Isolation, characterization and recombinant protein expression in Veggie-CHO: A serum-free CHO host cell line

Abstract

The dihydrofolate reductase-deficient Chinese hamster ovary cell line, DXB11-CHO, commonly used as a host cell for the production of recombinant proteins requires 7.5% serum-supplementation for optimal growth. Regulatory issues surrounding the use of serum in clinical production processes and the direct and indirect costs of using serum in large-scale production and recovery processes have triggered efforts to derive serum-independent host cell lines. We have successfully isolated a serum-free host that we named Veggie-CHO. Veggie-CHO was generated by adapting DXB11-CHO cells to growth in serum-free media in the absence of exogenous growth factors such as Transferrin and Insulin-like growth factor, which we have previously shown to be essential for growth and viability of DXB11-CHO cells. Veggie-CHO cells have been shown to maintain an average doubling time of 22 hr in continuous growth cultures over a period of three months and have retained the dihydrofolate reductase -deficient phenotype of their parental DXB11-CHO cells. These properties and the stability of its serum-free phenotype have allowed the use of Veggie-CHO as host cells for transfection and amplified expression of recombinant proteins. We describe the derivation a serum-free recombinant cell line with an average doubling time of 20 hr and specific productivity of 2.5 Units recombinant Flt-3L protein per 10e6 cells per day.

Characterization of yeastolate fractions that promote insect cell growth and recombinant protein production

“…Schlaeger (1996) and Heidemann et al (2000) have hypothesized that the hydrolysates provide cells with various low molecular weight nutrients that may be mimicked by an optimized initial free amino acid content of the protein-free medium. While Rasmussen et al (1998) and Burteau et al (2003) concluded that peptides in the hydrolysates could function as growth factors for CHO cells. Data from this work shows that F1 promoted Sf 9 cell growth most effectively in spite of its lowest concentration of free amino acids (Table 1).…”

Effects of clonal variation on growth, metabolism, and productivity in response to trophic factor stimulation: a study of Chinese hamster ovary cells producing a recombinant monoclonal antibody

“…A number of authors have suggested that IGF-I could substitute for insulin in cell culture medium, either by exogenous addition (Rasmussen et al 1998), or by engineering the cell lines to produce their own IGF-I (Hunt et al 1997;Li et al 2000;Sunstrom et al 1998;Sunstrom et al 2000). However, exogenous addition of IGF-I is complicated by the presence of IGF binding proteins (IGFBP) secreted by the cells.…”

Identification of novel small molecule enhancers of protein production by cultured mammalian cells

“…The clonal CHO cell line used as the host in our recombinant expression experiments was derived from previously described serum-free adapted CHO cells (Rasmussen et al, 1998). For 96-well plate screening, production temperatures and initial cell densities used had been tested and shown to be adequate for cell growth and response to control SMEs in preliminary experiments.…”