6 T1hT, anti-CD3 T3Q antibodies or fragments thereof growing in protein-free medium, thus the antibodies secreted by this cell lines.
The cell lines obtained by the method of the present invention growth in protein-free medium stably for at least 40 generations.
The recombinant cell line hR3 was obtained by transfection of the myeloma NSO cell line with the vector constructions to express the light and heavy chains of the humanized anti-EGF human receptor hR3 monoclonal antibody. The adaptation of this cell line to protein-free medium was carried out following the procedure Previously described, by two stage reduction of protein content of the medium.
These cells were cultured in RPMI-1640 medium supplemented with 10% of FBS. The FBS was replaced by adding the protein reach supplement, Nutridoma NS (Boheringer Manheinn) to RPMI-1640 protein-free medium when the protein concentration was 0.15 mg/mL. The reduction of the protein content in the initial medium was done by successive dilutions with PFHM-II protein-free medium (Gibco).
The recombinant cell line T1hT was obtained by transfection of the myeloma NSO cell line with the vector constructions to express the light and heavy chains of a humanized by epitope T suppression method anti-human CD6 monoclonal antibody.
The adaptation of this cell line to protein/free medium was carried out following the procedure described in the point 2, by two stage reduction of protein content of the medium.
These cells were initially cultured in RPMI-1640 medium supplemented with 10% of FBS. The reduction in the protein content was done by successive dilution of the initial medium with PFHM-II protein-free medium from Gibco.
The results of calculation of the critical concentrations and adaptation rates Vadapt. are showed in the
The recombinant cell line T3Q was obtained by transfection of the myeloma NSO cell line with the vector constructions to express the light and heavy chains of the humanized monoclonal antibody T3Q which recognize the CD3 receptor on human lymphocytes.
The adaptation of this cell line to protein/free medium was carried out following the procedure described in the point 2, by two stage reduction of protein content of the medium.
These cells were initially cultured in RPMI-1640 medium supplemented with 10% of FBS. The reduction in the protein content was done by successive dilution in the PFHM-II protein-free medium from Gibco.
The results of calculation of the critical concentrations and adaptation rates Vadapt. are showed in the
| Number | Date | Country | Kind |
|---|---|---|---|
| 239/2002 | Oct 2002 | CU | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10532269 | Apr 2005 | US |
| Child | 12759208 | US |
