Molecular size distribution in pentavalent (A, C, Y, W, X) meningococcal polysaccharide conjugate vaccine by HPSEC-UV-MALS-RI method- a conceivable stability indicating parameter.

Molecular size distribution (MSD) of polysaccharides serves as a key parameter that directly correlates to the immunogenicity of vaccine. MSD at meningococcal polysaccharide (A, C, Y and W) or conjugate bulk level is well established under detailed pharmacopeial and WHO guidelines. We report here, a newly developed method for determination of molecular size distribution of pentavalent Meningococcal conjugate vaccine comprising of A, C, Y, W and X (MenFive). Although serogroup specific molecular size could not be estimated here; lot to lot consistency monitoring, molecular aggregates distribution in final lot, are key takeaways of this method. Determination of MSD in pentavalent fill finished product was quite challenging. Various columns/detectors combination, buffers, physico-chemical conditions (temperature, 2-8 °C, 25 °C, 40 °C and 60 °C; flow rate, 0.3 mL to 0.8 mL), liquid/lyophilized formulations, were explored. Polymer-based packed columns were explored for estimation for MSD by aqueous size exclusion chromatography, using combinations of- Shodex OHPAK SB 807 HQ, Shodex OHPAK SB 806 HQ, G6000 PWXL, coupled with guard Shodex OHPAK SB-G-6B. MenFive showed heterogenous distribution of molecules ranging from 200 to 19000 kDa, indicating its complex nature. However, 1000-8000 kDa was dominant range, comprising of ≥ 50 % distribution of molecules, in both liquid as well as lyophilized formulations, with average molecular weight around 6000-6500 kDa. The molar mass distribution after slicing would provide an insight to the conformation of molecules through its presentation as HMW, LMW, aggregates and subsequently, the presence of dominant population of molecules of a particular molecular weight and its total contribution in the sample.

Method development and validation of unbound saccharide content (serogroup A, C, Y, W, X) in novel pentavalent meningococcal polysaccharide conjugate vaccine with two different carrier proteins.

Exclusive DOC-HCl formulations were developed for free polysaccharide content estimation in Meningococcal serogroup A, C, Y, W and X from pentavalent meningococcal vaccine (A, C, Y, W, X). The DOC precipitation method reported herein stands as an alternative to the ultra-filtration method for free polysaccharide estimation. DOC content was optimized for all the serogroups at a single concentration, where as effective acid concentration was altered as per serogroup. Briefly, two DOC-HCl formulations were developed for intended purpose, one for TT conjugated serogroups Men A & Men X where as other for CRM conjugated serogroups Men C, Men Y and Men W with effective HCl concentration of 23 mM and 193 mM for precipitation of Protein-DOC complex respectively. Furthermore, an exclusive buffer/DOC-HCl formulation for estimation of Men X free polysaccharide in fill finished product was developed. Accuracy of the method was proven at 12.5 %, 25 %, 50 % and 100 % of test specification where recoveries were found in the range of 70-130 %. In case of repeatability, intra assay variation ranged from 2 % to 7 % whereas inter assay variation was noted to be 2-14 %. Specificity studied revealed no interference of assay components such as sample excipients, DOC, acids. Critical quality and stability-indicating characteristics were measured. Monovalent polysaccharide standards of Men A, C, Y, W and X were developed and assigned the unitage concentration 1.01, 1.10, 1.09, 1.08 and 1.00 mg/mL respectively. Linearity curve was optimized from 0.17 to 27 µg/mL for Men A and C whereas from 0.33 to 27 µg/mL for Men Y and W considering free polysaccharide content estimation. The study suggests that DOC-HCl method meets all the criteria for free polysaccharide estimation in multivalent vaccines with additional advantages of high throughput and sized independent separation hence can be used for quality control testing.

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.

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.