a Biologics Process Development, Bristol-Myers Squibb , 38 Jackson Road, Devens , MA , USA.
MAbs. 2018 Apr;10(3):397-405. doi: 10.1080/19420862.2017.1421880. Epub 2018 Jan 18.
The elucidation of antibody higher order structure (HOS) is critical in therapeutic antibody development. Since HOS determines the protein bioactivity and chemo-physical properties, this knowledge can help to ensure that the safety and efficacy attributes are not compromised. Protein conformational array (PCA) is a novel method for determining the HOS of monoclonal antibodies. Previously, we successfully utilized an enzyme-linked immunosorbent assay (ELISA)-based PCA along with other bioanalytical tools to elucidate the structures of antibody aggregates. In this study, applying a new multiplex-based PCA with 48-fold higher throughput than the ELISA-based one we revealed structural differences between different antibody molecules and antibody structure changes affected by various processing conditions. The PCA analysis of antibody molecules clearly demonstrated significant differences between IgG1 and IgG4 subclasses in epitope exposure and folding status. Furthermore, we applied small angle X-ray scattering to decipher mechanistic insights of PCA technology and validate structural information obtained using PCA. These findings enhance our fundamental understanding of mAbs' HOS in general. The PCA analysis of antibody samples from various processing conditions also revealed that antibody aggregation caused significantly higher exposure of antibody epitopes, which potentially led to a "foreign" molecule that could cause immunogenicity. The PCA data correlated well with protein stability results from traditional methods such as size-exclusion chromatography and protein thermal shift assay. Our study demonstrated that high throughput PCA is a suitable method for HOS analysis in the discovery and development of therapeutic antibodies.
抗体高级结构（HOS）的阐明对于治疗性抗体的开发至关重要。由于 HOS 决定了蛋白质的生物活性和理化性质，因此这方面的知识有助于确保安全性和疗效不受影响。蛋白质构象阵列（PCA）是一种用于确定单克隆抗体 HOS 的新方法。此前，我们成功地利用基于酶联免疫吸附测定（ELISA）的 PCA 以及其他生物分析工具来阐明抗体聚集物的结构。在这项研究中，我们应用了一种新的基于多重检测的 PCA，其通量比之前基于 ELISA 的方法高 48 倍，从而揭示了不同抗体分子之间的结构差异以及不同处理条件对抗体结构的影响。抗体分子的 PCA 分析清楚地表明，IgG1 和 IgG4 亚类在表位暴露和折叠状态方面存在显著差异。此外，我们应用小角度 X 射线散射技术来深入了解 PCA 技术的机制，并验证使用 PCA 获得的结构信息。这些发现增强了我们对 mAbs 一般 HOS 的基本理解。来自各种处理条件的抗体样品的 PCA 分析还表明，抗体聚集导致抗体表位明显暴露，这可能导致潜在的免疫原性“异物”。PCA 数据与传统方法（如尺寸排阻色谱法和蛋白质热转移测定法）得到的蛋白质稳定性结果相关性良好。我们的研究表明，高通量 PCA 是用于治疗性抗体的发现和开发中的 HOS 分析的合适方法。
