Abstract：
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.
摘要：
在其重链的c末端连接有两个单链可变片段(scFv)的附加双特异性抗体(aBsAb)是双特异性治疗剂中的有希望的形式之一。与单克隆抗体（mAb）相比，疏水和柔性scFv片段的存在使aBsAb分子具有更高的分子疏水性和结构灵活性，从而使其净化更具挑战性。我们着手研究aBsAb的独特分子特性如何影响其在蛋白A色谱上的性能。我们表明，由于其高分子疏水性，aBsAb具有很高的色谱诱导聚集倾向，这不能通过添加常见的离液盐来改善。此外，离液盐的存在，如精氨酸盐酸盐（Arg-HCl），在蛋白A色谱期间延迟aBsAb洗脱，而不是促进在mAb蛋白A洗脱中广泛观察到的。然而，我们能够通过优化洗脱条件来克服聚集问题,并且在具有小于5%的高分子量(HMW)种类的蛋白A洗脱液中将aBsAb纯度从29%提高到93%。我们还表明，aBsAb的高分子柔性导致蛋白A洗脱后aBsAb分子的不同流体动力学大小，中和,再酸化,是pH依赖性的。这与mAb不同,在mAb中,即使再暴露于酸,它们的大小在中和后也不会改变。从其高分子疏水性和结构灵活性的角度清楚地解释了aBsAb在蛋白A色谱中的上述独特观察。
