Analytical Chemistry Group, Regeneron Pharmaceuticals, Inc, Tarrytown, NY, USA.
MAbs. 2022 Jan-Dec;14(1):2133674. doi: 10.1080/19420862.2022.2133674.
Therapeutic monoclonal antibodies (mAbs) have a propensity to host a large number of chemical and enzymatical modifications that need to be properly assessed for their potential impact on target binding. Traditional strategies of assessing the criticality of these attributes often involve a laborious and low-throughput variant enrichment step prior to binding affinity measurement. Here, we developed a novel competitive binding-based enrichment strategy followed by mass spectrometry analysis (namely, competitive binding-MS) to achieve high-throughput evaluation of potential critical quality attributes in therapeutic mAbs. Leveraging the differences in target binding capability under competitive binding conditions, the criticality of multiple mAb attributes can be simultaneously evaluated by quantitative mass spectrometry analysis. The utility of this new workflow was demonstrated in three mAb case studies, where different post-translational modifications occurring within the complementarity-determining regions were successfully interrogated for their impact on antigen binding. As this workflow does not require prior enrichment (e.g., by forced degradation or liquid chromatography fractionation) of the variants, it is particularly valuable during the mAb candidate developability assessment, where fast turn-around time is highly desired to assist candidate selection.: ACN: acetonitrile; ADCC: antibody-dependent cell-mediated cytotoxicity; AEX: anion exchange chromatography; bsAb: bispecific antibody; CDC: complement-dependent cytotoxicity; CDR: complementarity-determining region; CML: carboxymethylation; CQA: critical quality attribute; DDA: data-dependent acquisition; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; FA: formic acid; Fab: Fragment antigen-binding; FcRn: neonatal Fc receptor; HC: heavy chain; HIC: hydrophobic interaction chromatography; IAA: iodoacetamide; IEX: ion exchange chromatography; LC: light chain; mAb monoclonal antibody; msAb: monospecific antibody; MS: mass spectrometry; PBS: phosphate-buffered saline; pI: isoelectric point; PTM: post-translational modification; SCX: strong cation exchange chromatography; SEC: size exclusion chromatography; SPR: surface plasmon resonance; XIC: extracted ion chromatography.
治疗性单克隆抗体（mAbs）容易发生大量的化学和酶学修饰，需要对其对靶标结合的潜在影响进行适当评估。传统的评估这些属性的关键性的策略通常涉及在结合亲和力测量之前进行繁琐的、低通量的变异体富集步骤。在这里，我们开发了一种新的基于竞争结合的富集策略，随后进行质谱分析（即竞争结合-MS），以实现治疗性 mAbs 中潜在关键质量属性的高通量评估。利用竞争结合条件下靶标结合能力的差异，可以通过定量质谱分析同时评估多个 mAb 属性的关键性。该新工作流程的实用性在三个 mAb 案例研究中得到了证明，其中在互补决定区中发生的不同翻译后修饰被成功地用于研究其对抗原结合的影响。由于该工作流程不需要对变体进行预先富集（例如通过强制降解或液相色谱分级分离），因此在 mAb 候选物可开发性评估中特别有价值，在该评估中，快速周转时间是高度期望的，以帮助候选物选择。: ACN：乙腈；ADCC：抗体依赖性细胞介导的细胞毒性；AEX：阴离子交换色谱；bsAb：双特异性抗体；CDC：补体依赖性细胞毒性；CDR：互补决定区；CML：羧甲基化；CQA：关键质量属性；DDA：数据依赖采集；DMSO：二甲基亚砜；DTT：二硫苏糖醇；FA：甲酸；Fab：抗原结合片段；FcRn：新生儿 Fc 受体；HC：重链；HIC：疏水相互作用色谱；IAA：碘乙酰胺；IEX：离子交换色谱；LC：轻链；mAb 单克隆抗体；msAb：单特异性抗体；MS：质谱；PBS：磷酸盐缓冲盐水；pI：等电点；PTM：翻译后修饰；SCX：强阳离子交换色谱；SEC：尺寸排阻色谱；SPR：表面等离子体共振；XIC：提取离子色谱。
