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).