title: New capillary gel electrophoresis method for fast and accurate identification and quantification of multiple viral proteins in influenza vaccines.
abstract: Current methods for the identification and/or quantification of viral proteins in influenza virus and virosome samples suffer from long analysis times, limited protein coverage and/or low accuracy and precision. We studied and optimized capillary gel electrophoresis (CGE) in order to achieve faster and enhanced characterization and quantification of viral proteins. Sample preparation as well the composition of the gel buffer was investigated in order to achieve adequate protein separation in relatively short times. The total sample preparation (reduction and deglycosylation) could be carried out efficiently within two hours. Hydrodynamic injection, separation voltage, and capillary temperature were optimized in full factorial design. The final method was validated and showed good performance for hemagglutinin fragment 1 (HA1), hemagglutinin fragment 2 (HA2), matrix protein (M) and nucleoprotein (NP). The CGE method allowed identification of different virus strains based on their specific protein profile. B/Brisbane inactivated virus and virosome samples could be analyzed within one day. The CGE results (titers) were comparable to single radial immune-diffusion (SRID), but the method has the advantage of a much faster time to results. CGE analysis of A/Christchurch from upstream process demonstrated the applicability of the method to samples of high complexity. The CGE method could be used in the same analyte concentration range as the RP-HPLC method, but showed better precision and accuracy. Overall, the total analysis time for the CGE method was much shorter, allowing analysis of 100 samples in 4 days instead of 10 days for SRID.
authors.affiliation: Crucell Holland B.V., Archimedesweg 4-6, 2333 CN Leiden, The Netherlands. Electronic address: evantri@its.jnj.com.
authors.forename: Ewoud
authors.initials: E
authors.lastname: van Tricht
authors.affiliation: Crucell Holland B.V., Archimedesweg 4-6, 2333 CN Leiden, The Netherlands.
authors.forename: Lars
authors.initials: L
authors.lastname: Geurink
authors.affiliation: Crucell Holland B.V., Archimedesweg 4-6, 2333 CN Leiden, The Netherlands.
authors.forename: Bojana
authors.initials: B
authors.lastname: Pajic
authors.affiliation: Crucell Holland B.V., Archimedesweg 4-6, 2333 CN Leiden, The Netherlands.
authors.forename: Johan
authors.initials: J
authors.lastname: Nijenhuis
authors.affiliation: Crucell Holland B.V., Archimedesweg 4-6, 2333 CN Leiden, The Netherlands.
authors.forename: Harold
authors.initials: H
authors.lastname: Backus
authors.affiliation: Crucell Holland B.V., Archimedesweg 4-6, 2333 CN Leiden, The Netherlands.
authors.forename: Marta
authors.initials: M
authors.lastname: Germano
authors.affiliation: VU University Amsterdam, AIMMS Division of BioMolecular Analysis, Faculty of Sciences, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Govert W
authors.initials: GW
authors.lastname: Somsen
authors.affiliation: Kantisto BV, Callenburglaan 22, 3742 MV Baarn, The Netherlands; Uppsala University, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Analytical Pharmaceutical Chemistry, Biomedical Centre, PO Box 574, SE-751 23 Uppsala, Sweden; Australian Centre for Research on Separation Science ACROSS, School of Physical Sciences, Chemistry, University of Tasmania, Hobart, Australia.
authors.forename: Cari E
authors.initials: CE
authors.lastname: Sänger-van de Griend
mesh_terms: D019075:Electrophoresis, Capillary; D009980:Influenza A virus; D009981:Influenza B virus; D007252:Influenza Vaccines / Q000032:analysis*; D014764:Viral Proteins / Q000032:analysis*; D022701:Virosomes
chemical_list: D007252:Influenza Vaccines; D014764:Viral Proteins; D022701:Virosomes
keywords: CESDS; Capillary gel electrophoresis; Hemagglutinin; Influenza; Quantification; Viral proteins