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Yu M.,CAS Institute of Process Engineering | Yu M.,PLA Key Laboratory of Biopharmaceutical Process | Yu M.,University of Chinese Academy of Sciences | Li Y.,CAS Institute of Process Engineering | And 15 more authors.
Journal of Chromatography A | Year: 2014

Limited binding capacity and low recovery of large size multi-subunits virus-like particles (VLPs) in conventional agarose-gel based chromatographic supports with small pores have long been a bottleneck limiting the large scale purification and application of VLPs. In this study, four anion exchange media including DEAE-Sepharose FF (DEAE-FF), DEAE-Capto, gigaporous DEAE-AP-120. nm and DEAE-AP-280. nm with average pore diameters of 32. nm, 20. nm, 120. nm and 280. nm, respectively, were applied for purification of hepatitis B virus surface antigen (HBsAg) VLPs. Pore size effects of media on the VLPs adsorption equilibrium, adsorption kinetics, dynamic binding capacity (DBC), and recovery were investigated in detail. According to the confocal laser scanning microscopy observation, adsorption of the VLPs in DEAE-FF and DEAE-Capto was mostly confined to a thin shell on the outer surface of the beads, leaving the underlying pore space and the binding sites inaccessibly, while the large pores in gigaporous media enabled the VLPs to access to the interior pore spaces by diffusion transport efficiently. Compared to the most widely used DEAE-FF, gigaporous media DEAE-AP-280. nm gained about 12.9 times increase in static adsorption capacity, 8.0 times increase in DBC, and 11.4 times increase in effective pore diffusivity. Beyond increasing the binding capacity and enhancing the mass transfer, the gigaporous structure also significantly improved the stability of the VLPs during intensive adsorption-desorption process by lowing the multi-point interaction between the VLPs and binding sites in the pores. At 2.0. mg/mL-media loading quantity, about 85.5% VLPs were correctly self-assembled after the chromatography with DEAE-AP-280. nm media; oppositely about 85.2% VLPs lost their normal assembly with DEAE-FF due to irreversible disassembly. Comparative investigation was made to study the purifying performance of these four chromatographic media for actual VLPs purification from recombinant Hansenula polymorpha. DEAE-AP-280. nm media were demonstrated the best results showing the highest recovery of 68.33% and purification fold of 3.47, at 2.98. mg protein/mL-media loading quantity and a flow rate of 240. cm/h. © 2014 Elsevier B.V.

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