Chen Serene W, Hoi Kong Meng, Mahfut Farouq Bin, Yang Yuansheng, Zhang Wei
Downstream Processing Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore, Singapore.
Cell Line Development Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore, Singapore.
Bioresour Bioprocess. 2022 Jul 4;9(1):72. doi: 10.1186/s40643-022-00562-y.
Bispecific antibodies (bsAbs) are therapeutically promising due to their ability to bind to two different antigens. However, the bsAb byproducts and impurities, including mispaired homodimers, half-antibodies, light chain mispairings, antibody fragments and high levels of high molecular weight (HMW) species, all pose unique challenges to their downstream processing. Here, using two knob-into-hole (KiH) constructs of bsAbs as model molecules, we demonstrate the excellent removal of bsAb byproducts and impurities in a single Protein A chromatography under optimized conditions, including hole-hole homodimer mispaired products which are physicochemically very similar to the target bsAbs and still present even with the use of the KiH format, though at reduced levels. The removal occurs through the incorporation of an intermediate low-pH wash step and optimal elution conditions, achieving ~ 60% monomeric purity increase in a single Protein A step, without the introduction of sequence-specific bsAb modifications to specifically induce differential Protein A binding. Our results also suggest that the higher aggregation propensity of bsAbs may cause aggregation during the column process, hence an optimization of the appropriate loading amount, which may be lower than that of monoclonal antibodies (mAbs), is required. With the use of loading at 50% of 10% breakthrough (QB10) at 6-min residence time, we show that an overall high monomer purity of 92.1-93.2% can be achieved with good recovery of 78.4-90.6% within one capture step, which is a significant improvement from a monomer purity of ~ 30% in the cell culture supernatant (CCS). The results presented here would be an insightful guidance to all researchers working on the purification process development to produce bispecific antibodies, especially for knob-into-hole bispecific antibodies.
双特异性抗体（bsAbs）因其能够结合两种不同抗原而具有治疗前景。然而，bsAb的副产物和杂质，包括错配的同源二聚体、半抗体、轻链错配、抗体片段以及高分子量（HMW）物质的高水平存在，都给其下游加工带来了独特的挑战。在此，我们使用两种双特异性抗体的“旋钮入孔”（KiH）构建体作为模型分子，证明了在优化条件下，通过单一的蛋白A层析能够出色地去除bsAb的副产物和杂质，其中包括在物理化学性质上与目标bsAb非常相似的孔 - 孔同源二聚体错配产物，即便使用KiH形式这些产物仍会出现，不过含量有所降低。这种去除是通过加入中间的低pH洗涤步骤和优化洗脱条件实现的，在单一的蛋白A步骤中实现了约60%的单体纯度提高，且无需引入序列特异性的bsAb修饰来特意诱导不同的蛋白A结合。我们的结果还表明，bsAbs较高的聚集倾向可能会在层析过程中导致聚集，因此需要优化合适的上样量，该上样量可能低于单克隆抗体（mAbs）。在6分钟停留时间下以10%穿透率（QB10）的50%进行上样，我们表明在一次捕获步骤中能够实现92.1 - 93.2%的总体高单体纯度以及78.4 - 90.6%的良好回收率，这相较于细胞培养上清液（CCS）中约30%的单体纯度有了显著提高。本文呈现的结果将为所有致力于双特异性抗体纯化工艺开发的研究人员，尤其是针对“旋钮入孔”双特异性抗体的研究人员，提供具有深刻见解的指导。
