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Biotechnology and Applied Biochemistry (2002) 36, (219–226) (Printed in Great Britain)

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Evaluation of meningococcal C oligosaccharide conjugate vaccines by size-exclusion chromatography/multi-angle laser light scattering

Kornelia Jumel*, Mei M. Ho† and Barbara Bolgiano†¹

*National Centre for Macromolecular Hydrodynamics, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Sutton Bonington, LE12 5RD, U.K., and †Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts., EN6 3QG, U.K.

Key words: meningococcal C-CRM₁₉₇ conjugate, meningococcal C–tetanus toxoid conjugate.

Abbreviations used: CRM₁₉₇, a non-catalytic cross-reacting mutant (CRM) of diphtheria toxin; dn/dc, specific refractive-index increment; HPAEC-PAD, high-performance anion-exchange chromatography coupled with pulsed amperometric detection; MenC, meningococcal C saccharide; NeuAc, N-acetylneuraminic acid; NIBSC, National Institute for Biological Standards and Control; SEC/MALLS, HPLC size-exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS) and refractive-index (RI) detection; TT, tetanus toxoid (a formaldehyde-detoxified preparation of tetanus toxin).

¹To whom correspondence should be addressed (e-mail bbolgiano@nibsc.ac.uk).

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 (_w) values for the CRM₁₉₇ conjugates as follows: MenC-CRM₁₉₇-A, 75000 g · mol^-1 and MenC–CRM₁₉₇-B, 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 _w of 1.63×10⁶ 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.

Received 11 July 2002/16 August 2002; accepted 10 September 2002

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