HPCE monitoring of the N-glycosylation pattern and sialylation of murine erythropoietin produced by CHO cells in batch processes.
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.
Le Floch
François
F
Laboratoire des Sciences du Génie Chimique-CNRS, Institut National Polytechnique de Lorraine, ENSAIA, 2, avenue de la Forêt-de-Haye, BP 172, F-54505 Vandoeuvre-lès-Nancy, France.
Tessier
Bertrand
B
Chenuet
Sébastien
S
Guillaume
Jean-Marc
JM
Cans
Pierre
P
Marc
Annie
A
Goergen
Jean-Louis
JL
D000818:Animals; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods*; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D019075:Electrophoresis, Capillary / Q000379:methods*; D004921:Erythropoietin / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D051379:Mice; D019158:N-Acetylneuraminic Acid / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D013050:Spectrometry, Fluorescence / Q000379:methods
D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D004921:Erythropoietin; D019158:N-Acetylneuraminic Acid