6.3.3 Reduce the amount of enzyme present
Disulfide bond reduction can also be minimized if there is less enzyme available to catalyze the disulfide bond redox reaction. One approach is to introduce components that can react with the enzyme as competing reaction pathways to consume the enzyme so it becomes unavailable for disulfide bond reduction. As discussed in root-cause analysis section, O2 can react with NADPH; thus improving DO levels in HCCF can minimize disulfide bond reduction (Handlogten, Wang, & Ahuja, 2020; Mun et al., 2015). For instance, researchers found that keeping a minimum 30% DO level by air sparging can prevent disulfide bond reduction and maintain higher than 90% intact mAb. Besides O2, H2O2 and L-cystine with optimal concentration can also react with enzyme and reduce the available enzyme for disulfide bond reduction. Consequently, researchers induced micro-molar level H2O2 and L-cystine to the HCCF to eliminate disulfide bond reduction (Chung et al., 2017; Du et al., 2018). Another approach is to minimize cell lysis during CCF harvest step, consequently there will be less enzyme secreted into CCF. This can be achieved by minimizing cell shear force in harvest step, such as using a hermetic style centrifuge (Trexler-Schmidt et al., 2010), or controlling the depth filtration differential pressure lower than certain values (O’Mara et al., 2019).