Fast and highly selective determination of hemagglutinin content in quadrivalent influenza vaccine by reversed-phase high-performance liquid chromatography method

J Chromatogr A. 2017 Dec 15;1528:18-24. doi: 10.1016/j.chroma.2017.10.041. Epub 2017 Oct 18.


Seasonal inactivated quadrivalent influenza vaccines are currently formulated to include antigens from two strains of influenza A and a strain from each of the two circulating influenza B virus lineages. However, the applicability of the potency assay currently required for the release of vaccines has been hindered due to cross-reactivity between the two B strains. In this study, a reversed-phase high-performance liquid chromatography method previously developed for the separation and quantitative determination of the hemagglutinin content in trivalent influenza vaccine preparations was further extended and found to be adaptable for the assessment of all four hemagglutinin antigens present in quadrivalent influenza vaccines. Vaccines prepared from monovalent bulks and commercial quadrivalent products from the past three vaccination seasons in the Northern Hemisphere were tested with the new method. The results showed excellent resolution of all four hemagglutinins from frequently interfering formulation agents such as surfactants. This method provides a simple approach for fast evaluation of quality and hemagglutinin strain identification in influenza vaccines. It is also the only physicochemical method capable of distinguishing the B strains in quadrivalent influenza vaccines.

Keywords: Antibody-free strain identity; B strain separation; Hemagglutinin content; Quadrivalent influenza vaccines (QIV); Reversed-phased high-Performance liquid chromatography (RP-HPLC).

MeSH terms

  • Chemistry, Pharmaceutical / methods*
  • Chromatography, Reverse-Phase*
  • Hemagglutinins / analysis*
  • Influenza A virus / immunology
  • Influenza B virus / immunology
  • Influenza Vaccines / chemistry*
  • Influenza Vaccines / standards


  • Hemagglutinins
  • Influenza Vaccines