Quantitation of serogroups in multivalent polysaccharide-based meningococcal vaccines: optimisation of hydrolysis conditions and chromatographic methods

Abstract

Quantitative determination of the individual polysaccharide components in multivalent meningococcal vaccines is an important step in manufacturing and regulatory control. Current methods are complicated due to the use of multiple chromatographic setups and/or other analytical techniques for the four meningococcal serogroup polysaccharides (A, C, Y, W135). In addition, different methods are sometimes used depending on whether or not the polysaccharide is conjugated to a carrier protein. In an effort to simplify such analyses, hydrolysis conditions were determined for the optimal yield of each characteristic saccharide from the respective repeating units. One condition was identified for mannosamine-6-phosphate from MenA, one for neuraminic acid from MenC, and one for both glucose and galactose from MenY and MenW135, respectively. These conditions, initially assessed for monovalent solutions, were then confirmed for a quadrivalent solution. The monosaccharide products were separated, identified and quantitated using a single HPAEC-PAD protocol, with a customised multi-stage linear gradient eluent profile and one column setup, for determination of all four serogroup components. Comparison to calibration curves constructed from sets of monosaccharide or hydrolysed polysaccharide standards allowed for the quantitation of each characteristic serogroup monosaccharide in polysaccharide and polysaccharide-conjugate vaccines. When required, molecular size separation using a non-cellulosic centrifugal filter device effectively removed all interfering saccharide excipient without loss of serogroup polysaccharides. These methods were used to analyse multiple lots of a number of different monovalent or multivalent real polysaccharide-based vaccine products, in liquid or lyophilised powder formulations, with or without excipients. The methods were demonstrated to be highly reproducible and very useful for the evaluation of antigen content and lot-to-lot consistency of manufacture. The methods described here represent an increase in precision, level of accuracy and efficiency compared to current methods, and may be adaptable for evaluation of other types of polysaccharide-based vaccines.

Keywords: CV; Capsular polysaccharide; EP; European Pharmacopoeia; Excipient removal; Gal; Glc; Glycoconjugate; HPAEC-PAD; ManN-6-P; ManNAc-6-P; Meningococcal vaccine; N-acetylmannosamine-6-phosphate; N-acetylneuraminic acid; Neisseria meningitidis; Neu; Neu5Ac; PS-conj; Quantitation; Serogroup; TFA; coefficient of variation; galactose; glucose; high-pH anion-exchange chromatography with pulsed amperometric detection; mannosamine-6-phosphate; neuraminic acid; polysaccharide-conjugate; trifluoroacetic acid.