Analytical Procedures Used in the Control of Bacterial Glycoconjugate Vaccines

Abstract

Bacterial glycoconjugate vaccines consist of capsular polysaccharide chemically conjugated to a carrier protein. Vaccines against Haemophilus influenzae type b (Hib) have proven effective in preventing invasive infection by Hib in young children. Because the stability of the conjugate is essential for vaccine efficacy, physicochemical analyses are necessary to ensure the integrity of these vaccines. Analytical methods used in conjugate vaccine development and production vary among manufacturers due to different components and conjugation chemistries. In the recent past, control authorities have focused on chemical analyses and conventional immunodiffusion and enzyme-linked immunosorbent assays to evaluate the protein and polysaccharide components of glycoconjugate vaccines. We have developed one-dimensional 500 MHz proton nuclear magnetic resonance (NMR) spectroscopy to determine the identity and purity of the polysaccharide component. The NMR spectrum of polyribosyl ribitol phosphate (PRP) was consistent with the structure of the repeating unit, and any trace contaminants were readily detected. We have also developed gel-permeation chromatography to assess the structure and stability of two conjugate vaccines. The characteristic peak height relationship for each manufacturer's product was consistent, with no detectable changes when stored according to manufacturer's specifications. Vaccine stability was determined by examining samples subjected to high temperatures for prolonged periods of time, and aggregation and degradation of vaccines was detected using this method. These physicochemical analyses are essential because there is no suitable animal model that correlates with immunogenicity in man. Therefore, there is an urgent need to develop new analytical procedures and biological correlates of activity in order to control Hib vaccines and develop new glycoconjugate vaccines against other pathogenic bacteria. In this study, NMR and fast-protein liquid chromatography (FPLC) analyses were used to determine the identity, purity, and stability of two Hib conjugate vaccines licensed for use in the UK. Our results demonstrate that physicochemical methods can be used to evaluate the manufacturing consistency and stability of protein-polysaccharide conjugate vaccines.