Development of optimized transfectoma cell lines for production of chimeric antibodies in hollow fiber cell culture systems

Abstract

Methods for the selection of transfectoma cells that express large quantities of mouse-human chimeric antibodies have been develped. SP2/0 mouse myeloma cells were transfected with pSV2-gpt and pSV2-neo based immunoglobulin expression vectors. Double transfectants were selected using the xanthine-guanine phosphoribosyl transferase (gpt)and the neomycin (neo) selection marker genes. ELISA-based screening of transfectoma clones resulted in the isolation of IgG-producing transfectomas. Introduction of the kappa light-chain 3'-enhancer into the light-chain expression vector significantly increased immunoglobulin expression, but only when the enhancer was located at its physiological site, 9 kb downstream of the kappa constant region exon. With some of the transfectomas, final yields of up to 80 mg/L of chimeric IgG were obtained in conventional flask cultures using serum-free growth medium. A pilot-scale AcuSyst Maximizer hollow fiber cell culture system was used for the production of gram amounts of chimeric IgG. Results obtained with different transfectoma clones in conventional culture were not fully predictive for yields in the hollow fiber system. In contrast, differences in productivity between individual clones in the laboratory-scale Tecnomouse cell culture unit were comparable with those in the Maximizer system. Up to 200 mg of chimeric IgG were produced per day in one Maximizer bioreactor. (c) 1995 John Wiley & Sons, Inc.

Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions

“…In recent years, the increasing need for therapeutic proteins derived from mammalian cells such as chimeric antibodies (Bebbington et al, 1992;Robinson and Memmert, 1991;Schläpfer et al, 1995) has led to many developments in the field of animal cell technology. These multidisciplinary efforts have focused on improvement of bioreactor design, development of media formulations and optimization of feeding strategies in fed-batch and perfusion culture modes.…”

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“…There are a number of advantages to using hollow fiber bioreactors for the production of biologicals such as proteins (Liu et al, 1991;Ala-Uotila et al, 1994;Schlapfer et al, 1995), cells (Knazek et al, 1990;Stronek, 1999), and viruses (Ratner et al, 1978). These advantages are a direct result of cell retention and the high density cell growth that hollows fiber systems provide.…”

“…Media com-ponents and autocrine factors will partition across the membrane based on permeability, cellular consumption (or production), and diffusivity. As a result, the traditional research tools (such as T-flasks) for medium development or cell growth assessment are not useful for optimizing conditions for a hollow fiber system (Schlapfer et al, 1995). In addition, traditional hollow fiber systems are too cumbersome or expensive for routine screening purposes.…”

Screening Tool for Hollow‐Fiber Bioreactor Process Development

“…Data from 96-well plates or T-flask cultures are useful for prediction of cell line performance in stirred-tank reactors. However, these data do not correlate with cell growth and productivity in high-density hollow-fiber cultures (8). Research to understand the fundamental mechanisms that affect cell productivity in hollow-fiber culture is very complex, especially since the important factors are likely to be cell line dependent.…”

“…This lack of understanding is due, in part, to the lack of a good model system for fundamental research and process development. One approach to hollow-fiber research is to use a small system such as that shown in Figure 1 (8,9). However, these systems are still too expensive for routine development and cell growth takes several weeks to reach confluency.…”