Quantification and purification of 140s aphtovirus viral particles by liquid chromatography

SEKI, CRISTINA; DELGADO, MARIA; NAVARRO, A; ROBIOLO, BLANCA; PERIOLO, OSVALDO; RUSPI, DANIEL; PEREZ FILGUEIRA, DANIEL; MATTION, NORA; LA TORRE, JOSE

Jerez de la Frontera

Simposio; Session of the Standing Technical Committee of the European Commission for the Control of FMD. EuFMD Open Session 201; 2012

European Commission for the Control of FMD

QUANTIFICATION AND PURIFICATION OF 140S APHTOVIRUS VIRAL PARTICLES BY LIQUID CHROMATOGRAPHY C. Seki 1., M. Delgado 1, A. Navarro 1, B. Robiolo 1, O. Periolo 1, D. Ruspi 1, M. Perez Filgueira2, N. Mattion 1, J. La Torre 1 1 Centro de Virología Animal, ICT Milstein, CONICET, Saladillo 2468, Buenos Aires, Argentina 2 Instituto Nacional de Tecnología Agropecuaria, CICVyA, N Repetto y De Los Reseros s/n, Hurlingham (1686), Buenos Aires, Argentina. Introduction: Whole particle (140S) antigen payload in FMD vaccines is usually determined by ultracentrifugation of the viral suspensions on sucrose gradients (Vazquez et al., 1979). However, this method is time consuming, requires the use of expensive equipment and it is difficult to standardize. Therefore, the main objective of this work was to develop an alternative method based on liquid chromatography (LC), combined with the precise identification of each virus strain present in polyvalent vaccines by means of strain specific monoclonal antibodies (MAbs). Materials and methods Viral particles were quantified by LC on Sephacryl S® 400 columns. The methodology was standardized using heat-treated or unheated concentrated samples from inactivated FMDV A24/Cruzeiro suspensions. This virus strain generates a large amount of natural empty capsids (75S) which have been very useful, together with the 12S subunits, as size markers. Determination of antigenic mass by LC and sucrose gradients was compared using intact virus (140S) or heated samples (12S). The fractions were further analyzed using ELISA with strain specific MAbs. Results: The comparison between the antigenic payload calculated by LC and sucrose gradients gave a good correlation (r>0.9) for A24 strain. Similar results were obtained with the vaccine strains O1 Campos, A/Arg/2001 and C3 Indaial. Quantification of viral particles by LC allowed the detection of antigenic payloads as low as 5µg/ml. However, the use of ELISA with MAbs allowed the detection of lower amounts of 140S particles (5 ng/ml). Discussion: The results showed that LC is a suitable tool for quantification of FMDV 140 S particles during the vaccine production process as well as in the final product. We are also exploring the use of this method for industrial production of intact FMDV antigen free of undesirable contaminants, such as DNA and non-structural proteins.