Glutathione and thioredoxin systems contribute to recombinant monoclonal antibody interchain disulfide bond reduction during bioprocessing

Abstract

Antibody interchain disulfide bond reduction during biopharmaceutical manufacturing has received increased attention since it was first reported in 2010. Antibody reduction leads to loss of product and reduced product stability. It is therefore critical to understand the underlying mechanisms of reduction. To date, the thioredoxin system has been reported as the sole contributor to antibody reduction during bioprocessing. In this work, we show that the glutathione system, in addition to the thioredoxin system, is involved in reducing antibody molecules and the contributions of the two systems can vary depending upon the cell culture process. The roles of the glutathione and thioredoxin systems were evaluated for three molecules with different IgG subclass where reduction was observed during manufacturing: mAb A, mAb B, and mAb C representing an IgG , IgG , and IgG respectively. The expression of enzymes for both the thioredoxin and glutathione systems were confirmed in all three cell lines. Inhibitors were evaluated using purified mammalian reductases to evaluate their specificity. The optimized experimental conditions enabled both the determination of reductase activity contributed from as well as the amount of antibody reduced by each enzymatic system. Our results demonstrate that the underlying enzymatic mechanisms are different depending upon the cell culture process; one of the two systems may be the dominant mechanism, or both enzymatic systems may be involved. Specifically, the glutathione system was found to be the major contributor to mAb A reduction while the thioredoxin system was the major contributor to mAb C reduction. Intriguingly, mAb B experienced significant reduction from both enzymatic systems. In summary, we have demonstrated that in addition to the thioredoxin pathway, the glutathione system is a second major pathway contributing to antibody reduction and this knowledge can be leveraged to develop more specific antibody reduction mitigation strategies targeted at the dominant reduction mechanism. Biotechnol. Bioeng. 2017;114: 1469-1477. © 2017 Wiley Periodicals, Inc.

Online control of cell culture redox potential prevents antibody interchain disulfide bond reduction

“…Due to the importance of these therapeutics, considerable efforts have been focused on improving the manufacturing processes for these molecules in terms of both yield and product quality (Li, Vijayasankaran, Shen, Kiss, & Amanullah, 2010). A challenge that has received increased attention in recent years relates to the reduction of monoclonal antibody (mAb) interchain disulfide bonds during manufacturing processes (Handlogten, Zhu, & Ahuja, 2017; Hutterer et al, 2013; Kao, Hewitt, Trexler‐Schmidt, & Laird, 2010; O'Mara et al, 2019). Antibody reduction results in the loss of product, requires more complex purification processes, and can reduce the stability of the drug product (Chung et al, 2017; Mun et al, 2015).…”

Impact of enzymatic reduction on bivalent bispecific antibody fragmentation and loss of product purity upon reoxidation

“…Unlike BisAbs, monoclonal antibody (mAb) fragmentation and aggregate formation during manufacturing processes has been previously studied. Reduction of mAbs has been shown to be a result of cell lysis from shear forces during harvest operations, thereby releasing intracellular reductases that can act on the mAb (Handlogten, Zhu, & Ahuja, 2017; Hutchinson, Bingham, Murrell, Farid, & Hoare, 2006; Koterba, Borgschulte, & Laird, 2012; Trexler‐Schmidt et al, 2010). Once a mAb has been reduced, fragments may reform the intact antibody with no significant change in bioactivity (Wang, Liu, Cai, Huang, & Flynn, 2015).…”

“…We present the in vitro reduction and reoxidation properties of a set of BisAb formats (IgG‐scFv fusions) using the thioredoxin enzyme system to induce DSB reduction. The thioredoxin pathway was specifically selected because it is a well‐characterized enzymatic system that has been demonstrated to reduce mAbs during manufacturing operations (Handlogten et al, 2017; Koterba et al, 2012; Magnusson, Björnstedt, & Holmgren, 1997). Due to the reported link between mAb reduction and aggregation during the low pH viral inactivation step of the purification process, we additionally assessed the reoxidation behavior of enzyme‐reduced BisAbs with and without low pH treatment followed by room temperature storage, both of which occur during typical BisAb production.…”

Intracellular response to process optimization and impact on productivity and product aggregates for a high‐titer CHO cell process

“…The fed-batch processes were run in 3 L glass stirred tank bioreactors using proprietary media and nutrient feeds with an initial working volume of 1.5 L as previously described (Handlogten, Zhu, & Ahuja, 2017). The fed-batch processes were run in 3 L glass stirred tank bioreactors using proprietary media and nutrient feeds with an initial working volume of 1.5 L as previously described (Handlogten, Zhu, & Ahuja, 2017).…”