Abstract：
Charge heterogeneity is inherent to all therapeutic antibodies and arises from post-translational modifications (PTMs) and/or protein degradation events that may occur during manufacturing. Among therapeutic antibodies, the bispecific antibody (bsAb) containing two unique Fab arms directed against two different targets presents an additional layer of complexity to the charge profile. In the context of a bsAb, a single domain-specific PTM within one of the Fab domains may be sufficient to compromise target binding and could potentially impact the stability, safety, potency, and efficacy of the drug product. Therefore, characterization and routine monitoring of domain-specific modifications is critical to ensure the quality of therapeutic bispecific antibody products. We developed a Digestion-assisted imaged Capillary isoElectric focusing (DiCE) method to detect and quantitate domain-specific charge variants of therapeutic bispecific antibodies (bsAbs). The method involves enzymatic digestion using immunoglobulin G (IgG)-degrading enzyme of S. pyogenes (IdeS) to generate F(ab)2 and Fc fragments, followed by imaged capillary isoelectric focusing (icIEF) under reduced, denaturing conditions to separate the light chains (LCs) from the Fd domains. Our results suggest that DiCE is a highly sensitive method that is capable of quantitating domain-specific PTMs of a bsAb. In one case study, DiCE was used to quantitate unprocessed C-terminal lysine and site-specific glycation of Lys98 in the complementarity-determining region (CDR) of a bsAb that could not be accurately quantitated using conventional, platform-based charge variant analysis, such as intact icIEF. Quantitation of these PTMs by DiCE was comparable to results from peptide mapping, demonstrating that DiCE is a valuable orthogonal method for ensuring product quality. This method may also have potential applications for characterizing fusion proteins, antibody-drug conjugates, and co-formulated antibody cocktails.
摘要：
电荷异质性是所有治疗性抗体固有的,并且起因于可能在制造期间发生的翻译后修饰(PTM)和/或蛋白质降解事件。在治疗性抗体中，例如,包含针对两个不同靶标的两个独特Fab臂的双特异性抗体(bsAb)对电荷分布呈现额外的复杂性层。在bsAb的背景下，其中一个Fab域内的单结构域特异性PTM可能足以损害靶标结合，并可能潜在地影响稳定性，安全,效力，效力和药物产品的功效。因此,结构域特异性修饰的表征和常规监测对于确保治疗性双特异性抗体产品的质量至关重要.我们开发了一种消化辅助成像毛细管等电聚焦（DiCE）方法来检测和定量治疗性双特异性抗体（bsAbs）的结构域特异性电荷变体。该方法涉及使用化脓性链球菌（IdeS）的免疫球蛋白G（IgG）降解酶进行酶消化，以产生F（ab）2和Fc片段，其次是成像的毛细管等电聚焦（ICIEF）下降低，将轻链(LC)与Fd结构域分离的变性条件。我们的结果表明，DiCE是一种高度灵敏的方法，能够定量bsAb的结构域特异性PTM。在一个案例研究中，DiCE用于定量bsAb的互补决定区（CDR）中未加工的C端赖氨酸和Lys98的位点特异性糖基化，基于平台的电荷变异分析，比如完整的ICIEF。通过DiCE对这些PTM的定量与肽图谱的结果相当，证明DiCE是确保产品质量的一种有价值的正交方法。这种方法也可能具有表征融合蛋白的潜在应用。抗体-药物缀合物，和共同配制的抗体混合物。
