Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

Abstract

To investigate the effect of culture temperature on erythropoietin (EPO) production and glycosylation in recombinant Chinese hamster ovary (CHO) cells, we cultivated CHO cells using a perfusion bioreactor. Cells were cultivated at 37 degrees C until viable cell concentration reached 1 x 10(7) cells/mL, and then culture temperature was shifted to 25 degrees C, 28 degrees C, 30 degrees C, 32 degrees C, 37 degrees C (control), respectively. Lowering culture temperature suppressed cell growth but was beneficial to maintain high cell viability for a longer period. In a control culture at 37 degrees C, cell viability gradually decreased and fell below 80% on day 18 while it remained over 90% throughout the culture at low culture temperature. The cumulative EPO production and specific EPO productivity, q(EPO), increased at low culture temperature and were the highest at 32 degrees C and 30 degrees C, respectively. Interestingly, the cumulative EPO production at culture temperature below 32 degrees C was not as high as the cumulative EPO production at 32 degrees C although the q(EPO) at culture temperature below 32 degrees C was comparable or even higher than the q(EPO) at 32 degrees C. This implies that the beneficial effect of lowering culture temperature below 32 degrees C on q(EPO) is outweighed by its detrimental effect on the integral of viable cells. The glycosylation of EPO was evaluated by isoelectric focusing, normal phase HPLC and anion exchange chromatography analyses. The quality of EPO at 32 degrees C in regard to acidic isoforms, antennary structures and sialylated N-linked glycans was comparable to that at 37 degrees C. However, at culture temperatures below 32 degrees C, the proportions of acidic isoforms, tetra-antennary structures and tetra-sialylated N-linked glycans were further reduced, suggesting that lowering culture temperature below 32 degrees C negatively affect the quality of EPO. Thus, taken together, cell culture at 32 degrees C turned out to be the most satisfactory since it showed the highest cumulative EPO production, and moreover, EPO quality at 32 degrees C was not deteriorated as obtained at 37 degrees C.

Towards dynamic metabolic flux analysis in CHO cell cultures

“…To combat these negative effects, various strategies have been employed to reduce the levels of by-products and reduce the effects that these compounds have on CHO cell metabolism. For example, process parameters such as pH, temperature, CO 2 and osmolarity have been optimized [12][13][14], and complex feeding strategies have been devised using limited glucose and glutamine feeding, or feeding of other nutrients [15][16][17]. In addition, metabolism of CHO cells has been genetically engineered to enhance anaplerosis by overexpression of pyruvate carboxylase genes [18] and antiapoptotic genes such as Bcl-2 [19].…”

Towards the implementation of quality by design to the production of therapeutic monoclonal antibodies with desired glycosylation patterns

“…This is consistent with previous research that has shown that the genes involved in the synthesis of the precursor oligosaccharide on the membrane of the endoplasmic reticulum are upregulated during the transition between growth stage 0 (G 0 ) and growth stage 1 (G 1 ). 101 In addition, Ahn and collaborators 100 found that at temperatures below 32 C, branching and sialic acid content of EPO produced in CHO cells decreased when compared to the glycoforms produced at 37…”

“…[98][99][100] These research groups have proposed that decreased temperatures maintain cell viability over longer periods of time, and that protein glycosylation occurs more efficiently under these high-viability conditions. This is consistent with previous research that has shown that the genes involved in the synthesis of the precursor oligosaccharide on the membrane of the endoplasmic reticulum are upregulated during the transition between growth stage 0 (G 0 ) and growth stage 1 (G 1 ).…”

Application of Quality by Design Paradigm to the Manufacture of Protein Therapeutics

“…pH causes GnTI, ManII, GalT and SiaT to mislocalize in Golgi [156] Viability, growth phase and temperature EPO/CHO:  branching at 32°C<T<37°C  temperature decreases UDPGlcNAc and UDP-GalNAc concentration [157] Serum and lipid supplements IFN /CHO:  branching with lipid supplementation…”