Introduction
Recombinant proteins for human therapeutic use has increased significantly in the last 25 years. Disulfide bonds play an important role in maintaining and stabilizing the three-dimensional structure of those proteins (Dombkowski et al., 2014; Wang et al., 2015). Their reduction was observed now and then during a recombinant protein manufacturing (Trexler-Schmidt et al., 2010; Kao et al., 2010; Mullan et al., 2011). Such a reduction event was catastrophic as the product failed to meet the drug substance specifications and the bulk product was lost. The reduction has a substantial impact on the functional effects of a mAb, including variable effects on antigen binding and Fc function, with the potential to significantly impact mAb efficacy in vivo (Gurjar et al., 2019). The reduction mainly took place in the harvested cell culture fluids (HCCF). The release of intracellular reducing components was widely considered to be the cause of the observed reductions (Trexler-Schmidt et al., 2010; Kao et al., 2010). The release took place both in cell culture bioreactors and in the harvest process of cell culture fluids (CCF). However, the harvest process was widely considered to be major (Trexler-Schmidt et al.,2010; Kao et al., 2010). At the end of a production phase, the cell culture fluid was usually harvested either by disc stack centrifugation followed by depth filtration or by depth filtration only. The mechanical stresses from centrifugation and depth filtration were reported to cause cell lysis.
The disulfide bond reduction of monoclonal antibodies has been studied intensively. The thioredoxin reduction pathway, including thioredoxin reductase (TrxR) and NADPH, has been proposed as a primary contributor (Kao et al., 2010; Koterba et al., 2012). The monoclonal antibodies contain both intra chain disulfide bonds and inter chain disulfide bonds. The inter chain disulfide bonds are more susceptible to reduction than intra ones (Liu et al., 2012). For the two studied IgG1, the disulfide bonds between the light chain and heavy chain were more susceptible for reduction than those between two heavy chains. The upper disulfide bond of the two inter heavy chain disulfide bonds was more susceptible than the lower one (Liu et al., 2010). Reduction-susceptible disulfide bonds are frequently located at the surface of a protein (Wang et al., 2015). There were large differences in reduction sensitivities between different monoclonal antibodies (Hutterer et al.,2013; Wang et al., 2015). Moreover, the reduction power in CCF and HCCF could vary largely for different molecules and different processes as well (Hutterer et al., 2013). The reduction occurrence depends on molecule, cell culture process, and harvest process.
The various measures to prevent the disulfide reduction occurrence had been developed. Those measures included air sparging, air overlay, pH adjustment, cooling down, TrxR inhibitors and chemical additions. The added chemicals could be H2O2, EDTA, Cu2+,Hg2+, Zn2+, Co2+, Mn2+, and so on (Trexler-Schmidt et al.,2010; Chung et al., 2017; Mun et al., 2015; Chaderjian et al., 2005; Du et al, 2018). Among them, air sparging or air overlay is most often used due to its simplicity and minimal potential impact on product qualities (Mun et al., 2015). The implementation of the above measures in a recombinant protein manufacturing will inevitably increase its operation complexity somewhat and might impact its product qualities as well sometimes. A decrease in main peak and an increase in acidic peak were reported when air sparging was applied during a HCCF hold for a given monoclonal antibody (Mun et al., 2015). Such a decrease in main peak and an increase in acidic peak had been seen in our labs as well for some molecules (data not shown here). Besides that, the decrease in the filterability of HCCF was observed in our labs and at-scale manufacturing (data not shown here) when air sparging or air overlay was applied.
If air sparging only doesn’t work, the cooling HCCF down plus air sparging might be worth to be considered next. In the case that the cooling down plus air sparging was still not sufficient, the addition of a weak oxidant, such as Cu2+, might be a choice. Certainly such an addition would introduce additional complexity to a process. It is highly desirable to apply a reduction measure only if necessary and to apply no strong measure if a moderate one works. How to assess the level of the risk of disulfide bond reduction for a given process quantitatively? How to determine what measure is appropriate for a given process? This study reported a quantitative method to assess the risk of disulfide bond reduction for a given process. The outcome of the assessment could tell whether a reduction measure should be taken and what measure is appropriate if yes. Answer what measure (mild, moderate, or strong) shall be appropriate for a given process.