Development of physicochemical methods for analysis of pandemic influenza vaccine

Abstract

The utility of a size exclusion high-performance liquid chromatography (SE-HPLC) method, in the identification and quantitation of haemagglutinin (HA) protein in monovalent, inactivated influenza vaccines for the purposes of potency assessment is reported. This method is sufficiently eveloped to provide a distinct peak separation of HA from other vaccine constituents, and a correlation with HA potency determined by single radial-immunodiffusion (SRID) assay. Sensitivity of the method is demonstrated, with each HA peak accurately titrated to the HA content of the protein load injected. Highly reproducible chromatographic profiles (on a G4000SWxl column) were achieved, demonstrating vaccine protein integrity and stability. Potency assessments, determined by SE-HPLC peak area analysis, provided very good correlation with the quantitative HA protein values, determined by SRID, reported by the manufacturer and by Health Canada's Centre for Biologics Evaluation, Pandemic Influenza Division (PID). Peak resolution was further enhanced by expanding to a tandem SE-HPLC system, utilizing a G4000SWxl SE column coupled to a G3000SWxl column. HA was detected in the 11-17 minute (min) elution, collected in 30 second (sec) fractions, with the smallest, most distinct peak width detected to date at the 15.5 min fraction. This SE-HPLC method has considerable potential for widespread use as a physicochemical method for HA identification and quantification, in quality control testing for seasonal and pandemic influenza vaccines, and as a practical alternative for potency measures by the reagent dependent SRID assay. Increased resolution and further method development will facilitate the collection of separated fractions for further analysis, to correlate immunoactivity to HA type and content.