Genor Biopharma Co., Ltd., Shanghai, China.
Prep Biochem Biotechnol. 2012;42(2):183-202. doi: 10.1080/10826068.2012.654572.
Aggregate removal is one of the most important aspects in monoclonal antibody (mAb) purification. Cation-exchange chromatography (CEX), a widely used polishing step in mAb purification, is able to clear both process-related impurities and product-related impurities. In this study, with the implementation of quality by design (QbD), a process development approach for robust removal of aggregates using CEX is described. First, resin screening studies were performed and a suitable CEX resin was chosen because of its relatively better selectivity and higher dynamic binding capacity. Second, a pH-conductivity hybrid gradient elution method for the CEX was established, and the risk assessment for the process was carried out. Third, a process characterization study was used to evaluate the impact of the potentially important process parameters on the process performance with respect to aggregate removal. Accordingly, a process design space was established. Aggregate level in load is the critical parameter. Its operating range is set at 0-3% and the acceptable range is set at 0-5%. Equilibration buffer is the key parameter. Its operating range is set at 40 ± 5 mM acetate, pH 5.0 ± 0.1, and acceptable range is set at 40 ± 10 mM acetate, pH 5.0 ± 0.2. Elution buffer, load mass, and gradient elution volume are non-key parameters; their operating ranges and acceptable ranges are equally set at 250 ± 10 mM acetate, pH 6.0 ± 0.2, 45 ± 10 g/L resin, and 10 ± 20% CV respectively. Finally, the process was scaled up 80 times and the impurities removal profiles were revealed. Three scaled-up runs showed that the size-exclusion chromatography (SEC) purity of the CEX pool was 99.8% or above and the step yield was above 92%, thereby proving that the process is both consistent and robust.
聚集物去除是单克隆抗体（mAb）纯化中最重要的方面之一。阳离子交换色谱（CEX）是 mAb 纯化中广泛使用的一种抛光步骤，能够清除工艺相关杂质和产品相关杂质。在这项研究中，采用质量源于设计（QbD），描述了使用 CEX 稳健去除聚集物的工艺开发方法。首先进行了树脂筛选研究，并选择了一种合适的 CEX 树脂，因为它具有相对更好的选择性和更高的动态结合能力。其次，建立了 CEX 的 pH-电导率混合梯度洗脱方法，并对工艺进行了风险评估。第三，采用工艺特性研究评估了潜在重要工艺参数对聚集物去除工艺性能的影响。相应地，建立了工艺设计空间。负载中的聚集物水平是关键参数。其操作范围设定为 0-3%，可接受范围设定为 0-5%。平衡缓冲液是关键参数。其操作范围设定为 40 ± 5 mM 醋酸盐，pH 5.0 ± 0.1，可接受范围设定为 40 ± 10 mM 醋酸盐，pH 5.0 ± 0.2。洗脱缓冲液、负载量和梯度洗脱体积是非关键参数；它们的操作范围和可接受范围同样设定为 250 ± 10 mM 醋酸盐，pH 6.0 ± 0.2，45 ± 10 g/L 树脂和 10 ± 20%CV。最后，将工艺放大 80 倍，揭示了杂质去除情况。三次放大运行表明，CEX 池的尺寸排阻色谱（SEC）纯度为 99.8%或更高，收率高于 92%，从而证明该工艺既一致又稳健。
