Development of a Strategy of Influenza Virus Separation Based on Pseudoaffinity Chromatography on Short Monolithic Columns

Abstract

This research is devoted to the development and optimization of fine purification processes realized on short monolithic columns (CIM disks), using influenza vaccine and viruslike synthetic particles as model objects. The pseudoaffinity mode of liquid chromatography has been used as a tool for dynamic adsorption experiments. Viruslike particles, close to the dimensions of influenza viruses, were developed by means of main antigen of influenza viruses (hemeagglutinin) covalent binding to the outer aminated surface of synthetic latex particles. The natural receptor analogues of sialic acid were used as affinity ligands immobilized on the surface of the CIM disk by different ways to achieve a high adsorption capacity. Also, some other ligands were tested as possible candidates for virus capturing. The affinity binding parameters for influenza A virus were obtained by frontal elution method at optimized chromatographic conditions and immobilization schemes. The experimental data pointed out the possibility of selective isolation of hemeagglutinin from a mixture of vaccine proteins. The results obtained by fast affinity chromatography have shown functional and sterical correspondence viruslike synthetic models to influenza viruses. Additionally, the optimization of chromatographic conditions allowed isolation of influenza virus A while maintaining its virulence. The maximum value of adsorption capacity was registered for a monolithic disk, modified subsequently by chitosan and 2,6-sialyllactose and found to be equal to 6.9 x 10(12) virions/mL support.