Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow field-flow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (Rh) measured by online DLS with the Rh values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no self-associations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The Rg/Rh ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot.

C2P s r o The Campus Science Park Palachovo náměstí 726 2 625 00 Brno Czech Republic

Department of Immunology Faculty of Medicine and Dentistry Palacky University Olomouc Hnevotínska 3 775 15 Olomouc Czech Republic

Inst Clin Immunol and Allergol Charles University Prague Univ Hosp Hradec Kralove Sokolská 581 50005 Hradec Kralove Czech Republic

Institute of Physics AS Na Slovance 1999 2 182 00 Prague 8 Czech Republic

Malvern Panalytical Ltd Enigma Business Park Grovewood Road Great Malvern WR14 1XZ UK

Veterinary Research Institute Hudcova 70 621 00 Brno Czech Republic

Wyatt Technology Europe GmbH Hochstraße 12a 56307 Dernbach Germany

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