Method Validation for Determination of Total and Free Antigen Content in Meningococcal Vaccine Serotype A by HPAEC-PAD

Abstract

        The meningococcal vaccine is produced from the polysaccharide component of Neisseria meningitidis. Quantitative analysis of meningococcal vaccine serotype A antigen is an important test for quality control of vaccine to ensure adequate antigen content for effective immunization. This study was validated a method using High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). The method involved an anion-exchange column at 20°C, a mobile phase consisting of 44 mM NaOH and 280 mM sodium acetate, a flow rate of 1.0 mL/min, and an injection volume of 20 µL. Detection was performed using a PAD detector. The standard curve was linearity in the concentration range of 0.47 to 15.00 µg/mL, with a correlation coefficient (R) of 0.9983. The precision on the same day showed the relative standard deviation (RSD), was 0.22% for total antigen and 3.61% for free antigen, while intermediate precision showed the relative standard deviation of 1.31% and 2.93%, respectively. Furthermore, variations in analysts and instruments had no significant impact on the results, indicating the method’s robustness and reproducibility. The accuracy of measuring the standard substances used in the meningococcal vaccine was investigated, and the results showed that the percentage of recovery between 82.09% to 104.87%, indicating good accuracy. The method demonstrated specificity and was capable of quantifying antigen concentrations as low as 0.97 µg/mL in the vaccine. In conclusion, the HPAEC-PAD method is accurate, precise, and reliable, and is suitable for use as a standard method in the laboratory to ensure the production of high-quality meningococcal vaccines that meet standards.