Yu Deqiang, Song Yuanli, Huang Richard Y-C, Swanson Ryan K, Tan Zhijun, Schutsky Elizabeth, Lewandowski Angela, Chen Guodong, Li Zheng Jian
Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, USA.
Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, USA.
J Chromatogr A. 2016 Jul 29;1457:66-75. doi: 10.1016/j.chroma.2016.06.031. Epub 2016 Jun 11.
Antibody aggregate is a common issue in therapeutic antibodies, which may compromise product efficacy and cause adverse effects. Antibody aggregate level is normally controlled in bioprocessing by polishing steps after Protein A capture. This paper studied the Higher Order Structures (HOS) of antibody aggregates (dimer H1 and H2) and their adsorption on Protein A resin and thus elucidated the mechanism using Protein A capture for enhanced aggregate removal. The HOS of antibody aggregates and their complex with Protein A were characterized using HDX-MS combined with SEC-MALS, Protein Conformational Array (PCA), and molecular modeling. The aggregate size and Protein A binding ratio suggested that H2 has much more compact structure than H1. HDX-MS and PCA further revealed that H1 was formed by single Fab-Fab interaction while H2 formed by Fab-Fab and likely Fc-Fc interaction. On Protein A resin, both the molar binding ratio and the correlation between protein size and ligand distance support that each monomer can only bind one Protein A ligand, while each dimer can bind two ligands, thus resulting in stronger resin binding. Furthermore, dimer H2 binds stronger than dimer H1 due to its compact structure. By integrating biophysical analysis and molecular modeling with process development, this study revealed the antibody aggregate structures and the mechanism of aggregate removal using Protein A chromatography. It also provided a general strategy for in-depth product and process understanding in antibody and other biologics development.
抗体聚集体是治疗性抗体中的常见问题，它可能会损害产品疗效并导致不良反应。在生物加工过程中，抗体聚集体水平通常通过蛋白A捕获后的精制步骤来控制。本文研究了抗体聚集体（二聚体H1和H2）的高级结构（HOS）及其在蛋白A树脂上的吸附，从而阐明了利用蛋白A捕获增强聚集体去除的机制。使用氢氘交换质谱（HDX-MS）结合尺寸排阻色谱-多角度激光散射（SEC-MALS）、蛋白质构象阵列（PCA）和分子建模对抗体聚集体及其与蛋白A的复合物的高级结构进行了表征。聚集体大小和蛋白A结合率表明，H2的结构比H1紧凑得多。HDX-MS和PCA进一步揭示，H1由单个Fab-Fab相互作用形成，而H2由Fab-Fab以及可能的Fc-Fc相互作用形成。在蛋白A树脂上，摩尔结合率以及蛋白质大小与配体距离之间的相关性均支持每个单体只能结合一个蛋白A配体，而每个二聚体可以结合两个配体，从而导致更强的树脂结合。此外，二聚体H2由于其紧凑的结构而比二聚体H1结合更强。通过将生物物理分析和分子建模与工艺开发相结合，本研究揭示了抗体聚集体结构以及利用蛋白A色谱去除聚集体的机制。它还为深入了解抗体及其他生物制品开发中的产品和工艺提供了一个通用策略。
