Evaluation of structural modification induced activation of pneumococcal polysaccharide by GC-MS for the conjugate vaccine.

Polysaccharide (Ps) activation evaluation is an imperative quality attribute in a conjugate vaccine. Pneumococcal polysaccharide (PnPs) serotypes 5, 6B, 14, 19A and 23F were cyanylated for 3 and 8 min. The cyanylated and non-cyanylated polysaccharides were methanolysed and derivatized to assess the activation of each sugar by GC-MS. The activation of 22 and 27% serotype 6B and 11 and 36% in serotype 23 F Ps at 3 and 8 min respectively showed controlled conjugation kinetics with CRM197 carrier protein estimated by SEC-HPLC and optimal absolute molar mass by SEC-MALS. The Glc and Gal are the most commonly activated sugars of all PnPs serotypes while N-acetyl sugars PneuNAc, GalNAc and Rha in serotypes 5, 14 and 19A respectively showed >50% activation which contributes to conjugate aggregate formation at 8 min compared to 3 min cyanylation. The GC-MS analysis of structural modifications at functional groups entails important information to characterize the activated polysaccharide for consistent conjugate vaccine manufacturing.

Quantitation of free cyanide using ion exchange chromatography in Neisseria meningitidis serogroups A, C, W, Y and X conjugates used in vaccine manufacture.

Polysaccharide vaccines essentially used in the prevention of bacterial infections are known to be good immunogens when conjugated to an immunogenic protein using various cyanylating agents. Analysis of residual cyanide in polysaccharide conjugate vaccines is an ardent task due to the complexity of the sample matrices and the lack of suitable methods. We report a selective ion chromatography method with electrochemical detection using IonPac AS7 column for estimation of residual cyanide in meningococcal serogroups A, C, W, Y and X bulk conjugates in presence of other interfering ions. Gold electrode and Ag/AgCl reference electrode ensures sensitivity and reproducibility of cyanide quantitation. The calibration curve of the method is linear having r2 ≥0.990 over the concentration range 1.45 ng/mL to 93.10 ng/mL. The recovery of cyanide in bulk conjugates ranged between 96.0% and 108.9%. The limits of detection and quantitation were 0.50 ng/mL and 1.45 ng/mL which corresponds to 0.31 ng/µg and 0.91 ng/µg of polysaccharide respectively. The method validation and feasibility study were performed using Men W and Men X bulk conjugates respectively with in house residual cyanide specification due to unavailability of pharmacopeia guidelines. The method is reproducible and can accurately quantify residual cyanide in purified meningococcal bulk conjugates.

Evaluation of a sensitive GC-MS method to detect polysorbate 80 in vaccine preparation.

Polysorbates are the most versatile and common surfactants used as protein stabilizers. Analysis of residual polysorbate 80 (PS 80) in conjugate vaccine is challenging due to complexity of conjugate matrices and heterogeneity of the structure of the PS 80 analyte. The direct approach using high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) and gas chromatography-mass spectrometry (GC-MS) that is based on oleic acid methyl ester formation followed by transesterification have been evaluated to quantitate residual PS 80 in meningococcal serogroups A, C, W, Y and X bulk conjugates. HPLC-ELSD method was observed to be less sensitive in comparison to the GC-MS method. The GC-MS method showed promise for quantitation of residual polysorbate 80 with advantages of higher sensitivity, simple sample preparation and mass spectral characterization compared to methods reported to literature. The oleic acid methyl ester was solubilized in hexane and injected in GC-MS to separate on highly polar capillary CP-WAX 52 CB Column. The mass spectral analysis showed characteristic ions at m/z 180, 222 and 264. The method was validated with linearity r2 > 0.99 over the concentration range of 0.5 to 100 µg/mL with LOD and LOQ of 0.3 and 0.91 respectively using PS 80 standard. The GC-MS method provides a simple, fast and label free technique for the precise quantitation of residual PS 80 in meningococcal bulk conjugate vaccine sample, achieved with accuracy between 85-105%.