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Optimization and kinetic modeling of interchain disulfide bond reoxidation of monoclonal antibodies in bioprocesses.

ABSTRACT: Disulfide bonds play a crucial role in folding and structural stabilization of monoclonal antibodies (mAbs). Disulfide bond reduction may happen during the mAb manufacturing process, resulting in low molecular weight species and possible failure to meet product specifications. Although many mitigation strategies have been developed to prevent disulfide reduction, to the best of our knowledge, reforming disulfide bonds from the reduced antibody in manufacturing has not previously been reported. Here, we explored a novel rescue strategy in the downstream process to repair the broken disulfide bonds via in-vitro redox reactions on Protein A resin. Redox conditions including redox pair (cysteine/cystine ratio), pH, temperature, and reaction time were examined to achieve high antibody purity and a high reaction rate. Under the optimal redox condition, >90% reduced antibody could be reoxidized to form an intact antibody on Protein A resin in an hour. In addition, this study showed high flexibility on the range of the intact mAb fraction in the initial reduced mAb sample (the lower limit of intact mAb faction could be 14% based on the data reported in this study). Furthermore, a kinetic model based on elementary oxidative reactions was constructed to help optimize the reoxidation conditions and to predict product purity. Together, the deep understanding of interchain disulfide bond reoxidation, combined with the predictive kinetic model, provided a good foundation to implement a rescue strategy to generate high-purity antibodies with substantial cost savings in manufacturing processes.

SUBMITTER: Tang P

PROVIDER: S-EPMC7577745 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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Optimization and kinetic modeling of interchain disulfide bond reoxidation of monoclonal antibodies in bioprocesses.

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