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 CRM197 conjugates (MenC-CRM197-A and MenC-CRM197-B) and one tetanus toxoid (TT) conjugate (MenC-TT) was performed in PBS, pH 7.4, on TSK-Gel® (TosoHaas) analytical columns [CRM197 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 (Mw) values for the CRM197 conjugates as follows: MenC-CRM197-A, (almost equal) 75000 g · mol-1 and MenC-CRM197-B, (almost equal) 350000 g · mol^-1, suggesting that MenC-CRM197-A is a monomer (one carrier protein per conjugate molecule), while MenC-CRM197-B is largely composed of conjugates containing three or four CRM197 molecules. The MenC-TT conjugate eluted as a two-component system with Mw of 1.63×10^6 and 395000 g · 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.