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N ‐glycan analysis by CGE–LIF: Profiling influenza A virus hemagglutinin N ‐glycosylation during vaccine production
Author(s) -
Schwarzer Jana,
Rapp Erdmann,
Reichl Udo
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800042
Subject(s) - glycosylation , glycan , glycoprotein , hemagglutinin (influenza) , virus , influenza a virus , n linked glycosylation , neuraminidase , monoclonal antibody , biology , influenza vaccine , chinese hamster ovary cell , biochemistry , recombinant dna , virology , chemistry , antibody , gene , immunology , receptor
Glycoproteins, such as monoclonal antibodies as well as recombinant and viral proteins produced in mammalian cell culture play an important role in manufacturing of many biopharmaceuticals. To ensure consisting quality of the corresponding products, glycosylation profiles have to be tightly controlled, as glycosylation affects important properties of the corresponding proteins, including bioactivity and antigenicity. This study describes the establishment of a method for analyzing N ‐glycosylation patterns of mammalian cell culture‐derived influenza A virus glycoproteins used in vaccine manufacturing. It comprises virus purification directly from cell culture supernatant, protein isolation, deglycosylation, and clean‐up steps as well as “fingerprint” analysis of N ‐glycan pools by CGE‐LIF, using a capillary DNA‐sequencer. Reproducibility studies of CGE‐LIF, virus purification, and sample preparation have been performed. For demonstrating its applicability, the method was exemplarily used for monitoring batch‐to‐batch reproducibility in vaccine production, with respect to the glycosylation pattern of the membrane protein hemagglutinin of influenza A/PR/8/34 (H1N1) virus. This method allows characterization of variations in protein glycosylation patterns, directly by N ‐glycan “fingerprint” alignment.