ABSTRACT

Disulfide bond reduction, which commonly occurs during monoclonal antibody (mAb) manufacturing processes, can result in a drug substance with high levels of low molecular weight (LMW) species that may fail release specifications because the drug’s safety and the efficiency may be affected by the presence of this material. We previously studied disulfide reoxidation of mAbs and demonstrated that disulfide bonds could be reformed from the reduced antibody via redox reactions under an optimal redox condition on Protein A resin. The study here implements a redox system in a manufacturing setting to rescue the reduced mAb product and to further eliminate LMW issues in downstream processing. As such, we incorporate the optimized redox system as one of the wash buffers in Protein A chromatography to enable an on-column disulfide reoxidation to form intact antibody in vitro. Studies at laboratory scale (1 cm (ID) x 20 cm (Height), MabSelect SuRe LX) and pilot scale (30 cm (ID) x 20 cm (Height), MabSelect SuRe LX) were performed to demonstrate the effectiveness and robustness of disulfide formation with multiple mAbs using redox wash on Protein A columns. By applying this rescue strategy using ≤50 g/L-resin loading, the intact mAb purity was improved from <5% in the Protein A column load to >90% in the Protein A column elution with a product yield of >90%. Studies were also done to confirm that adding the redox wash has no negative impact on process yield or impurity removal or product quality. The rescued mAbs were confirmed to form complete interchain disulfide bonds, exhibiting comparable biophysical properties to the reference material. Furthermore, since the redox wash is followed by a bridging buffer wash before the final elution, no additional burden is involved in removing the redox components during the downstream steps. Due to its ease of implementation, significant product purity improvement, and minimal impact on other product quality attributes, we demonstrate that the on-column reoxidation using a redox system is a powerful, simple, and safe tool to recover reduced mAb during manufacturing. Moreover, the apparent benefits of using a high-pH redox wash may further drive the evolution of Protein A platform processes.

 抽象的

二硫键还原通常发生在单克隆抗体 (mAb) 制造过程中，可能会导致原料药中含有高含量的低分子量 (LMW) 物质，从而可能不符合放行规范，因为药物的安全性和效率可能会受到二硫键还原的影响。这种材料的存在。我们之前研究了 mAb 的二硫键再氧化，并证明二硫键可以在 Protein A 树脂上的最佳氧化还原条件下通过氧化还原反应从还原的抗体中重新形成。本研究在制造环境中实施了氧化还原系统，以挽救减少的 mAb 产品并进一步消除下游加工中的 LMW 问题。因此，我们将优化的氧化还原系统作为 Protein A 色谱中的洗涤缓冲液之一，以实现柱上二硫键再氧化，从而在体外形成完整的抗体。进行了实验室规模（1 cm（ID）x 20 cm（高度），MabSelect SuRe LX）和中试规模（30 cm（ID）x 20 cm（高度），MabSelect SuRe LX）的研究，以证明使用氧化还原清洗 Protein A 柱，与多种 mAb 形成二硫键。通过应用这种使用 ≤ 50 g/L 树脂负载的拯救策略，蛋白质 A 柱洗脱中完整 mAb 的纯度从 <5 id=25> 90% 提高，产品收率 > 90%。研究还证实添加氧化还原清洗剂不会对工艺产量、杂质去除或产品质量产生负面影响。获救的单克隆抗体被证实形成完整的链间二硫键，表现出与参考材料相当的生物物理特性。 此外，由于氧化还原洗涤之后是最终洗脱之前的桥接缓冲液洗涤，因此在下游步骤中去除氧化还原成分时不涉及额外的负担。由于其易于实施、产品纯度显着提高以及对其他产品质量属性的影响最小，我们证明使用氧化还原系统的柱上再氧化是在制造过程中回收还原的 mAb 的强大、简单且安全的工具。此外，使用高 pH 氧化还原洗涤的明显好处可能会进一步推动 Protein A 平台工艺的发展。