Evaluation of meningococcal C oligosaccharide conjugate vaccines by size-exclusion chromatography/multi-angle laser light scattering.
The mean molecular masses of three different meningococcal C saccharide (MenC)-protein conjugate vaccines and their constituent proteins were estimated using HPLC size-exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS) and refractive-index (RI) detection (SEC/MALLS). Chromatography of two CRM(197) conjugates (MenC-CRM(197)-A and MenC-CRM(197)-B) and one tetanus toxoid (TT) conjugate (MenC-TT) was performed in PBS, pH 7.4, on TSK-Gel (TosoHaas) analytical columns [CRM(197) is a non-catalytic cross-reacting mutant (CRM) of diphtheria toxin]. Analysis of the light-scattering signal measured at 18 angles simultaneously, using the RI signal as a measure of concentration, gave absolute weight-average-molecular-mass (M(w)) values for the CRM(197) conjugates as follows: MenC-CRM(197)-A, approximately 75,000 g x mol(-1) and MenC-CRM(197)-B, approximately 350,000 g x mol(-1), suggesting that MenC-CRM(197)-A is a monomer (one carrier protein per conjugate molecule), while MenC-CRM(197)-B is largely composed of conjugates containing three or four CRM(197) molecules. The MenC-TT conjugate eluted as a two-component system with (M(w)) of 1.63 x 10(6) and 395,000 g x mol(-1), suggesting that some cross-linked complexes contain up to six TT molecules. Comparison of results from MALLS/RI with those obtained using UV detection highlights the differences in size and relative composition of the various subpopulations of the MenC conjugates that can be obtained using different detection systems.
Jumel
Kornelia
K
National Centre for Macromolecular Hydrodynamics, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Sutton Bonington, LE12 5RD, UK.
Ho
Mei M
MM
Bolgiano
Barbara
B
D001426:Bacterial Proteins / Q000737:chemistry*; D002850:Chromatography, Gel / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D007834:Lasers; D008027:Light; D046911:Macromolecular Substances; D022401:Meningococcal Vaccines / Q000032:analysis / Q000737:chemistry*; D008970:Molecular Weight; D011135:Polysaccharides, Bacterial / Q000737:chemistry*; D012031:Refractometry / Q000379:methods*; D012542:Scattering, Radiation
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D022401:Meningococcal Vaccines; D011135:Polysaccharides, Bacterial; C037676:meningococcal group C polysaccharide; C410218:serogroup C meningococcal conjugate vaccine