Table 4 summarized the activation energy of two redox systems at different cysteine levels. 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 Phenformin hydrochloride 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. KEYWORDS: Monoclonal antibody, disulfide bond, redox/reduction/reoxidation, kinetic modeling Introduction Recombinant monoclonal antibodies (mAbs) constitute a prominent class of therapeutic proteins.1,2 A typical mAb has a molecular weight of approximately 150 kilodalton (kDa) and consists of two identical light chains and two identical heavy chains, linked by inter-heavy-heavy (HH) and inter heavy-light (HL) disulfide bonds.3C5 Disulfide bonds that connect two heavy chains or connect a light chain and a heavy chain are known as interchain disulfide bonds. Disulfide bonds that connect the two -sheets in a Phenformin hydrochloride single domain (constant domain or variable domain) are known as intrachain disulfide bonds. In a typical mAb, there are 12 intrachain disulfide bonds (one per each domain), two interchain disulfide bonds between light chain and heavy chain, and two to eleven interchain disulfide bonds between two heavy chains.3,6 Disulfide bonds stabilize proteins thermodynamically and mechanically. Improper disulfide bond formation and disulfide bond reduction can affect process performance, protein stability, and biological functionality.4,5,7C10 In recent years, the disulfide bond reduction after cell culture harvest has been observed more often for many large-scale mAb manufacturing processes, resulting in out-of-specification levels of low-molecular-weight (LMW) species and potential batch failure in manufacturing.7,11,12 Studies focusing Phenformin hydrochloride on preventing interchain disulfide bond breakage have been reported previously.11C17 Yet, mitigation methods do not always adequately prevent the disulfide reduction from occurring. In addition, these mitigation methods may require extra equipment and increase the manufacturing cost. Alternatively, reoxidizing the disulfide from the reduced antibody species to generate intact antibody products as a rescue strategy could be developed to address the disulfide bond reduction challenge. The rescue strategy can save the reduced mAb batches without sacrificing the manufacturing process flexibility or increasing the manufacturing cost. To our best knowledge, this is the first time that the approach of developing the rescue strategy in manufacturing process has been reported.8,16,18C21 As a post-translational modification, a disulfide bond is formed by reoxidizing two neighboring free cysteine residues.22C24 Though there is a vast body of knowledge of disulfide reoxidation for antibodies, the majority of these studies focused on the solution environment with limited investigational conditions.8,18,19,25,26 The existing studies cannot be directly and practically implemented in manufacturing process for three reasons. First, the reaction parameters were not optimized under typical manufacturing operation conditions to achieve high intact purity and fast reoxidation kinetics. Second, there is limited information on whether any downstream unit operation (such as Protein A chromatography, ion exchange chromatograph, and ultrafiltration/diafiltration) can be selected to implement the rescue strategies. Also, the manufacturability of using the rescue strategies at a large scale remains to be seen. Third, there is a lack of comprehensive characterizations to confirm the product comparability between the rescued mAb and the reference material. Thus, to develop practical industrial rescue strategies, the gap between the existing disulfide bond formation studies and the general mAb manufacturing process must be bridged. In this series of studies, we systematically evaluated how to implement the reduced mAb rescue strategy into the mAb manufacturing process to bridge the aforementioned gap. Our studies are discussed in two papers: 1) the proof-of-concept study evaluated the possibilities of rescuing reduced mAb, selected the unit operation step (Protein A chromatography) to include the rescue strategy and developed the kinetics models to optimize reaction parameters under typical manufacturing operation conditions; and 2) the developability and manufacturability study implemented a potential rescue strategy during Protein A chromatography and performed comprehensive characterizations of the recovered mAb, showing the capabilities of generating high-purity antibody products from the reduced form in the manufacturing process. The proof-of-concept study results are reported here, and developability and manufacturability study results are Mouse monoclonal to MLH1 discussed in a separate paper (Tan et al., mAbs, in press). This paper focuses on the fundamental understanding of disulfide bond reoxidation experiments demonstrated that a GSH/GSSG ratio similar to that found in the Phenformin hydrochloride ER.