Manufacturing Technology Association of Biologics, Shin-kawa, Chuo-ku, Japan.
Biomedical Engineering Center (YUBEC), Graduate School of Science and Technology for Innovation, Yamaguchi University, Ube, Japan.
Biotechnol Bioeng. 2024 Aug;121(8):2269-2277. doi: 10.1002/bit.28537. Epub 2023 Sep 10.
A continuous downstream process of monoclonal antibody was developed based on the process characterization. Periodic-counter current chromatography (PCCC) with two protein A columns was used for the capture step. For low pH virus inactivation (VI), a batch reactor was employed, which can work as a surge (buffer) tank. Flow-through chromatography (FTC) with two connected columns of different separation modes (anion-mixed mode and cation exchange) was designed as a polish step. After 24 h PCCC run, the collected pool was processed for VI. After adjusting pH and electric conductivity, the solution was fed to the two connected FTC columns for 24 h. Virus filter was also connected to the exit of the connected-column. PCCC and FTC were run in parallel. Six runs of different feed rates (0.5-10 L/day) and feed concentrations (1-3.2 g/L) were performed with protein A columns of 1-5 mL and FTC columns of 3-10 mL. The largest run (feed rate 10 L/day, feed concentration 2 g/L) was carried out at a GMP facility with 15 mL protein A columns and 100 mL FTC columns. Good recovery and purity values were obtained for all runs. The process was found to be flexible and stable for feed fluctuations. Only three surge or pool tanks were needed in addition to the final product pool tank.
基于工艺特性开发了单克隆抗体的连续下游工艺。使用两个蛋白 A 柱进行周期性逆流色谱（PCCC）捕获步骤。对于低 pH 值病毒灭活（VI），采用批式反应器，该反应器可用作缓冲罐。采用两种不同分离模式（阴离子混合模式和阳离子交换）的两个串联柱的流穿色谱（FTC）作为精制步骤。PCCC 运行 24 小时后，收集的池液进行 VI 处理。调整 pH 值和电导率后，将溶液进料至两个串联的 FTC 柱中 24 小时。连接柱的出口还连接有病毒过滤器。PCCC 和 FTC 并行运行。使用 1-5mL 的蛋白 A 柱和 3-10mL 的 FTC 柱，在不同的进料速率（0.5-10L/天）和进料浓度（1-3.2g/L）下进行了六次运行。在 GMP 设施中进行了最大运行（进料速率 10L/天，进料浓度 2g/L），使用了 15mL 的蛋白 A 柱和 100mL 的 FTC 柱。所有运行均获得了良好的回收率和纯度值。该工艺对于进料波动具有灵活性和稳定性。除最终产品池外，仅需三个缓冲罐或池罐。
