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A variety of different fermentation processes has been successfully employed to produce consistent protein-based biopharmaceuticals from genetically engineered animal cells. Chinese hamster ovary (CHO) cells were genetically modified to produce recombinant human soluble CD4, tissue plasminogen activator (tPA) or erythropoietin (EPO). Soluble CD4 was collected from extended perfused fermentations of several months' duration, during which some quantitative loss of DNA copy level, mRNA expression level, and fermentation titer were observed. In one extended run, a novel contaminant appeared in intermediates purified from later harvests. However, in all cases, the final soluble CD4 product was consistent in terms of purity and potency. Evaluation of genetic stability for tPA examined both biological traits at the cellular level as well as potency, purity and structure of product derived from cells at various levels of in vitro age; no significant cell age effects were observed. Similarly, evaluation of the EPO product showed that genetically-determined and process-determined traits such as potency, tryptic peptide mapping, and sialylation were consistent from lot to lot. These data exemplified how process design, process validation, and in-process and quality control assays can be used effectively to ensure the consistency of recombinant products derived from cell culture fermentations.

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Fluorescence in situ hybridization (FISH, 15) and high density, non-selective long term perfusion culture were used to study aspects of genetic stability and productivity in recombinant CHO cells. We analysed the distribution and structure of recombinant amplicons in chromosomes of CHO cells used for the production of proteins. In the presence, but not in the absence, of methotrexate (MTX) we found a high proportion of cells (40-60%) with multiple and/or unusually structured and extended chromosome regions containing amplified sequences. Removal of MTX from culture media resulted in the rapid decline in the frequency of cells containing amplified sequences exhibiting multiple and heterogeneous integrations. In cloned lines cultivated in the absence of MTX, a well defined signal motif on a specific chromosome, interpreted as the "master integration" unit, became increasingly abundant over time until almost all cells contained that signal motif. We used fluorescence in situ hybridization (FISH) with chemically modified DNA probes complementary to the integrated sequences to verify the stability of master integrations in cells cultured for extended periods in medium lacking MTX. In a second approach, we studied long-term stability of recombinant sequences during non-selective perfusion culture of CHO populations with non-cloned, MTX resistant heterogeneous populations of cells producing CD4IgG chimeric molecules. Due to the mode of transfection and primary selection the resulting cell lines consisted of subpopulations of cells derived from various independent integration events. The amplification and MTX selection procedure used consecutively generated multiple, structurally different amplicon types in the cell population, thus increasing the degree of heterogeneity. High density perfusion culture of these cells in the absence of MTX was used to maximize growth rates and was thought to select against cells with reduced growth rates, due to the highly amplified state of their introduced DNA, with concomitantly high productivity. However we found no evidence for such a selection; cells showed no reduction in copy number or total loss of amplified sequences at the end of the culture. More significantly, specific productivity of these cell lines grown under non-selective conditions did not decrease over the 100 days observation period.

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Mammalian cell suspension culture systems are being used increasingly in the biotechnology industry. This is due to their many advantages including simplicity and homogeneity of culture. Suspension systems are very adaptable (e.g., for microcarrier, microencapsulation, or other methods of culture). Their engineering is thoroughly understood and standardized at large scale, and automation and cleaning procedures are well established. Suspension systems offer the possibility of quick implementation of production protocols due to their ability to be scaled easily once the basic culture parameters are understood. The only main disadvantage of the suspension culture systems to date is their inapplicability for the production of human vaccines from either primary cell lines or from normal human diploid cell lines (Hayflick et al., 1987 and references therein). One of the great advantages of suspension culture is the opportunity it provides to study interactions of metabolic and production phenomena in chemostat or turbidostat steady-state systems. Furthermore, in suspension culture systems from which cell number and cell mass measurements are easy to obtain, rigorous and quantitative estimations of the effects of growth conditions or perturbations of metabolic homeostasis can be made. Such studies can speed up the development of optimal processes. With our increasing understanding of factors influencing expression in mammalian cells (Cohen and Levinson, 1988; Santoro et al., 1988) and the direct application of new methods in suspension culture (Rhodes and Birch, 1988), its usefulness and importance is likely to increase in the future. In this chapter, we have described some of the potential uses of the various suspension culture systems and have covered most of the established technology and literature. Due to the rapid developments and needs in the biotechnology industry and the versatility of suspension culture systems, it is probable that many more variations on this theme will evolve in the near future at both the pilot and production scales.

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