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.