Doctoral School of the Rzeszow University of Technology/PL, Poland.
Department of Chemical and Process Engineering, Rzeszów University of Technology, Rzeszów/PL, Poland.
J Chromatogr A. 2024 Sep 27;1733:465256. doi: 10.1016/j.chroma.2024.465256. Epub 2024 Aug 10.
A design procedure for the separation of charge variants of a monoclonal antibody (mAb) was developed, which was based on the coupling of cation-exchange chromatography (CEX) and anion-exchange chromatography (AEX) under high loading conditions. The design of the coupled process was supported by a dynamic model. The model was calibrated on the basis of band profiles of variants determined experimentally for the mAb materials of different variant compositions. The numerical simulations were used to select the coupling configuration and the loading conditions that allowed for efficient separation of the mAb materials into three products enriched with each individual variant: the acidic (av), main (mv) and basic (bv) one. In the CEX section, a two-step pH gradient was used to split the loaded mass of mAb into a weakly bound fraction enriched with av and mv, and a strongly bound fraction containing the bv-rich product. The weakly bound fraction was further processed in the AEX section, where the mv-rich product was eluted in flowthrough, while the av-rich product was collected by a step change in pH. The choice of flow distribution and the number of columns in the CEX and AEX sections depended on the variant composition of the mAb material. For the selected configurations, the optimized mAb loading density in the CEX columns ranged from 10 to 26 mg mL, while in the AEX columns it was as high as 300 or 600 mg mL, depending on the variant composition of the mAb material. By proper selection of the loading condition, a trade-off between yield and purity of the products could be reached.
开发了一种用于分离单克隆抗体 (mAb) 电荷变异体的设计程序，该程序基于在高负载条件下将阳离子交换色谱 (CEX) 和阴离子交换色谱 (AEX) 偶联。该偶联过程的设计得到了动力学模型的支持。该模型是根据不同变异体组成的 mAb 材料的实验确定的变异体带谱进行校准的。数值模拟用于选择允许有效分离 mAb 材料成三种产物的偶联配置和加载条件，这三种产物分别富含每种单独的变异体：酸性 (av)、主要 (mv) 和碱性 (bv)。在 CEX 部分，使用两步 pH 梯度将加载的 mAb 质量分离成富含 av 和 mv 的弱结合部分和富含 bv 的强结合部分。弱结合部分在 AEX 部分进一步处理，其中富含 mv 的产物在流通过程中洗脱，而富含 av 的产物通过 pH 阶跃收集。CEX 和 AEX 部分的流量分配和柱数的选择取决于 mAb 材料的变异体组成。对于所选的配置，CEX 柱中优化的 mAb 加载密度范围为 10 至 26 mg mL，而在 AEX 柱中高达 300 或 600 mg mL，具体取决于 mAb 材料的变异体组成。通过适当选择加载条件，可以在产物的收率和纯度之间取得平衡。
