Denaturation of inactivated FMDV in ion exchange chromatography: Evidence by differential scanning calorimetry analysis

Abstract Removing impurities by purification is important for livestock vaccines to avoid side effects and ensure sufficient immunogenic protection on animals. In this study, three anion-exchange media with similar particle size and ligand density except pore size were applied and compared for purification of inactivated foot-and-mouth disease virus (FMDV) antigen. The dynamic binding capacity for DEAE-POROS (214 nm) and DEAE–650 M (106 nm) were 11.53 and 10.03 mg/mL, while that for DEAE-FF (32 nm) was less than 1/10 of the previous two. The recovery of inactivated FMDV after chromatographic process of these three media was 68.42%, 66.32% and 54.46%, respectively, showing a decrement as the decrease of pore size of media. Possible denaturation of the FMDV on the surfaces of anion exchange media was analyzed by differential scanning calorimetry (DSC). FMDV is known to be prone to dissociate into smaller subunits 12S. In solution, this process is reflected by T m 1 of DSC, which is at about 48.52 °C. When inactivated FMDV were absorbed on DEAE-FF, the T m 1 became 41.73 °C, indicating the increased possibility of dissociation. The T m 1 for DEAE–650 M and DEAE-POROS was 44.04 °C and 45.37 °C, showing less dissociation and improved stability than on DEAE-FF. After DEAE-POROS chromatography, 94% FMDV recovery with 7.7-fold purification was achieved, further polishing by size exclusion chromatography led totally 173-fold increase in purity with average overall recovery of 79%.