Bromelain-cleaved hemagglutinin production from cell culture-derived influenza viruses enhances vaccine potency quantification by single radial immunodiffusion assay.

Single Radial Immunodiffusion (SRID) assay remains the gold standard for determining influenza vaccine potency. Accurate SRID assays require high-purity antigens and strain-specific antisera that closely match circulating viruses. However, mutations in influenza viruses propagated in traditional egg-based systems affect antigenicity and assay accuracy. Therefore, we optimized bromelain-cleaved hemagglutinin (BHA) production from cell culture-derived influenza viruses, specifically targeting A/H3N2 and B/Victoria lineage strains. We employed a sequential enzymatic digestion strategy and successfully generated highly purified BHA with improved antigen yield and minimal neuraminidase contamination. The resulting antisera demonstrated strong, strain-specific reactivity. Subsequent SRID assays confirmed that homologous antigen-antiserum pairs derived from cell culture-derived materials provided significantly more accurate hemagglutinin quantification than the heterologous or egg-derived combinations. These findings highlight the need to match antigen and antiserum sources. Further, cell culture-derived reagents could be used to enhance assay reliability, advancing influenza vaccine standardization and quality control in cell-based vaccine production. By demonstrating the importance of antigen-antiserum matching and optimizing BHA production from cell culture-derived influenza viruses, this study establishes a practical foundation for improving the reliability of SRID-based potency testing and advancing the standardization of next-generation influenza vaccines.