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PedvaxHIB is a pediatric vaccine that protects children from severe disease caused by the gram-negative bacterium Haemophilus influenzae type b (Hib). The vaccine is made by chemically conjugating Hib capsular polysaccharide to the outer membrane protein complex of Neisseria meningitidis. The protein-conjugated vaccine has proven to be extremely effective in preventing invasive Hib disease in infants and young children. This paper presents the nuclear magnetic resonance (NMR) methodology for the quantitative characterization of derivatized polysaccharide and its validation closely following ICH guidelines. The assay has been shown to be precise and accurate (relative standard deviation [RSD]

Purified capsular polysaccharide preparations from Streptococcus pneumoniae that are used for vaccine production typically contain residual levels of C-polysaccharide (C-Ps). Residual C-Ps is typically found in one of two forms, either chemically linked to the capsular polysaccharide (bound) or present by itself (free). Two analytical methods have been developed and applied to determine the relative percentages of the two C-Ps forms present in various capsular polysaccharide preparations. Both methods differentiate the two forms of C-Ps according to the difference of their hydrodynamic sizes. One method is based on labeling C-Ps with a fluorescent tag and separating the two forms of C-Ps by high-performance size exclusion chromatography with on-line refractive index and fluorescence detection, and the other method is based on measuring self-diffusion rates of the two forms of C-Ps by nuclear magnetic resonance (NMR) and quantifying each form with deconvolution. Both methods were evaluated for relative accuracy, precision, and ease of application, and they were found to provide comparable results for a large number of pneumococcal polysaccharide preparations. These analyses, combined with other quantitative NMR measurement of total C-Ps in the polysaccharide powder, provide a more refined means of evaluating the amount of each form of C-Ps in polysaccharide preparations targeted for vaccine production.

Colonization of implanted medical devices by coagulase-negative staphylococci such as Staphylococcus epidermidis is mediated by the bacterial polysaccharide intercellular adhesin (PIA), a polymer of beta-(1-->6)-linked glucosamine substituted with N-acetyl and O-succinyl constituents. The icaADBC locus containing the biosynthetic genes for production of PIA has been identified in both S. epidermidis and S. aureus. Whereas it is clear that PIA is a constituent that contributes to the virulence of S. epidermidis, it is less clear what role PIA plays in infection with S. aureus. Recently, identification of a novel polysaccharide antigen from S. aureus termed poly N-succinyl beta-(1-->6)-glucosamine (PNSG) has been reported. This polymer was composed of the same glycan backbone as PIA but was reported to contain a high proportion of N-succinylation rather than acetylation. We have isolated a glucosamine-containing exopolysaccharide from the constitutive over-producing MN8m strain of S. aureus in order to prepare polysaccharide-protein conjugate vaccines. In this report we demonstrate that MN8m produced a high-molecular-weight (>300,000 Da) polymer of beta-(1-->6)-linked glucosamine containing 45-60% N-acetyl, and a small amount of O-succinyl (approx 10% mole ratio to monosaccharide units). By detailed NMR analyses of polysaccharide preparations, we show that the previous identification of N-succinyl was an analytical artifact. The exopolysaccharide we have isolated is active in in vitro hemagglutination assays and is immunogenic in mice when coupled to a protein carrier. We therefore conclude that S. aureus strain MN8m produces a polymer that is chemically and biologically closely related to the PIA produced by S. epidermidis.