Effects of molecule hydrophobicity and structural flexibility of appended bispecific antibody on Protein A chromatography

化学 双特异性抗体 色谱法 灵活性(工程) 分子 亲和层析 抗体 组合化学 有机化学 单克隆抗体 数学 生物 统计 免疫学
作者
Xinhui Wang,Nattha Ingavat,Jia Min Liew,Nuruljannah Dzulkiflie,Han Ping Loh,Yee Jiun Kok,Xuezhi Bi,Yuansheng Yang,Wei Zhang
出处
期刊:Journal of Chromatography A [Elsevier BV]
卷期号:1731: 465206-465206 被引量:2
标识
DOI:10.1016/j.chroma.2024.465206
摘要

Appended bispecific antibody (aBsAb) with two single chain variable fragments (scFv) linked at the c-terminus of its heavy chains is one of the promising formats in bispecific therapeutics. The presence of hydrophobic and flexible scFv fragments render aBsAb molecules higher molecule hydrophobicity and structural flexibility compared to monoclonal antibody (mAb), thus making its purification more challenging. We set out to investigate how the unique molecular properties of aBsAb affect its performance on Protein A chromatography. We showed that aBsAb has a high propensity for chromatography-induced aggregation due to its high molecule hydrophobicity, and this couldn't be improved by the addition of common chaotropic salts. Moreover, the presence of chaotropic salts, such as arginine hydrochloride (Arg-HCl), retarded aBsAb elution during Protein A chromatography rather than facilitating which was widely observed in mAb Protein A elution. Nevertheless, we were able to overcome the aggregation issue by optimizing elution condition and improved aBsAb purity from 29 % to 93 % in Protein A eluate with a high molecular weight (HMW) species of less than 5 %. We also showed that the high molecular flexibility of aBsAb leads to different hydrodynamic sizes of the aBsAb molecule post Protein A elution, neutralization, and re-acidification, which are pH dependent. This is different from mAbs where their sizes do not change post neutralization even with re-exposure to acid. The above unique observations of aBsAb in Protein A chromatography were clearly explained from the perspectives of its high molecular hydrophobicity and structural flexibility.
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