Journal of Chromatography A, 1218 (2011) 2432–2437 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Reversed phase monolithic analytical columns for the determination of HA1 subunit of influenza virus haemagglutinin Lidija Urbas a , Boˇ stjan Koˇ sir a , Matjaˇ z Peterka a,c , Boris Pihlar b , Aleˇ s ˇ Strancar a , Miloˇ s Barut a,c,∗ a BIA Separations d.o.o., Teslova 30, SI-1000 Ljubljana, Slovenia b Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aˇ skerˇ ceva 5, SI-1000 Ljubljana, Slovenia c The Centre of Excellence for Biosensors, Instrumentation and Process Control - COBIK, Velika pot 22, SI-5250 Solkan, Slovenia article info Article history: Available online 28 December 2010 Keywords: Monolithic column Chromatography HPLC analysis Influenza Haemagglutinin abstract Monoliths are chromatographic stationary phases, which were specially designed for efficient purifi- cation of large biomolecules, like proteins, viruses and DNA. In this work, the small scale monolithic butyl (C4) and styrene-divinyl benzene (SDVB) columns were applied for reversed phase analyses of various degraded influenza viruses. The binding of the HA1 subunit of haemagglutinin to the monolithic columns was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blot. The working linear range was determined as 1.60 × 10 10 viral particles/mL to at least 1.64 × 10 11 viral particles/mL, the limit of detection was found to be 2.56 × 10 9 virus particles/mL and the limit of quantification was 5.12 × 10 9 virus particles/mL. The analytical HPLC method developed with the H1N1 virus was also applicable for the analytics of the HA1 subunit of H3N2 influenza virus and the influenza B virus. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Monoliths are continuous stationary phases, cast as homo- geneous columns of various dimensions in a single piece [1]. Monolithic chromatographic supports are known for their abil- ity to effectively purify large molecules in a short time. Structural and hydrodynamic characteristics of monoliths make them bene- ficial for purification of proteins, DNA and viruses [2,3]. Convective Interaction Media (CIM) ion-exchange monoliths have been applied for the concentration or purification of plant viruses [4–6], phages [7,8], rotaviruses [6], hepatitis A virus and feline calicivirus [9], as well as for measles and mumps virus [10]. According to the preliminary results, they have also shown to be applicable for the purification of the influenza virus [2]. Besides ion-exchange monoliths, pseudo-affinity methacrylate monoliths have been reported for the purification of the influenza A virus as well [11]. Influenza virus is an enveloped virus. Single stranded RNA is stored inside of a lipid membrane, which contains two main glycoproteins on the outside layer; haemagglutinin (HA) and neu- raminidase (NA) [12]. For the preparation of influenza vaccines various purification steps are employed. Virus titers are determined ∗ Corresponding author at: BIA Separations d.o.o. Teslova 30 SI-1000, Ljubljana, Slovenia. Tel.: +386 1 426 56 49; fax: +386 1 426 56 50. E-mail address: milos.barut@monoliths.com (M. Barut). for different samples, which are collected from various process steps (in-process control) as well as in the final vaccine (final con- trol). Methods applied are either based on the infectivity of the virus, like the tissue culture infectious dose (TCID 50 ) [13,14], or applicable for determining virus particles like the haemagglutina- tion assay [14,15] or single radial immunodiffusion (SRID) [16,17]. The latter is highly sensitive however it is labor intensive and mostly suitable for relatively pure vaccine products [18]. It also requires the production of reference antigens and specific anti- bodies, which is costly and time-consuming [19]. For this reason various chromatographic methods for determining the concentra- tion of the influenza virus and profiling of its proteins started to emerge. Phelan and Cohen managed to separate the major pro- teins of the detergent-disrupted influenza virus by RP-HPLC on conventional porous media, however, individual protein recoveries were low [20], as was the case with the ion-exchange chromatogra- phy [21]. Size exclusion chromatography showed higher potential to separate viral proteins with recoveries >90% [21]. A 2D HPLC method was reported for quantitative detection of virus proteins in vaccines, utilizing SEC and a non-porous silica RP column [22]. Non-porous C18 silica-based column gave satisfactory results in terms of sensitivity, protein recovery and sensibility as opposed to conventional porous silica particle based C4 column [23]. Kapteyn et al. developed an RP-HPLC method using a perfusion particle- based POROS column. The method is based on measuring the peak area of HA1, the hydrophilic subunit of HA, which is proportional to the amount of the analyzed HA [24,25]. 0021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2010.12.082