Screening for Antibody Reduction at the Clone Selection Stage to Prevent Reduction During Manufacturing

Therapeutic monoclonal antibodies (mAbs) occupy a large part of the biopharmaceutical market; over 50 new mAbs are presently undergoing late-stage clinical trials, including human and humanized IgG1, 2 and 4 molecules, bispecifics, antibody-drug conjugates, and peptide-mAb fusion proteins.1 One of the associated challenges with the production of therapeutic mAbs is the reduction of the interchain disulfide bonds, which causes a loss of product, increased manufacturing complexity, and reduced drug product stability.5 Antibody reduction seems to be unpredictable and is typically not observed until the initial scale-up run. If left uncontrolled, reduction will be observed in all subsequent large-scale runs due to cell lysis caused by shear forces used to separate the supernatant from the cells during continuous centrifuge harvest operations. Small-scale centrifugation uses bench top centrifuges, which are more gentle than those used in the scale-up runs where reduction is most often observed. Cell lysis releases intracellular components, including reductases from the thioredoxin and glutathione enzymatic systems which have been shown to act on the antibody and reduce disulfide bonds.5 While we observe reduction in the large-scale manufacturing processes, small-scale reactors do not typically indicate disulfide bond reduction unless we artificially lyse the cells using a freeze-thaw method or a capillary shear device.16 However, freeze-thaw of the harvested cell culture fluid (HCCF), long-term storage at 5°C of the HCCF, or oxygen exposure from air surface transfer may cause the reducing components to lose activity. We investigated methods to propose a reduction assay to either identify clones that are unlikely to result in antibody reduction, or offer reduction mitigation strategies prior to the first scale-up run if a clone is likely to have reduced product. Thus, loss of product and delays to the project timeline can be prevented. Prior research suggests a correlation between reductase activity and antibody reduction. We continued this research and focused on identifying a correlation between antibody reduction and either the levels of expression or activities of the enzymes of the thioredoxin and glutathione systems. However, we did not find a correlation between either reductase expression or reductase activity and mAb reduction. NADPH is a cofactor for the glutathione reductase (GR) and the thioredoxin reductase (TrxR) and the total amount or ratio of NADP+/NADPH may correlate with mAb reduction but neither was quantified as part of this work. Interestingly, we found correlations between reductase expression and viability, VCD, titer, percent product aggregates, lactate, and pCO2. Additionally, we found a correlation between reductase activity and percent product aggregates. This data suggests that the intracellular redox environment is important for both cell growth and product quality. Future studies should focus on screening top clones from projects that do and do not have a history of reduction to determine variability in reductase expression and activity. Future studies should also look at NADPH

Screening for Antibody Reduction at the Clone Selection Stage to Prevent Reduction During Manufacturing

Therapeutic monoclonal antibodies (mAbs) occupy a large part of the biopharmaceutical market; over 50 new mAbs are presently undergoing late-stage clinical trials, including human and humanized IgG1, 2 and 4 molecules, bispecifics, antibody-drug conjugates, and peptide-mAb fusion proteins.1 One of the associated challenges with the production of therapeutic mAbs is the reduction of the interchain disulfide bonds, which causes a loss of product, increased manufacturing complexity, and reduced drug product stability.5 Antibody reduction seems to be unpredictable and is typically not observed until the initial scale-up run. If left uncontrolled, reduction will be observed in all subsequent large-scale runs due to cell lysis caused by shear forces used to separate the supernatant from the cells during continuous centrifuge harvest operations. Small-scale centrifugation uses bench top centrifuges, which are more gentle than those used in the scale-up runs where reduction is most often observed. Cell lysis releases intracellular components, including reductases from the thioredoxin and glutathione enzymatic systems which have been shown to act on the antibody and reduce disulfide bonds.5 While we observe reduction in the large-scale manufacturing processes, small-scale reactors do not typically indicate disulfide bond reduction unless we artificially lyse the cells using a freeze-thaw method or a capillary shear device.16 However, freeze-thaw of the harvested cell culture fluid (HCCF), long-term storage at 5°C of the HCCF, or oxygen exposure from air surface transfer may cause the reducing components to lose activity. We investigated methods to propose a reduction assay to either identify clones that are unlikely to result in antibody reduction, or offer reduction mitigation strategies prior to the first scale-up run if a clone is likely to have reduced product. Thus, loss of product and delays to the project timeline can be prevented. Prior research suggests a correlation between reductase activity and antibody reduction. We continued this research and focused on identifying a correlation between antibody reduction and either the levels of expression or activities of the enzymes of the thioredoxin and glutathione systems. However, we did not find a correlation between either reductase expression or reductase activity and mAb reduction. NADPH is a cofactor for the glutathione reductase (GR) and the thioredoxin reductase (TrxR) and the total amount or ratio of NADP+/NADPH may correlate with mAb reduction but neither was quantified as part of this work. Interestingly, we found correlations between reductase expression and viability, VCD, titer, percent product aggregates, lactate, and pCO2. Additionally, we found a correlation between reductase activity and percent product aggregates. This data suggests that the intracellular redox environment is important for both cell growth and product quality. Future studies should focus on screening top clones from projects that do and do not have a history of reduction to determine variability in reductase expression and activity. Future studies should also look at NADPH