Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Abstract

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10(9) cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.

The response of GS‐NS0 myeloma cells to pH shifts and pH perturbations

“…This occurs even when there is considerable glucose remaining in the culture medium. Only with pH shifts above pH 8.0 did lactate continue to accumulate in the medium after 48 h. This has also been observed by Zhou et al (1996Zhou et al ( , 1997 who investigated nutrient-feeding strategies for batch culturing of a similar GS-NS0 cell line. They reported a similar shift in lactate metabolism from production to consumption and postulated that this switch occurred when the cells became pyruvate limited; lactate being consumed to supply pyruvate.…”

“…Although these changes were kept to a minimum, the calculated pCO 2 values decreased from 146 mm Hg at pH 6.5 to 2 mm Hg at pH 9.0 and the calculated bicarbonate concentration increased from 1.13 g/L at pH 6.5 to 4.17 g/L at pH 9.0. Generally, data concerning the independent effect of pCO 2 on GS-NS0 cells is scarce, although Zhou et al (1996) and Aunins and Hensler (1993) reported that an increase in pCO 2 to 120 mm Hg accompanied by increasing osmolarity did not affect the culture performance of a similar GS-NS0 cell line. In addition, Kimura and Miller (1996) have investigated the effect of pCO 2 on CHO cells.…”

Transcriptome and proteome analysis of Chinese hamster ovary cells under low temperature and butyrate treatment

“…Increased productivity has also been realized through intensive bioprocess engineering research. The employment of fedbatch culture ( (Bibila et al, 1994;Bibila and Robinson, 1995;Xie and Wang, 1996;Zhou et al, 1996Zhou et al, , 1997a, reviewed in (Wlaschin and Hu, 2006)) has been instrumental in In recent years, both transcriptome and proteome analytical tools have gained popularity in cell culture applications, as reviewed in Gupta and Lee (2007). These applications have included the study of cell lines of varying productivities (Khoo et al, 2007;Seth et al, 2007a,b), as well as different culture conditions known to impact recombinant protein production (Baik et al, 2006;Shen and Sharfstein, 2006;De Leon Gatti et al, 2007;Yee et al, 2008a,b).…”

Achievement of high cell density and high antibody productivity by a controlled-fed perfusion bioreactor process

“…Correspondence to: J.-D. Yang for large-scale antibody production using this fed-batch approach (Bibila and Robinson, 1995;Seaver, 1997;Zhou et al, 1996Zhou et al, , 1997b.…”