Department of Chemical Engineering and Biotechnology, University of Cambridge , Cambridge, CB2 3RA, United Kingdom.
MedImmune Ltd. , Aaron Klug Building, Granta Park, Cambridge, CB21 6GH, United Kingdom.
Anal Chem. 2017 Feb 21;89(4):2361-2368. doi: 10.1021/acs.analchem.6b04158. Epub 2017 Feb 3.
Antibodies are an important class of drugs, comprising more than half of all new FDA approvals. Therapeutic antibodies must be chemically stable both in storage and in vivo, following administration to patients. Deamidation is a major degradation pathway for all natural and therapeutic proteins circulating in blood. Here, the linkage between deamidation propensity and structural dynamics is investigated by examining two antibodies with differing specificities. While both antibodies share a canonical asparagine-glycine (NG) motif in a structural loop, this is prone to deamidation in one of the antibodies but not the other. We found that the hydrogen-exchange rate at the adjacent two amides, often the autocatalytic nucleophiles in deamidation, correlated with the rate of degradation. This previously unreported observation was confirmed upon mutation to stabilize the deamidation lability via a generally applicable orthogonal engineering strategy presented here. We anticipate that the structural insight into chemical degradation in full-length monoclonal antibodies and the high-resolution hydrogen-exchange methodology used will have broad application across biochemical study and drug discovery and development.
抗体是一类重要的药物，占所有新的 FDA 批准药物的一半以上。治疗性抗体在储存和给予患者后在体内都必须具有化学稳定性。去酰胺化是所有在血液中循环的天然和治疗性蛋白质的主要降解途径。在这里，通过检查两种具有不同特异性的抗体来研究去酰胺化倾向和结构动力学之间的联系。虽然这两种抗体在结构环中都具有典型的天冬酰胺-甘氨酸（NG）基序，但其中一种抗体中的这个基序容易发生去酰胺化，而另一种抗体则不会。我们发现，相邻两个酰胺的氢交换率（通常是去酰胺化中的自催化亲核试剂）与降解速率相关。这一以前未报道过的观察结果通过突变得到了证实，通过这里提出的一种通用的正交工程策略来稳定去酰胺化的不稳定性。我们预计，对全长单克隆抗体中化学降解的结构见解以及所使用的高分辨率氢交换方法将在生物化学研究以及药物发现和开发中具有广泛的应用。
