N-glycan analysis by CGE-LIF: profiling influenza A virus hemagglutinin N-glycosylation during vaccine production

Electrophoresis. 2008 Nov;29(20):4203-14. doi: 10.1002/elps.200800042.

Abstract

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.

Publication types

  • Evaluation Study

MeSH terms

  • Animals
  • Cell Culture Techniques
  • Cells, Cultured
  • Chromatography, High Pressure Liquid
  • Electrophoresis, Capillary / methods
  • Electrophoresis, Polyacrylamide Gel
  • Fluorescent Dyes
  • Glycosylation
  • Hemagglutinin Glycoproteins, Influenza Virus / analysis*
  • Hemagglutinin Glycoproteins, Influenza Virus / immunology
  • Hemagglutinin Glycoproteins, Influenza Virus / metabolism*
  • Influenza A Virus, H1N1 Subtype / chemistry
  • Influenza A Virus, H1N1 Subtype / immunology
  • Influenza A virus / chemistry*
  • Influenza A virus / immunology
  • Influenza Vaccines / analysis*
  • Polysaccharides / analysis*
  • Reproducibility of Results
  • Spectrometry, Mass, Electrospray Ionization

Substances

  • Fluorescent Dyes
  • Hemagglutinin Glycoproteins, Influenza Virus
  • Influenza Vaccines
  • Polysaccharides