Effect of phase inversion and separation on hepatitis B core antigen extraction from unclarified bacterial feedstock using aqueous two-phase system

Abstract

Conventional purification methods for hepatitis B core antigen (HBcAg) such as sucrose-gradient ultracentrifugation and size-exclusion chromatography are tedious and time-consuming. Therefore, a new approach using aqueous two-phase system (ATPS) was employed in this study to purify HBcAg from unclarified Escherichiacoli feedstock. Phase inversion and separation are important factors in protein partitioning, particularly in ATPS. Therefore, the objectives of this study were to first generate phase diagrams for various MW PEGs and salts, followed by determination of phase inversion and separation effects on the partitioning of HBcAg in the selected systems. Based on the phase diagrams generated in this study, polyethylene-glycol (PEG) 6000–sodium sulphate system required the lowest concentration of phase components for biphasic formation compared to other systems. When unclarified feedstock was introduced into the systems, it was observed that most of the HBcAg were partitioned to the top PEG rich phase. Moreover, it was shown that phase inversion and separation had significant effects on partitioning of HBcAg. Vigorous inversion method using a vortex mixer was favoured over gentle inversion method using a rotary shaker used in this study due to its higher HBcAg recovery. Furthermore, centrifugation was an ideal method to use for phase separation as this method shortened the overall phase separation time with better recovery and purification factor compared to systems that were left on the bench to achieve phase separation. The percent recovery and purification factor obtained were 68% and 1.93 respectively when the systems were vortexed for 1min and centrifuged at 1000g for 3min at 4°C. When these results were compared with those obtained using sucrose gradient ultracentrifugation, the percent recovery obtained using the current method was more than 20 times higher. However, the latter purification methods produced higher purity of HBcAg of more than 90% compared to that obtained in the present study (∼71%). When the purified HBcAg was analysed with enzyme-linked immunosorbent assay (ELISA), it was observed that the antigenicity was still preserved and comparable to those purified with sucrose gradient ultracentrifugation.