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KMID : 0545120030130060926
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Journal of Microbiology and Biotechnology
2003 Volume.13 No. 6 p.926 ~ p.936
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Characterization of Humanized Antibody Produced by Apoptosis-Resistant CHO Cells under Sodium Butyrate-Induced Condition
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KIM, NO SOO
CHANG, KERN HEE/CHUNG, BO SUP/KIM, SUNG HYUN/KIM, JUNG HOE/LEE, GYUN MIN
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Abstract
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Overexpression of human Bcl-2 protein in recombinant Chinese hamster ovary (rCHO) cells producing humanized antibody (SH2-0.32) considerably suppressed sodium butyrate (NaBu)-induced apoptosis during batch culture by using commercially available serum-free medium. which extended the culture longevity.
Due to the extended culture longevity provided by the anti-apoptotic effect of Bcl-2 overexpression. The final anfbody concentration of 14C6-bcl-2 culture (Bcl-2 high producer. 23 ㎍ m l^(-1) ) was 2 times higher than that of the SH2-0.32-Δbcl-2 culture (cells transfected with hcl-2-deficient plasmid, 10.5 ㎍ ml^(-1 ) in the presence of NaBu. To determine the effect of NaBu/Bcl-2 overexpression on the molecular integrity of protein products. antibodies purified from 14C6-bcl-2 and SH2-0.32-Abcl-2 cultures in the presence of NaBu were characterized by using various molecular assay systems. For comparison. antibody purified from the parental rCHO cell culture (SH2-0.32) in the abscnce of NaBu was also characterized. No significant changes in molecular weight of antibodies could be observed by SDS-PAGE. From GlycoSep-N column analysis, it was found that the core oligosaccharide structure (GlcNAc₂,Man₃,ClcNAc₂) was not affected by NaBu/Bcl-2 overexpression, while the microheterogeneity of N-linked oligosaccharide structure was slightly affected. Compared with the antibody produced in the absence of NaBu, the proportion of neutral oligosaccharides was increased from 10% ( 14C6-bcl-2) to 16% (SH2-0.32-ΔbcI-2) in the presence of NaBu. which was accompanied by the reduced proportion of acidic oligosaccharides. cspccially of monosialylated and disial ylated forms.
The changes in microheterogeneousoligofomal structures of antibody in turn affected the mobility of antibody isoforms in isoelectric focusing (IEF). resulting in the occurrence of some more basic antibody isofornis produced in the presence of NaBu. However, the antigen-antibody binding properties were not changed by alteration of glycosylation pattern. The competitive enzyme-linked immunosorbent assay (ELISA) showed that the antibody produced by NaBu/Bcl-2 overexpression maintained its antigen-antibody binding propcrties with binding affinity of about 2.5 ×10¹" MI. Taken together, no significant effects of NaBu/Bcl-2 ovcrexprcssion on the molecular integrity of´ antibodies. produccd by using scrim-free medium, could be observed by the molecular assay systems.
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