title: Development and validation of high-performance size exclusion chromatography methods to determine molecular size parameters of Haemophilus influenzae type b polysaccharides and conjugates.
abstract: Current vaccines against Haemophilus influenzae type b (Hib) consist of the polyribosyl ribitol phosphate (PRP) capsular polysaccharide chemically conjugated to a carrier protein. Among the various biological and physical analyses to be performed on these vaccines, the determination of the molecular size of the polysaccharide preparations throughout the conjugation process is particularly relevant. Comparison of results from high-performance size exclusion chromatography (HPSEC) with those routinely obtained using conventional gel permeation chromatography (CGPC) methods highlights the correlation between the two methods for determining the values of the chromatographic distribution coefficient (KD) of native and activated polysaccharides. The resulting data showed that the KD value is sufficient to characterize these polysaccharides using an HPSEC method. However, additional molecular size parameters (i.e., molar mass and hydrodynamic radius) are necessary for a reliable characterization of the tetanus conjugate (PRP-T), certainly due to the lattice-like structure of the conjugate. In practice, an absolute detection system in HPSEC composed of a low-angle light scattering detector, a viscometer, and a refractive index (RI) detector was used. As demonstrated, these HPSEC methods are rapid, accurate, and reproducible for the polysaccharides and their glycoconjugates and provide a relevant and more informative alternative to the current CGPC methods.
authors.affiliation: Analytical Research Department, SanofiPasteur, 69280 Marcy l'Étoile, France. Electronic address: jerome.thiebaud@sanofipasteur.com.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Thiébaud
authors.affiliation: Analytical Research Department, SanofiPasteur, 69280 Marcy l'Étoile, France.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Fanget
authors.affiliation: Analytical Research Department, SanofiPasteur, 69280 Marcy l'Étoile, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Jaudinaud
authors.affiliation: Discovery Department, Sanofi Pasteur, 69280 Marcy l'Étoile, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Fourrichon
authors.affiliation: Chief Medical Office, Sanofi Pasteur, 69280 Marcy l'Étoile, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Sabouraud
authors.affiliation: Analytical Research Department, SanofiPasteur, 69280 Marcy l'Étoile, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Talaga
authors.affiliation: Strategic Alignment and Expertise Department, Sanofi Pasteur, 69280 Marcy l'Étoile, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Uhlrich
mesh_terms: D002236:Carbohydrate Conformation; D002850:Chromatography, Gel / Q000379:methods* / Q000592:standards; D002851:Chromatography, High Pressure Liquid; D006001:Glycoconjugates / Q000737:chemistry*; D019741:Haemophilus influenzae type b / Q000737:chemistry*; D010316:Particle Size; D011134:Polysaccharides / Q000737:chemistry*
chemical_list: D006001:Glycoconjugates; D011134:Polysaccharides
keywords: Capsular polysaccharide; Glycoconjugate vaccines; HPSEC; Haemophilus influenzae type b; Method validation; Molecular size; PRP; Static light scattering