Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang Key Laboratory of Smart Biomaterials, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310058, China.
Hisun Biopharmaceutical Co., Ltd., Hangzhou 311404, China.
J Chromatogr A. 2023 Sep 27;1707:464292. doi: 10.1016/j.chroma.2023.464292. Epub 2023 Aug 9.
The multicolumn counter-current solvent gradient purification (MCSGP) method has proven effective in addressing the issue of elution profile overlap for difficult-to-separate proteins, leading to improved purity and recovery. However, during the MCSGP process, the flow rate and proportion of loaded proteins undergo changes, causing a significant discrepancy between the elution profiles of batch process design and the actual MCSGP process. This mismatch negatively impacts the purity and recovery of the target protein. To address this challenge, an improved process design (reDesign) was proposed with the first-run MCSGP to mimic the actual continuous process and obtain elution profiles that closely resemble the real ones. The reDesign was demonstrated with both a model protein mixture and a sample of monoclonal antibody (mAb) with charge variants. For model protein mixture, the reDesign-based MCSGP process (reMCSGP) showed a remarkable improvement in recovery, increasing from 83.6% to 97.8% while maintaining a purity of more than 95%. For mAb sample, the recovery of reMCSGP improved significantly to 93.9%, surpassing the performance of normal MCSGP processes at a given purity level of more than 84%. In general, the new process design strategy developed in this work could generate a more representative elution profile that closely mirrors actual conditions in continuous processes, which enhances the separation performance of MCSGP.
多柱逆流溶剂梯度净化（MCSGP）方法已被证明在解决难以分离的蛋白质洗脱轮廓重叠问题方面非常有效，从而提高了纯度和回收率。然而，在 MCSGP 过程中，加载蛋白质的流速和比例会发生变化，导致批量过程设计和实际 MCSGP 过程的洗脱轮廓之间存在显著差异。这种不匹配会对目标蛋白质的纯度和回收率产生负面影响。为了解决这个挑战，提出了一种改进的过程设计（reDesign），使用第一次运行的 MCSGP 来模拟实际的连续过程，并获得与真实洗脱轮廓非常相似的洗脱轮廓。该 reDesign 已在模型蛋白混合物和具有电荷变异的单克隆抗体（mAb）样品中得到验证。对于模型蛋白混合物，基于 reDesign 的 MCSGP 过程（reMCSGP）在回收率方面有显著提高，从 83.6%提高到 97.8%，同时保持纯度高于 95%。对于 mAb 样品，reMCSGP 的回收率显著提高到 93.9%，超过了在给定纯度水平（高于 84%）下的正常 MCSGP 过程的性能。总的来说，这项工作中开发的新过程设计策略可以生成更具代表性的洗脱轮廓，更接近连续过程中的实际情况，从而提高 MCSGP 的分离性能。
