HPCE Monitoring of the N-Glycosylation Pattern and Sialylation of Murine Erythropoietin Produced by CHO Cells in Batch Processes

• Published in: Biotechnology progress Vol. 20; no. 3; pp. 864 - 871
• Main Authors: Floch, François Le, Tessier, Bertrand, Chenuet, Sébastien, Guillaume, Jean-Marc, Cans, Pierre, Marc, Annie, Goergen, Jean-Louis
• Format: Journal Article
• Language: English
• Published: USA American Chemical Society 01.05.2004; American Institute of Chemical Engineers
• Subjects: Animals; Biological and medical sciences; Bioreactors; Biotechnology; Cell Culture Techniques - methods; Cricetinae; Culture Media, Serum-Free; Electrophoresis, Capillary - methods; Erythropoietin; Erythropoietin - analysis; Erythropoietin - chemistry; Erythropoietin - metabolism; Fluorescence; Fundamental and applied biological sciences. Psychology; Gel electrophoresis; Glycosylation; Hydrolysis; Lasers; Mice; N-Acetylneuraminic Acid - analysis; N-Acetylneuraminic Acid - chemistry; N-Acetylneuraminic Acid - metabolism; oligosaccharides; Protein Engineering - methods; Q1; Recombinant Proteins - analysis; Recombinant Proteins - biosynthesis; Spectrometry, Fluorescence - methods; Glycosylation; Batchwise; Erythropoietin; Operating process; CHO cell line; Monitoring
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp0343211

Using capillary electrophoresis coupled to laser‐induced fluorescence (HPCE‐LIF), it was possible to profile N‐linked oligosaccharides from EPO, including species containing sialic acid, during the course of batch cultures performed either in serum‐free or serum‐containing medium. Although an unusual high heterogeneity of the N‐linked oligosaccharides was observed by both SDS‐PAGE and HPCE analysis, the patterns of mEPO glycans after desialylation by mild acid hydrolysis were found to be quite constant over the course of the cultures either with or without serum supplementation. In contrast, when the protein was analyzed by HPCE without acidic desialylation, fingerprints of N‐linked oligosaccharides changed with time in serum‐containing conditions. This phenomenon appeared to be mainly due to the desialylation of mEPO as a result of a sialidase activity released upon cell lysis. These results demonstrate that though a higher EPO titer was obtained in serum supplemented conditions, sialylation of EPO was severely affected by the presence of serum in the culture medium.