Vaccine 29 (2011) 7182–7187 Contents lists available at ScienceDirect Vaccine j ourna l ho me pag e: www.elsevier.com/locate/vaccine Foot and mouth disease (FMD) virus: Quantification of whole virus particles during the vaccine manufacturing process by size exclusion chromatography Marcelo A. Spitteler a , Ignacio Fernández a , Erika Schabes a , Alejandro Krimer b , Emmanuel G. Régulier a , Mariela Guinzburg a , Eliana Smitsaart a , M. Susana Levy a,∗ a BIOGENESIS-BAGO S.A. Ruta Panamericana km 38,5 (B1619IEA) Garin, Buenos Aires, Argentina b Instituto Nacional de Tecnología Industrial (INTI), Av. General Paz 5445 (B1650KNA) San Martín, Buenos Aires, Argentina a r t i c l e i n f o Article history: Available online 7 June 2011 Keywords: Size exclusion chromatography (SEC) Foot and mouth disease (FMD) vaccine FMDV 140S (146S) a b s t r a c t Foot and mouth disease (FMD) is a highly infectious viral disease that affects cattle, sheep, goats and swine causing severe economic losses worldwide. The efficacy of inactivated vaccines is critically depen- dent on the integrity of foot and mouth disease virus (FMDV) particles. The recommended method to quantify the active ingredient of vaccines is the 140S quantitative sucrose density gradient analysis. This method has been an immensely valuable tool over the past three decades but it is highly operator depen- dent and difficult to automate. We developed a method to quantify FMDV particles during the vaccine manufacturing process that is based on separation of components by size-exclusion chromatography and measurement of virus by absorption at 254 nm. The method is linear in the 5–70 g/mL range, it is applicable to different FMDV strains, and has a good correlation with the 140S test. The proposed method uses standard chromatographic media and it is amenable to automation. The method has potential as a process analytical technology and for control of final product by manufacturers, international vaccine banks and regulatory agencies. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Foot-and-mouth disease (FMD) is an acute systemic viral infec- tion that affects food producing animals, such as cattle, sheep, goats and swine. Despite its very low mortality rate, the highly conta- gious nature of FMD, associated productivity losses and economic impact make it one of the most serious diseases of the livestock industry. The disease is endemic in many parts of the world. OIE peri- odically publishes disease distribution and outbreak maps; the FMD sanitary status has a profound economic impact in coun- tries with meat trade depending economies [1]. Effective vaccines and stringent control programs have eradicated the disease in most developed countries but, regardless of strict international trade policies, major outbreaks have occurred relatively recently in Europe (2000, 2001) and in Japan (2000, 2010). Despite of continu- ous efforts to develop recombinant subunit vaccines that would not require the propagation of the pathogen in large scale, only whole- virus inactivated vaccines manufactured in BSL-3 facilities are as yet available in the market [2]. It is estimated that over a billion ∗ Corresponding author. Tel.: +54 3327 448354; fax: +54 3327 448347. E-mail address: Susana.levy@biogenesisbago.com (M.S. Levy). vaccine doses are manufactured annually worldwide. In addition, frozen antigens are stored in national and international antigen banks. The efficacy of inactivated virus vaccines – which are routinely used as part of eradication programs and in emergency contexts – is highly dependent on virus integrity [3,4]. FMDV a non-enveloped virus with icosahedral symmetry and approximately 30 nm diam- eter is extremely labile in vitro, whole particles dissociate into monomers at temperatures above 56 ◦ C and pH below 6 [5]. The 140S quantitative sucrose density gradient analysis is the recom- mended method [6,7] to quantify virus antigen and, on that basis, formulate vaccines. The 140S method, as developed by Barteling and Meloen [8], has provided over the past three decades a reli- able method for virus concentration measurement. The method consists of ultracentrifugation of the sample in a sucrose concen- tration gradient from about 20 to 45%. The sucrose gradients are prepared layering sucrose solutions of decreasing concentration either by hand or using simple gradient mixers. More recently, lab- oratories have resorted to robotized gradient formers that quickly produce smooth slopes with better reproducibility. A number of international efforts have been attempted in order to standard- ize the method but there is as yet neither a harmonized protocol nor an international FMDV standard. The technical complexities of the method and the requirement of specialized items of equipment have probably contributed to this situation. 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.05.078