4.2 Physical factors.
Environment pH and temperature are two best known factors that influence enzyme reaction rate. As a consequence, mAb disulfide bond reduction level is higher when cell culture pH is closer to neutral pH since the NADPH oxidization rate was higher under a neutral pH condition (pH 7.0) than acidic pH condition (e.g. pH 6.5) (A. Holmgren, 1979; P. Xie et al., 2016). In addition, Low temperature could also slow the enzyme reaction rate and eventually reduced the disulfide bond reduction level (Hutterer et al., 2013; H. Liu, Nowak, Shao, Ponniah, & Neill, 2016; O’Mara et al., 2019). Furthermore, short HCCF storage time can eliminate the disulfide bond reduction level as the reaction time was reduced (Mun et al., 2015; O’Mara et al., 2019). However, it should be noted that if the storage time is long enough, the reduced mAb can re-oxidize and form intact mAb in HCCF as the enzymes lose their activities (Mun et al., 2015).