Attribute Sciences, Process Development, Amgen Inc., Thousand Oaks, California 91320, United States.
Discovery Protein Science, Amgen Research, Amgen Inc., Burnaby, BC V5A1 V7, Canada.
Anal Chem. 2023 Oct 24;95(42):15477-15485. doi: 10.1021/acs.analchem.2c05554. Epub 2023 Oct 9.
The binding affinity of monoclonal antibodies (mAbs) for their intended therapeutic targets is often affected by chemical and post-translational modifications in the antigen binding (Fab) domains. A new two-dimensional analytical approach is described here utilizing native size exclusion chromatography (SEC) to separate populations of antibodies and bound antibody-antigen complexes for subsequent characterization of these modifications by reversed-phase (RP) liquid chromatography-mass spectrometry (LC-MS) at the intact antibody level. Previously, we utilized peptide mapping to measure modifications impacting binding. However, in this study, the large size of the modification (N-glycosylation) allowed assessing its impact from small amounts (∼20 ug) of intact antibody, without the need for peptide mapping. Here, we apply the native SEC-based competitive binding assay to quickly and qualitatively investigate the effects of Fab glycosylation of four antispike protein mAbs that were developed for use in the treatment of COVID-19 disease. Three of the mAbs were observed to have consensus N-glycosylation sites (N-X-T/S) in the Fab domains, a relatively rare occurrence in therapeutic mAbs. The goal of the study was to characterize the levels of Fab glycosylation present, as well as determine the impact of glycosylation on binding to the spike protein receptor binding domain (RBD) and the ability of the mAbs to inhibit RBD-ACE2 interaction at the intact antibody level, with minimal sample treatment and preparation. The three mAbs with Fab N-glycans were found to have glycosylation profiles ranging from full occupancy at each Fab (in one mAb) to partially glycosylated with mixed populations of two, one, or no glycan moieties. Competitive SEC analysis of mAb-RBD revealed that the glycosylated antibody populations outcompete their nonglycosylated counterparts for the available RBD molecules. This competitive SEC binding analysis was applied to investigate the three-body interaction of a glycosylated mAb blocking the interaction between endogenous binding partners RBD-ACE2, finding that both glycosylated and nonglycosylated mAb populations bound to RBD with high enough affinity to block RBD-ACE2 binding.
单克隆抗体（mAbs）与它们预期的治疗靶标的结合亲和力通常受到抗原结合（Fab）结构域中化学和翻译后修饰的影响。这里描述了一种新的二维分析方法，利用天然尺寸排阻色谱（SEC）分离抗体群体和结合的抗体-抗原复合物，然后通过反相（RP）液相色谱-质谱（LC-MS）在完整抗体水平上对这些修饰进行后续表征。以前，我们利用肽图来测量影响结合的修饰。然而，在这项研究中，修饰的较大尺寸（糖基化）允许从少量（约 20ug）完整抗体中评估其影响，而无需肽图。在这里，我们应用基于天然 SEC 的竞争性结合测定法，快速定性地研究了四种抗刺突蛋白 mAbs 的 Fab 糖基化对 COVID-19 疾病治疗的影响。这四种 mAb 中的三种在 Fab 结构域中具有共识的 N-糖基化位点（N-X-T/S），这在治疗性 mAb 中相对罕见。研究的目的是表征存在的 Fab 糖基化水平，以及确定糖基化对结合刺突蛋白受体结合域（RBD）的影响，以及 mAb 在完整抗体水平上抑制 RBD-ACE2 相互作用的能力，同时尽量减少样品处理和准备。发现三种具有 Fab N-聚糖的 mAb 具有从每个 Fab 完全占据的糖基化谱（在一种 mAb 中）到部分糖基化与两种、一种或没有糖基部分的混合群体。mAb-RBD 的竞争性 SEC 分析表明，糖基化抗体群体与未糖基化的抗体群体竞争，争夺可用的 RBD 分子。这种竞争性 SEC 结合分析被应用于研究糖基化 mAb 阻断内源性结合伴侣 RBD-ACE2 相互作用的三体相互作用，发现糖基化和未糖基化的 mAb 群体都以足够高的亲和力与 RBD 结合，从而阻断 RBD-ACE2 结合。
