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Development of dual functions negative chromatography adsorbent for the purification of hepatitis B core antigen virus-like particle from Escherichia coli homogenate
thesisposted on 15.05.2017, 04:53 by Lee, Micky Fu Xiang
Advancement in genetic engineering has allowed the expression of virus-like particle (VLP) in various recombinant hosts that can be used as vaccine to replace the attenuated or inactivated virus. However, the large particle size of VLP has rendered its adsorption onto the outer surface of adsorbent, hence reduce the recovery of VLP using bind and elute mode chromatography. Furthermore, the harsh condition during binding and elution processes could also damage the structure of VLP. Whereas in negative chromatography, small size impurities are retained by the adsorbent and the large size VLPs are allowed to flow through the column. As a result, the limitations in VLP purification using bind and elute mode chromatography can be avoided in negative chromatography. This work demonstrates the development of poly[oligo(ethylene glycol) methacrylate](POEGMA) grafted anion exchange adsorbent for the negative chromatography of hepatitis B VLP(HB-VLP). Firstly, the size selective adsorption of the adsorbent was performed on a feedstock consisted of 3 different sizes of protein. The large size HB-VLP was mostly (88%) excluded from the adsorbent along with 79% of medium size BSA, while the small size insulin was retained in the column. Further increased in temperature led to the collapse of the POEGMA chain, thus enhanced the adsorption of medium size BSA, while retaining the HB-VLP exclusion. POEGMA grafted anion exchange adsorbent was used for negative chromatography of HB-VLP from E. coli clarified lysate. The anion exchange adsorbents were grafted with POEGMA of different chain lengths; namely SQ (shorter chain length) and LQ (longer chain length). A shorter chain length of POEGMA (SQ) allowed a better penetration of host cell proteins (HCPs) through the POEGMA layer. Furthermore, the exclusion of HB-VLP from the surface of SQ allowed more HCPs to be adsorbed which resulted in 86% removal of impurities. The single step negative chromatography of HB-VLP has shown better performance compared to previous studies of anion exchange chromatography of HB-VLP. The grafted POEGMA conformation changes with temperature. The increase in temperature increased the purity and recovery of HB-VLP in flowthrough. The collapsed of POEGMA chains at higher temperature enhanced the HCPs adsorption while retaining its HB-VLP exclusion. This work demonstrates for the first time the role of thermo-responsive polymer in the preparative negative chromatography of HB-VLP. SQ was further benchmarked against inert layer coated anion exchange adsorbents (InertShell and InertLayer 1000), two negative chromatography adsorbent prototypes. The HB-VLP purity obtained under similar operation condition for SQ (62%) was much higher than the inert layer coated adsorbents. Preheat-treatment was found to deteriorate the performance of negative chromatography. Using a lower feed concentration has no significant effect over SQ. Therefore, SQ is more capable in handling higher feed concentration compared to InertLayer 1000. In general, the POEGMA grafted adsorbent is capable to remove substantial amount of impurities (HCPs) and suitable for primary step. Extra purification step is required to remove the remained HCPs and the residual DNA which may co-flowthrough with HB-VLP. Further work is recommended to enhance the shielding effect of POEGMA on HB-VLP while allowing more HCPs for adsorption.