On-column disulfide bond formation of monoclonal antibodies during Protein A chromatography eliminates low molecular weight species and rescues reduced antibodies

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.

Antibody disulfide bond reduction and recovery during biopharmaceutical process development—A review

“…Furthermore, this case study demonstrated that the redox wash has no negative impact on process yield or product quality. Extensive characterization of the reoxidized antibody confirmed a complete formation of interchain disulfide bonds and comparable biophysical properties to the reference material (Tan et al, 2020).…”

“…Comprehensive evaluation using redox wash system in the platform Protein A chromatography (Tan et al, 2020). The study was performed using three mAb harvest cell cultures according to the design including three arms.…”

“…Figure 4a illustrates a comprehensive study plan, in which HCCFs of three mAbs (mAb‐1, mAb‐2, and mAb‐3) were used for the study (Tan et al, 2020). Each HCCF was divided into two pools, which underwent two treatments and storage conditions: “good HCCF” (air sparging + 4°C) and “bad HCCF” (nitrogen sparging + room temperature [19–25°C]).…”

“…While the thiol‐disulfide exchange reaction has been widely studied to provide deeper understandings of disulfide bond formation relations in cells (Cappel & Gilbert, 1988; Østergaard, Tachibana, & Winther, 2004; Tu et al, 2000), there are very few research in literature that applied this reaction to recover reduced mAb in the manufacturing process. In a recent study, researchers recovered the reduced mAb by using redox reagent buffer containing cysteine/cystine as wash buffer in Protein A chromatography step (Figure S4) (Tan et al, 2020; Tang et al, 2020). Based on the kinetic model developed in this study, cystine concentration and pH were found to be the most critical factors that affected reduced mAb recovery.…”

“…Many research studies have been conducted to analyze and understand the function of disulfide bond structure on antibody stability, and there are several reviews on this topic (Correia, 2010; Liu & May, 2012; Trivedi et al, 2009). At the same time, scientists have made substantial progress in identifying the root cause of disulfide bond reduction during mAb manufacturing process, and developing mitigation strategies to minimize the reduction as well as to recover the reduced mAb during the downstream process (Du et al, 2018; Hutterer et al, 2013; Kao et al, 2010; Mun et al, 2015; Tan et al, 2020; Tang et al, 2020; Trexler‐Schmidt et al, 2010). However, to the best of the authors' knowledge, there are limited review papers summarizing the latest development of disulfide bond reduction mitigation strategies that provide a comprehensive understanding of the application of these mitigation strategies across downstream steps.…”