Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, The Netherlands.
Netherlands Proteomics Center, Utrecht, The Netherlands.
MAbs. 2023 Jan-Dec;15(1):2175312. doi: 10.1080/19420862.2023.2175312.
The development of increasingly complex antibody formats, such as bispecifics, can lead to the formation of increasingly complex high- and low-molecular-weight by-products. Here, we focus on the characterization of high molecular weight species (HMWs) representing the highest complexity of size variants. Standard methods used for product release, such as size exclusion chromatography (SEC), can separate HMW by-products from the main product, but cannot distinguish smaller changes in mass. Here, for the identification of the diverse and complex HMW variants of a trivalent bispecific CrossMAb antibody, offline fractionation, as well as production of HMW by-products combined with comprehensive analytical testing, was applied. Furthermore, HMW variants were analyzed regarding their chemical binding nature and tested in functional assays regarding changes in potency of the variants. Changes in potency were explained by detailed characterization using mass photometry, SDS-PAGE analysis, native mass spectrometry (MS) coupled to SEC and bottom-up proteomics. We identified a major portion of the HMW by-products to be non-covalently linked, leading to dissociation and changes in activity. We also identified and localized high heterogeneity of a by-product of concern and applied a CD3 affinity column coupled to native MS to annotate unexpected by-products. We present here a multi-method approach for the characterization of complex HMW by-products. A better understanding of these by-products is beneficial to guide analytical method development and proper specification setting for therapeutic bispecific antibodies to ensure constant efficacy and patient safety of the product through the assessment of by-products.
越来越复杂的抗体形式（如双特异性抗体）的发展可能导致越来越复杂的高分子量和低分子量副产物的形成。在这里，我们专注于对代表大小变异体最高复杂度的高分子量物质（HMWs）进行特征描述。用于产品放行的标准方法，如尺寸排阻色谱法（SEC），可以将 HMW 副产物与主要产品分离，但无法区分质量上较小的变化。在这里，为了鉴定三价双特异性 CrossMAb 抗体的多样化和复杂的 HMW 变体，我们应用了离线分级以及生产 HMW 副产物并结合全面分析测试的方法。此外，还对 HMW 变体进行了化学结合性质分析，并在功能测定中测试了变体效价的变化。通过使用质量光度法、SDS-PAGE 分析、与 SEC 结合的天然 MS 和自上而下的蛋白质组学进行详细表征，解释了效价的变化。我们确定了大部分 HMW 副产物是非共价连接的，导致解离和活性变化。我们还鉴定并定位了一种关注的副产物的高度异质性，并应用 CD3 亲和柱结合天然 MS 来注释意外的副产物。我们在这里提出了一种用于复杂 HMW 副产物的特征描述的多方法方法。更好地了解这些副产物有助于指导分析方法的开发，并为治疗性双特异性抗体设定适当的规范，以通过评估副产物确保产品的疗效和患者安全性保持一致。
