title: High throughput quantification of capsular polysaccharides for multivalent vaccines using precipitation with a cationic surfactant.
abstract: The increasing requirement for multivalent vaccines containing diverse capsular polysaccharides has created an unmet need for a fast and straightforward assay for polysaccharide titer. We describe a novel and robust assay for the quantitation of anionic capsular polysaccharides. The binding of hexadecyltrimethyammonium bromide (Hb) to anionic capsular polysaccharides results in a precipitation reaction wherein the suspension turbidity is proportional to polysaccharide titer. The turbidity can be quickly measured as absorbance across a range of wavelengths that resolve scattering light. Carbohydrates comprised of repeating units of one to seven monosaccharides with phosphodiester groups, uronic acids, and sialic acids all reacted strongly and there does not appear to be specificity with respect to the particular anionic moiety. The assay is compatible with an array of common buffers across a pH range of 3.0-8.75 and with NaCl concentration exceeding 400 mM. Interference from DNA can be eliminated with a short incubation step with DNase. With these treatments, the assay has been employed in samples as complex as fermentation broth. A two-log dynamic range has been established with a mean relative standard deviation less than 10% across this range although inferior performance has been observed in fermentation broth. The precipitation assay enables the rapid quantitation of anionic polysaccharides. The resulting procedure can robustly measure the titer of myriad anionic capsular polysaccharides (CPS) in 96 samples in less than 30 min using low toxicity reagents and routine laboratory equipment. This development will greatly reduce the effort required to measure polysaccharide titer and yield during process development of polysaccharide vaccines.
authors.affiliation: The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK; Purification Process Development, Pfizer, 1 Burtt Road, Andover, MA 01810, USA. Electronic address: Aaron.Noyes@pfizer.com.
authors.forename: Aaron
authors.initials: A
authors.lastname: Noyes
authors.forename: Austin
authors.initials: A
authors.lastname: Boesch
authors.forename: Ranga
authors.initials: R
authors.lastname: Godavarti
authors.forename: Nigel
authors.initials: N
authors.lastname: Titchener-Hooker
authors.forename: Jonathan
authors.initials: J
authors.lastname: Coffman
authors.forename: Tarit
authors.initials: T
authors.lastname: Mukhopadhyay
mesh_terms: D001428:Bacterial Vaccines / Q000737:chemistry* / Q000276:immunology; D002412:Cations / Q000378:metabolism*; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000378:metabolism; D011232:Chemical Precipitation; D057166:High-Throughput Screening Assays / Q000379:methods*; D011134:Polysaccharides / Q000032:analysis*; D013053:Spectrophotometry / Q000379:methods; D013501:Surface-Active Agents / Q000378:metabolism*; D064166:Vaccine Potency
chemical_list: D001428:Bacterial Vaccines; D002412:Cations; D002593:Cetrimonium Compounds; D011134:Polysaccharides; D013501:Surface-Active Agents; D000077286:Cetrimonium
keywords: Anionic capsular polysaccharides; CMC; CPS; HTPD; Hb; Hexadecyltrimethylammonium bromide; High throughput process development; I; Mn C; Mn Y; RSD; Sugar quantitation; Titer; Vaccine; capsular polysaccharide; critical micelle concentration; hexadecyltrimethylammonium bromide; high throughput process development; ionic strength; meningococcal serotype C capsular polysaccharide; meningococcal serotype Y capsular polysaccharide; relative standard deviation