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.

Effect of trifluoroacetic acid on the antigenicity of capsular polysaccharides obtained from various Streptococcus pneumoniae serotypes.

Determining the safety, antigenicity, and immunogenicity by in vitro and in vivo studies is a prerequisite for the development of new vaccines. And this study investigated it for a vaccine made from Streptococcus pneumoniae serotypes 2, 5, 12F, 18C, and 22F. The crude CPS was purified and partially depolymerized by conventional and trifluoroacetic acid methods. 1H NMR analysis confirmed the identity of the depolymerized CPS which gave similar profiles to reference polysaccharides, except for serotype 18C which was de-O-acetylated during TFA treatment. The antigenicity of the depolymerized CPS prepared by either method was comparable to that of the native CPS for serotypes 2, 5, 18C, and 22F based on multiplex bead based competitive inhibition assay. This study demonstrated a relationship between antigenicity and immunogenicity, which offers more suitable candidates for conjugation. It was found that after partial depolymerization process, the CPS with optimal molecular size resulted in higher antigenicity. The immunogenicity of S. pneumoniae serotype 2 conjugates in mice was evaluated by opsonophagocytic assay and a multiplex bead-based assay, wherein on day 42 after immunization, the total and functional IgG titer was found to be increased by 32-fold.

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.

Development of competitive inhibition ELISA as an effective potency test to analyze human rabies vaccines and assessment of the antigenic epitope of rabies glycoprotein.

The potency of all modern tissue culture human rabies vaccines is measured based on the National Institute of Health (NIH) potency test that is laborious, time-consuming, involves large test variations and requires sacrifice of large number of animals. To circumvent these limitations, several researchers and WHO expert working groups have discussed development of alternative in vitro methods to replace the NIH potency test. Although several immunochemical methods have been proposed to quantify rabies glycoprotein (G-protein) using multiple murine monoclonal antibodies, we report an In vitro competitive inhibition ELISA (CIA) method based on the use of a neutralizing rabies glycoprotein site III directed novel therapeutic human rabies monoclonal antibody (RAB1) that shows equivalence to the mice NIH potency test in recognition of neutralization site of the glycoprotein. In vitro potency testing of WHO 7th International Standard for rabies vaccine (IS) by CIA using RAB1 and In-house reference standard (IHRS) as a standard to assess its suitability for the assessment of validation parameters showed accurate and precise values with <15% coefficient variance. The method was validated using 5PL standard curve with linearity r2 > 0.98 and LLOQ of 0.125 IU/mL indicating sensitivity of the method. The method was found to be precise, robust and accurate to quantitate intact rabies glycoprotein in final vaccine and showed a strong correlation (Pearson's r = 0.81) with the NIH potency values of licensed Vero cell rabies vaccine. The CIA test using RAB1 was able to accurately quantitate degradation of rabies vaccine and assess loss in antigenicity of lyophilized and reconstituted liquid rabies vaccine under thermal stress conditions. The method was able to differentiate between potent and reduced potency vaccine samples. The new in vitro competitive inhibition ELISA method using RAB1 thus can be a valid alternative to the NIH test.