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 Sep 14;9(1):98. doi: 10.1186/s40643-022-00590-8.
Bispecific antibodies (bsAbs), though possessing great therapeutic potential, are extremely challenging to obtain at high purity within a limited number of scalable downstream processing steps. Complementary to Protein A chromatography, polishing strategies play a critical role at removing the remaining high molecular weight (HMW) and low molecular weight (LMW) species, as well as host cell proteins (HCP) in order to achieve a final product of high purity. Here, we demonstrate using two knob-into-hole (KiH) bsAb constructs that two flow-through polishing steps utilising Capto Butyl ImpRes and Capto adhere resins, performed after an optimal Protein A affinity chromatography step can further reduce the HCP by 17- to 35-fold as well as HMW and LMW species with respect to monomer by ~ 4-6% and ~ 1%, respectively, to meet therapeutical requirement at 30-60 mg/mL-resin (R) load. This complete flow-through polishing strategy, guided by Design of Experiments (DoE), eliminates undesirable aggregation problems associated with the higher aggregation propensity of scFv containing bsAbs that may occur in the bind and elute mode, offering an improved ease of overall process operation without additional elution buffer preparation and consumption, thus aligning well with process intensification efforts. Overall, we demonstrate that through the employment of (1) Protein A chromatography step and (2) flow-through polishing steps, a final product containing < 1% HMW species, < 1% LMW species and < 100 ppm HCP can be obtained with an overall process recovery of 56-87%.
双特异性抗体（bsAbs）虽然具有巨大的治疗潜力，但要在有限数量的可扩展下游加工步骤中获得高纯度的产品极具挑战性。作为蛋白A层析的补充，精制策略在去除残留的高分子量（HMW）和低分子量（LMW）物质以及宿主细胞蛋白（HCP）方面起着关键作用，以实现高纯度的最终产品。在此，我们使用两种“旋钮入孔”（KiH）bsAb构建体证明，在最佳的蛋白A亲和层析步骤之后进行的两个利用Capto Butyl ImpRes和Capto adhere树脂的流通精制步骤，可以进一步将HCP降低17至35倍，以及相对于单体将HMW和LMW物质分别降低约4-6%和1%，以满足30-60 mg/mL树脂（R）负载下的治疗要求。这种由实验设计（DoE）指导的完整流通精制策略消除了与含单链抗体片段（scFv）的bsAbs在结合和洗脱模式下可能出现的较高聚集倾向相关的不良聚集问题，在无需额外制备和消耗洗脱缓冲液的情况下提高了整体工艺操作的简便性，因此与工艺强化努力非常契合。总体而言，我们证明通过采用（1）蛋白A层析步骤和（2）流通精制步骤，可以获得最终产品中HMW物质<1%、LMW物质<1%且HCP<100 ppm的产品，整体工艺回收率为56-87%。
