Effect of Production Method and Gene Amplification on the Glycosylation Pattern of a Secreted Reporter Protein in CHO Cells

Abstract

We have investigated the independent effects of selective gene amplification (using the dhfr amplifiable selection marker) and culture operating strategy (batch vs repeated fed-batch vs semicontinuous perfusion) on the glycosylation of a recombinant reporter protein (secreted alkaline phosphatase, SEAP) produced in transfected Chinese hamster ovary (CHO) cells. HPLC analyses coupled with susceptibility to various exoglycosidases were used to determine the N-glycosylation profile of SEAP samples. The dhfr amplified cell line yielded an almost 10-fold increase in specific productivity as compared to that of the unamplified cell line. The glycosylation pattern of the reporter protein produced in batch bioreactor cultures of the amplified cell line showed only slight differences as compared to the glycosylation pattern of the protein from batch bioreactor cultures of the unamplified cell line. In contrast, analysis of SEAP glycosylation structures from the protein isolated from semicontinuous perfusion cultures indicated that both relative glycan content and extent of sialylation were increased as compared to samples isolated from repeated fed-batch cultures. These results suggest that the slow growing perfusion cultures produce more completely glycosylated proteins than the faster growing repeated fed-batch cultures.

Relationship between tissue plasminogen activator production and specific growth rate in Chinese Hamster Ovary cells cultured in mannose at low temperature

“…This should be experimentally demonstrated by studying the effect of mannose concentration and culture temperature on mRNA stability and it is out of the scope of this work. An inverse relationship between l and productivity rate of other recombinant proteins has been observed for different cell lines in a variety of different cultures conditions (Lipscomb et al 2005). However, in disagreement with our results, Yokota and Tanji (2008) in CHO cells cultures under varied serum concentrations found a positive dependence between the l and the specific production rate of recombinant protein.…”

The combined effect of sodium butyrate and low culture temperature on the production, sialylation, and biological activity of an antibody produced in CHO cells

“…Documented reports have shown that culture process variables can considerably affect the sialic acid levels of recombinant proteins [22,23]. A higher sialylation of biopharmaceutical proteins is advantageous because glycoproteins lacking terminal sialic acid are generally ineffective in vivo because of rapid clearance by asialoglycoprotein receptors [33,34].…”

“…Thus, it is urgent to understand how cell culture conditions affect Neu5Gc levels. Extensive efforts have been made to describe the effects of environmental factors on the total sialic acid content of a glycoprotein [22,23]. However, there are very few literature reports describing the impact of culture conditions on glycoprotein Neu5Gc levels.…”

Engineering CHO cell growth and recombinant protein productivity by overexpression of miR-7

“…Cell growth and viability was measured on a Guava Benchtop Cytometer after staining with Viacount TM (Guava Technologies). SEAP activity was measured using the phosphatase assay as described previously (Lipscomb et al, 2005). Samples were diluted to ensure readings fell within the appropriate absorbance limits.…”