Zhang Jun, Woods Christopher, He Feng, Han Mei, Treuheit Michael J, Volkin David B
Process Development , Amgen Inc. , Thousand Oaks , California 91320 , United States.
Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center , University of Kansas , Lawrence , Kansas 66049 , United States.
Biochemistry. 2018 Sep 18;57(37):5466-5479. doi: 10.1021/acs.biochem.8b00575. Epub 2018 Sep 5.
Protein therapeutics, monoclonal antibodies (mAbs) in particular, are large, structurally complex molecules that are prone to numerous modes of degradation during their production and long-term storage. Physical degradation via protein aggregation is a major concern when developing protein therapeutic candidates for clinical use. A dimer is perhaps the simplest element of protein aggregation, and thus, a better understanding of protein dimers in terms of their structures, intermolecular interactions, and chemical nature will help in the development of rational strategies for reducing aggregation propensity. In this study, two different mAb dimers were generated from an IgG2 monoclonal antibody solution, i.e., a native dimer generated under long-term storage and a thermal dimer from a thermal stress condition. Both IgG2 dimers were characterized in terms of their chemical and physical properties, bioactivity, and conformational dynamics. The native IgG2 dimer was formed mainly through noncovalent association. It displayed minimal differences in biophysical properties and higher-order structure compared to the monomer yet showed compromised in vitro potency, likely because of steric hindrance. In contrast, the thermal IgG2 dimer was mainly disulfide-linked, but even so, no new non-native disulfide bonds were detected by peptide mapping. Two regions within the Fc-C2 domain of the thermal IgG2 dimer exhibited significantly increased flexibility as measured by hydrogen-deuterium exchange mass spectrometry, and notably, these regions are connected by an intrachain disulfide bond under natively folded conditions. These findings provide a better understanding of dimer formation under long-term storage and thermal stress conditions for this IgG2 mAb, and possible aggregation mechanisms are discussed.
蛋白质疗法，尤其是单克隆抗体（mAbs），是大型的、结构复杂的分子，在其生产和长期储存过程中容易出现多种降解方式。在开发用于临床的蛋白质治疗候选药物时，通过蛋白质聚集进行的物理降解是一个主要问题。二聚体可能是蛋白质聚集中最简单的元素，因此，从其结构、分子间相互作用和化学性质方面更好地理解蛋白质二聚体，将有助于制定合理的策略来降低聚集倾向。在本研究中，从一种IgG2单克隆抗体溶液中产生了两种不同的mAb二聚体，即长期储存条件下产生的天然二聚体和热应激条件下产生的热二聚体。对这两种IgG2二聚体的化学和物理性质、生物活性以及构象动力学进行了表征。天然IgG2二聚体主要通过非共价缔合形成。与单体相比，它在生物物理性质和高级结构上显示出最小的差异，但体外效力受损，可能是由于空间位阻。相比之下，热IgG2二聚体主要是通过二硫键连接，但即便如此，通过肽图分析未检测到新的非天然二硫键。通过氢-氘交换质谱法测定，热IgG2二聚体的Fc-C2结构域内的两个区域表现出显著增加的灵活性，值得注意的是，在天然折叠条件下，这些区域通过链内二硫键相连。这些发现有助于更好地理解这种IgG2 mAb在长期储存和热应激条件下的二聚体形成，并讨论了可能的聚集机制。
