Regeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.
Biotechnol Bioeng. 2023 Jun;120(6):1605-1613. doi: 10.1002/bit.28379. Epub 2023 Apr 20.
In the production of monoclonal antibodies (mAbs) intended for use in humans, it is a global regulatory requirement that the manufacturing process includes unit operations that are proven to inactivate or remove adventitious agents to ensure viral safety. Viral inactivation by low pH hold (LPH) is typically used to ensure this viral safety in the purification process of mAbs and other biotherapeutics derived from mammalian cell lines. To ascertain the effectiveness of the LPH step, viral clearance studies have evaluated LPH under worst-case conditions of pH above the manufacturing set point and hold duration at or below the manufacturing minimum. Highly acidic conditions (i.e., pH < 3.60) provide robust and effective enveloped virus inactivation but may lead to reduced product quality of the therapeutic protein. However, when viral inactivation is operated above pH 3.60 to ensure product stability, effective (>4 log reduction factor) viral inactivation may not be observed under these worst-case pH conditions in viral clearance studies. A multivariate design of experiments was conducted to further characterize the operating space for low pH viral inactivation of a model retrovirus, xenotropic murine leukemia virus (X-MuLV). The statistically designed experiment evaluated the effect of mAb isotype, pH, temperature, acid titrant, sodium chloride (NaCl) concentration, virus spike timing, and post-spike filtration on X-MuLV inactivation. Data from the characterization study were used to generate predictive models to identify conditions that reliably achieve effective viral inactivation at pH ≥ 3.60. Results of the study demonstrated that NaCl concentration has the greatest effect on virus inactivation in the range studied, and pH has a large effect when the load material has no additional NaCl. Overall, robust and effective inactivation of X-MuLV at pH 3.65-3.80 can be achieved by manipulating either the pH or the NaCl concentration of the load material. This study contributes to the understanding of ionic strength as an influential parameter in low pH viral inactivation studies.
在生产用于人类的单克隆抗体 (mAb) 时，全球监管要求制造过程包括已证明可灭活或去除外来因子以确保病毒安全性的单元操作。通过低 pH 保持 (LPH) 进行病毒灭活通常用于确保 mAb 和其他源自哺乳动物细胞系的生物治疗剂的纯化过程中的病毒安全性。为了确定 LPH 步骤的有效性，病毒清除研究评估了在制造设定点以上的最坏 pH 值和低于制造最低值的保持时间下的 LPH。酸性条件（即 pH < 3.60）提供了强大而有效的包膜病毒灭活，但可能导致治疗蛋白的产品质量下降。然而，当病毒灭活在 pH 3.60 以上操作以确保产品稳定性时，在病毒清除研究中，在这些最坏情况 pH 条件下可能不会观察到有效的 (>4 log 减少因子) 病毒灭活。进行了多变量实验设计，以进一步描述模型逆转录病毒、嗜性鼠白血病病毒 (X-MuLV) 的低 pH 病毒灭活的操作空间。经过统计学设计的实验评估了 mAb 同种型、pH 值、温度、酸滴定剂、氯化钠 (NaCl) 浓度、病毒峰值时间和峰值后过滤对 X-MuLV 灭活的影响。特征研究的数据用于生成预测模型，以确定在 pH ≥ 3.60 时可靠实现有效病毒灭活的条件。研究结果表明，在研究范围内，NaCl 浓度对病毒灭活的影响最大，当负载材料没有额外的 NaCl 时，pH 值的影响很大。总体而言，通过操纵负载材料的 pH 或 NaCl 浓度，可以在 pH 3.65-3.80 下实现 X-MuLV 的强大而有效的灭活。该研究有助于理解离子强度作为低 pH 病毒灭活研究中一个有影响力的参数。
