Destruction of assembly structures has been identified as a major cause for activity loss of virus and virus-like particles during their chromatographic process. A deep insight into the denaturation process at the solid-liquid interfaces is important for rational design of purification. In this study, in-situ differential scanning calorimetry (DSC) was employed to study the dissociation process of inactivated foot-and-mouth disease virus (FMDV) during ion exchange chromatography (IEC) at different levels of pH. The intact FMDV known as 146S and the dissociation products were quantified by high performance size exclusion chromatography (HPSEC) and the thermo-stability of 146S on-column was monitored in-situ by DSC. Serious dissociation was found at pH 7.0 and pH 8.0, leading to low 146S recoveries of 12.3% and 43.7%, respectively. The elution profiles from IEC and HPSEC combined with the thermal transition temperatures of 146S dissociation (Tm1) from DSC suggested two denaturation mechanisms that the 146S dissociation occurred on-column after adsorption at pH 7.0 and during elution step at pH 8.0. By appending different excipients including sucrose, the improvement of 146S recovery and reduced dissociation was found highly correlated to increment of 146S stability on-column detected by DSC. The highest recovery of 99.9% and the highest Tm1 of 54.49 °C were obtained at pH 9.0 with 20% (w/v) sucrose. According to chromatographic behaviors and Tm1, three different dissociation processes in IEC were discussed. The study provides a perspective to understand the denaturation process of assemblies during chromatography, and also supplies a strategy to improve assembly recovery.

中文翻译：

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离子交换色谱介质固液表面上生物大分子变性的机理-以灭活的口蹄疫病毒为例。

组装结构的破坏被认为是病毒和病毒样颗粒在色谱过程中活性丧失的主要原因。深入了解固液界面处的变性过程对于合理设计纯化至关重要。在这项研究中，原位差示扫描量热法（DSC）用于研究灭活的口蹄疫病毒（FMDV）在离子交换色谱（IEC）的不同pH值下的解离过程。通过高效尺寸排阻色谱法（HPSEC）对完整的FMDV（称为146S）和解离产物进行定量，并通过DSC现场监测146S在柱上的热稳定性。在pH 7.0和pH 8.0时会发生严重的离解，分别导致146S回收率低，分别为12.3％和43.7％。来自IEC和HPSEC的洗脱曲线与DSC的146S解离（Tm1）的热转变温度相结合，提出了两种变性机理，即146S离解发生在pH 7.0吸附后和pH 8.0洗脱步骤的柱上。通过添加包括蔗糖在内的不同赋形剂，发现146S回收率的提高和解离的减少与DSC检测到的146S柱上稳定性的增加高度相关。在pH 9.0时，蔗糖浓度为20％（w / v），回收率最高，为99.9％，Tm1最高，为54.49°C。根据色谱行为和Tm1，讨论了IEC中三种不同的解离过程。该研究为了解色谱过程中装配体的变性过程提供了一个视角，并提供了改善装配体回收率的策略。