Yu Mengran, Zhang Songping, Zhang Yan, Yang Yanli, Ma Guanghui, Su Zhiguo
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.
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; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, 300072, PR China.
J Chromatogr A. 2015 Apr 3;1388:195-206. doi: 10.1016/j.chroma.2015.02.048. Epub 2015 Feb 21.
Chromatographic purification of virus-like particles (VLPs) is important to the development of modern vaccines. However, disassembly of the VLPs on the solid-liquid interface during chromatography process could be a serious problem. In this study, isothermal titration calorimetric (ITC) measurements, together with chromatography experiments, were performed on the adsorption and disassembling of multi-subunits hepatitis B virus surface antigen virus-like particles (HB-VLPs). Two gigaporous ion-exchange chromatography (IEC) media, DEAE-AP-280 nm and DEAE-POROS, were used. The application of gigaporous media with high ligand density led to significantly increased irreversible disassembling of HB-VLPs and consequently low antigen activity recovery during IEC process. To elucidate the thermodynamic mechanism of the effect of ligand density on the adsorption and conformational change of VLPs, a thermodynamic model was proposed. With this model, one can obtain the intrinsic molar enthalpy changes related to the binding of VLPs and the accompanying conformational change on the liquid-solid interface during its adsorption. This model assumes that, when intact HB-VLPs interact with the IEC media, the total adsorbed proteins contain two states, the intact formation and the disassembled formation; accordingly, the apparent adsorption enthalpy, ΔappH, which can be directly measured from ITC experiments, presents the sum of three terms: (1) the intrinsic molar enthalpy change associated to the binding of intact HB-VLPs (ΔbindHintact), (2) the intrinsic molar enthalpy change associated to the binding of HB-VLPs disassembled formation (ΔbindHdis), and (3) the enthalpy change accompanying the disassembling of HB-VLPs (ΔconfHdis). The intrinsic binding of intact HB-VLPs and the disassembled HB-VLPs to both kinds of gigaporous media (each of which has three different ligand densities), were all observed to be entropically driven as indicated by positive values of ΔbindHintact and ΔbindHdis; while the nagative ΔconfHdis values suggested a spontenous enthalpy-driven process for the forming of HB-VLPs disassembled formation at all conditions studied. As ligand density increases, ΔconfHdis became more negative, which was in agreement with the findings from chromatography experiments, that higher ligand density leads to more serious disassembling of HB-VLPs. Results from thermodynamic studies provided us insight understanding on the mechanism of adsorption and conformational change of VLPs, as well as the effect of ligand densities on the structural stability of VLPs during IEC process.
病毒样颗粒（VLPs）的色谱纯化对现代疫苗的开发至关重要。然而，在色谱过程中，VLPs在固液界面的解离可能是一个严重问题。在本研究中，对多亚基乙型肝炎病毒表面抗原病毒样颗粒（HB-VLPs）的吸附和解离进行了等温滴定量热法（ITC）测量以及色谱实验。使用了两种大孔离子交换色谱（IEC）介质，DEAE-AP-280 nm和DEAE-POROS。高配体密度大孔介质的应用导致HB-VLPs的不可逆解离显著增加，从而在IEC过程中抗原活性回收率较低。为了阐明配体密度对VLPs吸附和构象变化影响的热力学机制，提出了一个热力学模型。通过该模型，可以获得与VLPs结合以及其在吸附过程中在液固界面伴随的构象变化相关的本征摩尔焓变。该模型假设，当完整的HB-VLPs与IEC介质相互作用时，总吸附蛋白包含两种状态，完整形式和解离形式；因此，可从ITC实验直接测量的表观吸附焓ΔappH呈现为三个项的总和：（1）与完整HB-VLPs结合相关的本征摩尔焓变（ΔbindHintact），（2）与HB-VLPs解离形式结合相关的本征摩尔焓变（ΔbindHdis），以及（3）HB-VLPs解离伴随的焓变（ΔconfHdis）。完整HB-VLPs和解离的HB-VLPs与两种大孔介质（每种介质具有三种不同的配体密度）的本征结合，均观察到由ΔbindHintact和ΔbindHdis的正值表明是熵驱动的；而负的ΔconfHdis值表明在所有研究条件下，形成HB-VLPs解离形式是一个自发的焓驱动过程。随着配体密度增加，ΔconfHdis变得更负，这与色谱实验结果一致，即更高的配体密度导致HB-VLPs更严重的解离。热力学研究结果为我们深入理解VLPs的吸附和构象变化机制，以及IEC过程中配体密度对VLPs结构稳定性的影响提供了帮助。
