Conclusions
The reduction power of HCCF varied largely with cell clones, cell culture conditions, growth stages, and harvest processes. The harvest processes and cell clones showed strong effects on the NADPH concentrations but not on the TrxR activities in HCCF. The HADPH concentration is correlated with the reduction power of HCCF instead of the TrxR activity. NADPH was found both in intact cells and in the supernatant of CCF. The amount of NADPH in intact cells was much more than that in the supernatant of CCF. The method and operation condition of a clarification affected the level of cell lysis in harvest processes and impacted the release of NADPH and reducing agents in HCCF. The amount of NADPH per cell varied largely with the stage of a cell culture. Due to multiple influencing factors and their complexities, it is hard to predict the reduction power of HCCF quantitatively. Moreover, large differences in the reduction sensitivities of recombinant molecules existed as well.
The method for the quantitative risk assessment of disulfide bond reduction was developed. The cell lysate at different dilutions was used to evaluate the reduction sensitivity of recombinant proteins. NADPH concentration and DCPIP decolor rate were used to quantify the reduction power of HCCF and the reduction sensitivity of recombinant proteins. The difference between the reduction power of HCCF and the reduction sensitivity of recombinant protein determined whether the reduction might occur.