Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology , 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
J Phys Chem B. 2017 Aug 31;121(34):8085-8093. doi: 10.1021/acs.jpcb.7b05603. Epub 2017 Aug 21.
Antibodies are therapeutic proteins that are becoming indispensable for the treatment of serious diseases. Their efficacies depend on their folded structures, the loss of which, through unfolding or aggregation, should be closely monitored during their manufacture, storage, and dosing for safe usage. In downstream processes, exposure of the crude antibody solution to acid, followed by neutralization, is an established standard protocol for antibody purification and inactivation of viruses in antibody preparations. Nevertheless, this treatment also triggers an unwanted side reaction, i.e., antibody aggregation. The aggregates continuously evolve at neutral pH. It remains unclear whether the aggregates progressively incorporate the native, folded, monomers into themselves, enabling aggregate growth as seen in amyloid fibrils. In the present study, the diffusion of fluorescently labeled monoclonal humanized immunoglobulin G1 monomers in aggregates produced by pH-shift stress was tracked by fluorescence correlation spectroscopy. This method was used in addition to monitoring aggregate formation by dynamic light scattering. The diffusing velocities of the monomers indicated that the folded monomers were not involved in aggregate formation, in contrast with unfolded monomers. On the basis of the results, we propose a bifurcated pathway for the refolding and aggregation of antibodies triggered by a pH shift from acidic to neutral. In the scheme, the refolded monomers survive until their unfolding. The insights in this study will contribute to the manufacture of aggregation-resistant therapeutic antibodies.
抗体是治疗蛋白，对于严重疾病的治疗已经不可或缺。它们的疗效取决于其折叠结构，在制造、储存和给药过程中，应密切监测其折叠结构的丧失，通过展开或聚集。在下游工艺中，粗抗体溶液暴露于酸中，随后进行中和，是抗体纯化和抗体制剂中病毒灭活的标准标准方案。然而，这种处理也会引发不必要的副反应，即抗体聚集。在中性 pH 下，聚集物会不断演变。目前尚不清楚聚集物是否会逐渐将天然折叠的单体纳入自身，从而使聚集物像淀粉样纤维一样生长。在本研究中，通过荧光相关光谱法追踪 pH 转换应激产生的聚集物中荧光标记的单克隆人源化 IgG1 单体的扩散。除了通过动态光散射监测聚集物形成外，还使用了这种方法。单体的扩散速度表明折叠单体不参与聚集物的形成，而未折叠单体则参与聚集物的形成。基于这些结果，我们提出了一种由 pH 从酸性到中性的转变引发的抗体重折叠和聚集的分支途径。在该方案中，重折叠的单体在展开之前一直存活。本研究的结果将有助于制造抗聚集的治疗性抗体。
