Improving stability of virus-like particles by ion-exchange chromatographic supports with large pore size: advantages of gigaporous media beyond enhanced binding capacity.
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-120nm and DEAE-AP-280nm with average pore diameters of 32nm, 20nm, 120nm and 280nm, 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-280nm 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 2.0mg/mL-media loading quantity, about 85.5% VLPs were correctly self-assembled after the chromatography with DEAE-AP-280nm 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-280nm media were demonstrated the best results showing the highest recovery of 68.33% and purification fold of 3.47, at 2.98mg protein/mL-media loading quantity and a flow rate of 240cm/h.
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; PLA Key Laboratory of Biopharmaceutical Process and Formulation Engineering, Beijing 100190, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
Mengran
M
Yu
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; PLA Key Laboratory of Biopharmaceutical Process and Formulation Engineering, Beijing 100190, PR China.
Yan
Y
Li
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; PLA Key Laboratory of Biopharmaceutical Process and Formulation Engineering, Beijing 100190, PR China. Electronic address: spzhang@ipe.ac.cn.
Songping
S
Zhang
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; PLA Key Laboratory of Biopharmaceutical Process and Formulation Engineering, Beijing 100190, PR China.
Xiunan
X
Li
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; PLA Key Laboratory of Biopharmaceutical Process and Formulation Engineering, Beijing 100190, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
Yanli
Y
Yang
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; PLA Key Laboratory of Biopharmaceutical Process and Formulation Engineering, Beijing 100190, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
Yi
Y
Chen
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; PLA Key Laboratory of Biopharmaceutical Process and Formulation Engineering, Beijing 100190, PR China.
Guanghui
G
Ma
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; PLA Key Laboratory of Biopharmaceutical Process and Formulation Engineering, Beijing 100190, PR China. Electronic address: zgsu@ipe.ac.cn.
Zhiguo
Z
Su
D000327:Adsorption; D002852:Chromatography, Ion Exchange; D004058:Diffusion; D006514:Hepatitis B Surface Antigens / Q000302:isolation & purification; D010843:Pichia; D016062:Porosity; D012685:Sepharose / Q000737:chemistry; D058425:Vaccines, Virus-Like Particle / Q000302:isolation & purification; D014771:Virion / Q000302:isolation & purification*
D006514:Hepatitis B Surface Antigens; D058425:Vaccines, Virus-Like Particle; D012685:Sepharose
Binding capacity; Gigaporous media; Ion-exchange chromatography; Pore size effect; Stability; Virus-like particles (VLPs)