Development of a Strategy of Influenza Virus Separation Based on Pseudoaffinity Chromatography on Short Monolithic Columns I. Kalashnikova, N. Ivanova, and T. Tennikova* Institute of Macromolecular Compounds, Russian Academy of Sciences, St. Petersburg, Russia This research is devoted to the development and optimi- zation 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 candi- dates 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 chro- matographic conditions allowed isolation of influenza virus A while maintaining its virulence. The maximum value of adsorption capacity was registered for a mono- lithic disk, modified subsequently by chitosan and 2,6- sialyllactose and found to be equal to 6.9 × 10 12 virions/ mL support. Influenza remains, due to its annual death rate and potential to cause pandemics, a major public health concern. The efforts to control the annual spread of influenza have focused on prophylactic vaccinations. Human influenza vaccines are tradition- ally produced in embryonated hen’s eggs and specific cell cultures. The latter crude material containing virus requires thorough purification from cell components (the components of nonviral origin) without lost of biological activity of antigenic determinates during vaccine manufacturing. Different modes of liquid chromatography, namely, ion ex- change, size exclusion, and others, as well as their combination, are widely used at influenza virus purification. 1-3 The most delicate and highly selective virus isolation can be realized using affinity chromatography. Recently obtained data illustrated that monolithic material on the base of a copolymer of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) seemed to be suitable for construction of biorecognizing systems in two formats, 4,5 both for isolation of viruslike particles by fast affinity chromatography and for their sensitive detection (diagnostics) using miccroarray technique. Besides that, the chromatography on short methacrylate-based monolithic columns (Convective Interaction Media, CIM, disks), demonstrating unique hydrody- namic and separation properties, 6-8 seems to be preferable to overcome many critical disadvantages of conventional separation techniques concerning large bioobjects’ separation. 9-14 Because of superior mass transfer and open porous structure (totally permeable for a flowing liquid large pores), the monoliths will be able to provide very fast biospecific pair formation involving viruses that reduces the risk of product degradation. It is known that the distinct terminal sialic acid species (N- acetylneuraminic acid) on the cell membrane surface serves as a hemeagglutinin-binding receptor to induce the penetration of the interior of influenza viruses by membrane fusion. 15,16 In this * To whom correspondence should be addressed. E-mail: tennikova@mail.ru. (1) Kalbfuss, B.; Wolff, M.; Morenweiser, R.; Reichl, U. Biotechnol. Bioeng. 2007, 96, 932-944. (2) Opitz, L.; Salaklang, J.; Bu ¨ttner, H.; Reichl, U.; Wolff, M. Vaccine 2007, 25, 939-947. (3) Zhao, R.; Fang, C.; Yu, X.; Liu, Y.; Luo, J.; Shangguan, D.; Xiong, S.; Su, T.; Liu, G. J. Chromatogr., A 2005, 1064, 59-66. (4) Kalashnikova, I.; Ivanova, N.; Evseeva, T.; Menshikova, A.; Vlakh, E.; Tennikova, T. J. Chromatogr. A 2007, 1144, 40-47. (5) Kalashnikova, I.; Ivanova, N.; Tennikova, T. Anal. Chem. 2007, 9, 5173- 5181. (6) Josic, Dj.; Buchacher, A. J. Biochem. Biophys. Methods 2001, 49, 153-174. 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Chem. 2008, 80, 2188-2198 2188 Analytical Chemistry, Vol. 80, No. 6, March 15, 2008 10.1021/ac702258t CCC: $40.75 © 2008 American Chemical Society Published on Web 02/14/2008