A new seed-train expansion method for recombinant mammalian cell lines
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A new approach has been developed and used to minimize the timeand more carefully monitor and control the seed-train expansionprocess of recombinant mammalian cell lines. The process uses 50or 100 ml cryo-bags that contain frozen cells at high cell densities of 20 × 106 ml-1 (100 ml bags) or 40 × 106 cells ml-1 (50 ml bags). The frozen bag cell suspension is thawed and transferred directly into a bioreactorthat has been modified such that pH, DO and temperature can becontrolled at the initial volume of two liters (the working volume eventually increases to 12 l). The successful use of thesecryo-bags and the modified `inoculation' bioreactor to initiate anew seed train expansion of rBHK or rCHO cells is described herein. The interval between cell thawing and the accumulation ofsufficient cell mass to inoculate a production reactor is reducedby at least 25 to 30 days compared to the conventional method that begins with the thaw of 1–2 ml cryo-vials. This `one-step'technology leads to a much more consistent scale-up by reducingmanual operations and avoiding subjective decisions during the scale-up phase. The cell metabolic rates and product integritywere similar to the control experiments. Furthermore, it was found that it is not necessary to include a wash step to removeDMSO prior to the inoculation.
- Avis, KE, Wagner, CM (1999) Cryopreservation – Applications in Pharmaceuticals and Biotechnology. Drug Manufacturing Technology Series. Interpharm Press, Denver, Colorado
- Chuppa, S, Tsai, Y-S, Yoon, S, Shackleford, S, Rozales, C, Bhat, R, Tsay, G, Matanguihan, C, Konstantinov, K, Naveh, D (1997) Fermentor temperature as a tool for control of high-density perfusion cultures of mammalian cells. Biotechnol Bioeng 55: pp. 328-338
- Diener, B, Utesch, D, Beer, N, Dürk, H, Oesch, F (1993) A method for the cryopreservation of liver parenchymal cells for studies of xenobiotics. Cryobiology 30: pp. 116-127
- Fennema, OR, Powrie, WD, Marth, EH (1973) Low-temperature Preservation of Food and Living Matter. Marcel Dekker, Inc., New York
- Franks F (1985) Biophysics and Biochemistry at Low Temperatures. Cambridge University Press.
- Franks, F (1991) Freeze-drying: From empiricism to predictability: The significance of glass transitions. Karger, Basel. Dev Biol Stand 74: pp. 9-19
- Grout, B, Morris, J, McLellan, M (1990) Cryopreservation and the maintenance of cell lines. Trends Biotechnol 8: pp. 293-297
- Heidemann, R, Zhang, C, Qi, H, Rule, J, Rozales, C, Park, S, Chuppa, S, Ray, M, Michaels, J, Konstantinov, K, Naveh, D (2000) The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells. Cytotechnology 32: pp. 157-167
- Heldman, DR, Singh, RP (1981) Food Process Engineering. The AVI Publishing Company, Inc., Westport, Connecticut
- Lindl T & Bauer J (1989) Zell-und Gewebekultur. 2. Aufl. Gustav Fischer Verlag.
- Morgan, SJ, Darling, DC (1993) Animal Cell Culture. Bios Scientific Publishers, Oxford
- Ninomiya, N, Shirahata, S, Murakami, H, Sugahara, T (1991) Largescale, high density freezing of hybridomas and its application to high-density culture. Biotechnol Bioeng 38: pp. 1110-1113
- Re, A, Vijayaraghhavan, K, Basade, M, He, S, Gulati, S (1998) Long-term cryopreservation: Successful trilineage engraftment after autologous bone marrow transplantation with bone marrow cryopreserved for seven years. J Hematotherapy 7: pp. 185-188
- Regidor, C, Posada, M, Monteagudo, D, Garauled, C, Somolinos, N, Forés, R, Briz, M, Fernández, M (1999) Stem cell transplantation. Exp Hematology 27: pp. 380-385
- Siegel, WH Cryopreservation of Mammailian Cell Cultures. In: Avis, KE, Wagner, CM eds. (1999) Cryopreservation – Applications in Pharmaceuticals and Biotechnology. Drug Manufacturing Technology Series. Interpharm Press, Denver, Colorado
- Thrift J, Tsai Y, Lowe B, Ng M, Michaels J, Hey J & Konstantinov, K (2000) Development and Optimization of Protein-free Cell Banking Technology. Proceedings of the Cell Culture Engineering VII Foundation Meeting, Santa Fee, NM, 5–10 February.
- Whitaker SC, Francis R & Siegel RC (1998) Validation of Continuously Perfused Cell Culture Processes for Production of Monoclonal Antibodies. In Kelly BD and Ramelmeier A (eds) Validation of Biopharmaceutical Manufacturing Processes. American Chemical Society, pp. 28–43.
- Wu, L, Sun, J, Wang, L, Woodman, K, Koutalistras, N, Horvat, M, Sheil, AGR (2000) Cryopreservation of primary porcine hepatocytes for use in bioartificial liver support systems. Transplantation Proc 32: pp. 2271-2272
- Wu, L, Sun, J, Wang, L, Woodman, K, Koutalistras, N, Horvat, M, Sheil, AGR (2001) Cryopreserved porcine hepatocytes in a liver biodialysis system. Transplantation Proc 33: pp. 1950-1951
- Zachariou M, Mazer J, Park H & Olson C (2001) Method for Evaluating Target Protein Quality from Fermenter. US Patent 6,214,975 B1.
- A new seed-train expansion method for recombinant mammalian cell lines
Volume 38, Issue 1-3 , pp 99-108
- Cover Date
- Print ISSN
- Online ISSN
- Kluwer Academic Publishers
- Additional Links
- cell banking
- cryo bags
- inoculation bioreactor
- one step inoculation
- seed-train expansion
- Industry Sectors
- Author Affiliations
- 1. Biotechnology Unit, Bayer Corporation, Berkeley, CA, USA
- 2. Bayer AG, D-42117, Wuppertal, Germany