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Evaluation of meningococcal C oligosaccharide conjugate vaccines by size‐exclusion chromatography/multi‐angle laser light scattering
Author(s) -
Jumel Kornelia,
Ho Mei M.,
Bolgiano Barbara
Publication year - 2002
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20020066
Subject(s) - chemistry , conjugate , size exclusion chromatography , chromatography , molecular mass , high performance liquid chromatography , diphtheria toxin , toxin , biochemistry , mathematical analysis , mathematics , enzyme
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 ( M̄ w ) values for the CRM 197 conjugates as follows: MenC‐CRM 197 ‐A, ≈75000 g·mol −1 and MenC–CRM 197 ‐B, ≈350000 g·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 M̄ w 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.