Method Development and Validation of Identity Test for Pneumococcal Polysaccharides and CRM197 Carrier Protein in Pneumococcal Conjugate Vaccine by Slot Blot Assay

Abstract

Background: Identity testing is a crucial process that should be conducted on the pneumococcal conjugate vaccine to verify its identity for quality control purposes. The vaccine consists of purified polysaccharides from each serogroup of Streptococcus pneumoniae, conjugated with diphtheria cross-reactive material (CRM₁₉₇), as specified in the registration dossier. Therefore, this testing ensures that recipients can trust the quality, efficacy, and safety of the vaccine. The identity test of the pneumococcal conjugate vaccine is a newly developed identity testing method by the Institute of Biological Products.

Objective: To develop and validate a standard method for identity testing of pneumococcal polysaccharides and the CRM₁₉₇ carrier protein using the slot blot assay to verify the identity of the pneumococcal conjugate vaccine.

Methods: The optimum conditions were determined based on the specific reaction between the antigen and antibody, making them suitable for testing. Additionally, method validation was confirmed for two parameters including specificity and intermediate precision.

Results: The results of the optimum conditions for the identity test of pneumococcal polysaccharides and the carrier protein diphtheria Cross Reactive Material (CRM₁₉₇) in pneumococcal conjugate vaccines, using the slot blot assay, demonstrated that each antigen was specific to its corresponding serotype antibody, leading to the formation of an antigen-antibody complex. Therefore, this method must be performed according to the specified protocol to accurately verify the identity of the vaccine. Validation results revealed high specificity: pneumococcal conjugate vaccine samples displayed distinct dark purple bands, indicating positive identification, while meningococcal vaccine samples showed a negative result, represented by a white band on the nitrocellulose membrane. Furthermore, intermediate precision was confirmed by two independent analysts, both of them obtained consistent results. In both cases, dark purple bands appeared on the nitrocellulose membrane, indicating positive identification. Therefore, this method successfully passed validation and can be adopted as a standard procedure.

Conclusions: The study results revealed that the identity test for pneumococcal polysaccharides and the CRM₁₉₇ carrier protein using the slot blot assay met the validation requirements. Consequently, it is suitable for use as a standard method for the registration of the pneumococcal conjugate vaccine in Thailand.