title: Comparative glycomics analysis of influenza Hemagglutinin (H5N1) produced in vaccine relevant cell platforms.
abstract: Hemagglutinin (HA) is the major antigen in influenza vaccines, and glycosylation is known to influence its antigenicity. Embryonated hen eggs are traditionally used for influenza vaccine production, but vaccines produced in mammalian and insect cells were recently licensed. This raises the concern that vaccines produced with different cell systems might not be equivalent due to differences in their glycosylation patterns. Thus, we developed an analytical method to monitor vaccine glycosylation through a combination of nanoLC/MS(E) and quantitative MALDI-TOF MS permethylation profiling. We then used this method to examine glycosylation of HAs from two different influenza H5N1 strains produced in five different platforms, including hen eggs, three different insect cell lines (High Five, expresSF+ and glycoengineered expresSF+), and a human cell line (HEK293). Our results demonstrated that (1) sequon utilization is not necessarily equivalent in different cell types, (2) there are quantitative and qualitative differences in the overall N-glycosylation patterns and structures produced by different cell types, (3) ∼20% of the N-glycans on the HAs produced by High Five cells are core α1,3-fucosylated structures, which may be allergenic in humans, and (4) our method can be used to monitor differences in glycosylation during the cellular glycoengineering stages of vaccine development.
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
authors.forename: Yanming
authors.initials: Y
authors.lastname: An
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Rininger
authors.forename: Donald L
authors.initials: DL
authors.lastname: Jarvis
authors.forename: Xianghong
authors.initials: X
authors.lastname: Jing
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Ye
authors.forename: Jared J
authors.initials: JJ
authors.lastname: Aumiller
authors.forename: Maryna
authors.initials: M
authors.lastname: Eichelberger
authors.forename: John F
authors.initials: JF
authors.lastname: Cipollo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002642:Chick Embryo; D002645:Chickens; D054794:Glycomics; D006031:Glycosylation; D057809:HEK293 Cells; D006389:Hemagglutinins, Viral / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000737:chemistry* / Q000276:immunology; D053124:Influenza A Virus, H5N1 Subtype / Q000737:chemistry* / Q000276:immunology; D007252:Influenza Vaccines / Q000096:biosynthesis; D007251:Influenza, Human / Q000276:immunology / Q000517:prevention & control; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry; D061987:Sf9 Cells; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera; D015053:Zygote / Q000821:virology
chemical_list: D006389:Hemagglutinins, Viral; D007252:Influenza Vaccines; D011134:Polysaccharides