Size Distribution Analysis of Influenza Virus Particles Using Size Exclusion Chromatography

J Chromatogr A. 2016 Sep 23;1465:117-25. doi: 10.1016/j.chroma.2016.08.056. Epub 2016 Aug 26.


Size exclusion chromatography is a standard method in quality control of biopharmaceutical proteins. In contrast, vaccine analysis is often based on activity assays. The hemagglutination assay is a widely accepted influenza quantification method, providing no insight in the size distribution of virus particles. Capabilities of size exclusion chromatography to complement the hemagglutination assay are investigated. The presented method is comparatively robust regarding different buffer systems, ionic strength and additive concentrations. Addition of 200mM arginine or sodium chloride is necessary to obtain complete virus particle recovery. 0.5 and 1.0M arginine increase the hydrodynamic radius of the whole virus particles by 5nm. Sodium citrate induces virus particle aggregation. Results are confirmed by dynamic light scattering. Retention of a H1N1v strain correlates with DNA contents between 5ng/mL and 670ng/mL. Quantitative elution of the virus preparations is verified on basis of hemagglutination activity. Elution of hemagglutination inducing compounds starts at a flow channel diameter of 7000nm. The universal applicability is demonstrated with three different influenza virus samples, including an industrially produced, pandemic vaccine strain. Size distribution of the pandemic H1N1v 5258, H1N1 PR/8/34, and H3N2 Aichi/2/68 preparations spreads across inter- and intra-particle volume and extends to the secondary interaction dominated range. Thus, virus particle debris seems to induce hemagglutination. Fragments generated by 0.5% Triton™ X-100 treatment increase overall hemagglutination activity.

Keywords: Aggregation; Analytical size exclusion chromatography; Fragmentation; Hemagglutination assay; Pandemic H1N1; Process analytical techniques.

MeSH terms

  • Animals
  • Arginine / chemistry
  • Chromatography, Gel*
  • DNA / analysis
  • DNA / chemistry
  • Dogs
  • Dynamic Light Scattering
  • Hemagglutination Tests
  • Influenza A Virus, H1N1 Subtype / physiology*
  • Influenza A Virus, H3N2 Subtype / physiology*
  • Influenza Vaccines / immunology
  • Influenza Vaccines / isolation & purification*
  • Madin Darby Canine Kidney Cells
  • Octoxynol / chemistry
  • Sodium Chloride / chemistry
  • Spectrometry, Fluorescence
  • Virion / isolation & purification*


  • Influenza Vaccines
  • Sodium Chloride
  • Octoxynol
  • DNA
  • Arginine