Evaluation of GC-MS for identification and characterization of pneumococcal serotype 24A, 24B, and 24F capsular polysaccharide.

Analysis of pneumococcal polysaccharides (PnPs) has been an arduous task, especially in similar serotypes. Pneumococci invades the host immune response by modulating capsule structure with small genetic changes making them indistinguishable from similar serotypes by conventional modes of analysis. The new serotype 24F causing invasive pneumococcal-resistant infection is an analytical challenge for its analysis as related serotypes 24A and 24B Ps share a common backbone. The difference in the branched chain which contains arabinitol and ribitol in 24F and 24B respectively are stereoisomers making their identification even more challenging. The composition analysis by GC-MS revealed distinct peaks for arabinitol in 24F and 24A Ps and ribitol in Pn 24B serotype polysaccharide. The mass spectral analysis confirmed their identification along with a heterologous cross-reactivity which confirmed anti-Pn-24F mAb reactive to Pn 24B than Pn 24A. The quantitative analysis of pneumococcal 24A, 24B and 24F using GC-MS showed sensitive analysis over the concentration range 3.125-200 µg/mL with regression coefficient >0.99 making ideal modality for the characterization, identification, and quantitation of pneumococcal 24A, 24B and 24F similar serotypes.

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.

Simultaneous purification and depolymerization of Streptococcus pneumoniae serotype 2 capsular polysaccharides by trifluoroacetic acid.

Development of an effective purification process in order to provide low cost and high-quality vaccine is the necessity of glycoconjugate vaccine manufacturing industries. In the present study, we have attempted to develop a method for simultaneous purification and depolymerization process for capsular polysaccharides (CPS) derived from Streptococcus pneumoniae serotype 2. Trifluoroacetic acid (TFA) was used to precipitate impurities which were then removed by centrifugation. It was observed that the TFA treatment could simultaneously depolymerize the CPS and purify it. The purified and depolymerized CPS was analyzed for its purity, structural identity and conformity, molecular size, antigenicity to meet desired quality specifications. The obtained results showed that the purification and depolymerization of S. pneumoniae serotype 2 CPS did not affect the antigenicity of CPS.

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%.