Denis Drapeau

Adaptation of Mammalian Cells to Growth in Serum-Free Media

A three-step protocol is described for adapting an anchorage-dependent, serum-dependent recombinant mammalian cell lineage to high density serum-free suspension culture. The objective is a cell lineage that is well-suited for the manufacture of a recombinant protein. The first step of the protocol generates an anchorage-independent cell lineage by culturing trypsin-treated cells in spinner flasks using serum-containing medium. The second step adapts the lineage to serum-free medium through a series of serum reduction steps in the presence of defined growth-promoting additives. The third step adapts the lineage to high-cell-density conditions by culturing the cells in a bioreactor in a manner that allows development of tolerance to growth-inhibiting substances released by the cells. Examples are presented for the use of this protocol for recombinant CHO cells.

Extracellular insulin degrading activity creates instability in a CHO-based batch-refeed continuous process

In a batch-refeed continuous process involving a recombinant Chinese hamster ovary cell line, a brief upset was Occasionally observed during which cell growth halted and cell viability dropped. This was found to be associated with depletion of insulin from the medium early during the affected passage. Insulin depletion was found to be primarily the result of insulin degrading activity released by the cells during the preceding passage.

Development of a Large Scale Process for the Production of Recombinant Truncated Factor VIII in CHO Cells under Cell Growth Arrest Conditions

A large scale biopharmaceutical process has been developed for the production of a genetically engineered variant of coagulation factor VIII. Factor VIII is the coagulation protein that is defective or missing in hemophilia A. Two challenging techniques, ceil growth arrest and perfusion, have been applied to ensure a high production yield. Key development aspects of this process will be presented. A B-domain deleted variant of the factor VIII gene, the factor VIII SQ gene, has been constructed and integrated in Chinese Hamster Ovary CHO DG44 DHFR− cells. The cells are cultivated in serum-free medium which has been developed to optimize the factor VIII SQ production. The cultivation and purification processes have been selected to reduce the deleterious actions of proteases present in the cultivation process. Factor VIII SQ has structural and functional characteristics comparable to full length factor VIII and has been licensed in Europe and USA under the name ReFacto (Genetics Institute).

Cell Stability, Animal

Factors that Affect Genetic Stability of Endogenous Genes Oncogenic Transformation, Aneuploidy, and Genetic Stability Molecular Regulation of Genetic Stablity Genotypic and Phenotypic Stability of Recombinant Cell Lines Stable Integration of Heterologous Genes in Animal Cell Genomes Phenotypic Heterogeneity and Population Dynamics Genetic and Epigenetic Manipulation of Cell Phenotypes Consequences of Genetic Instability on Recombinant Protein Quality Genotypic Characterization and Validation of Genetic Stability Gross Structural Evaluation of Genes and Transcripts Sequence Analysis of Genes and Transcripts Encoding the Product Genetic Stability Bibliography Keywords: cell storage; cell lines; transformation