Reduction risk assessment for a given manufacturing
The reduction power of HCCF and the reduction sensitivity of a recombinant protein are the two determining factors for a reduction occurrence. If the power and the sensitivity can be quantified using the same indicator, their comparison will be straightforward and the difference between them will tell the likelihood of a reduction occurrence for a given manufacturing. The following reduction risk assessment approach is formulated.
The quantification of the reduction power of HCCF: The reduction power of HCCF is correlated with the levels of its NADPH, reducing agents, and cell lysis. The NADPH can be quantified by using its specific kits. The level of the reducing agents can be measured by using DCPIP or other appropriate redox sensitive dyes and indicators. DCPIP is a weak oxidant and its aqueous solution has a blue color, but decolors if being reduced. The higher the reducing agent level is, the faster the decoloring rate will be. The NADPH concentration and the DCPIP decolor rate are correlated with the cell lysis level. The reduction power of a HCCF can be quantified either by the concentration of NADPH or the OD of decolored DCPIP.
The quantification of the reduction sensitivity of a recombinant protein: Dilute the cell lysate of a given molecule in a series for example 100%, 80%, 60%, 40%, and 20%, determine their NADPH concentration and their OD of decolored DCPIP, spike the diluted samples with the purified target molecule to a final concentration of 1 mg/mL, overlay the spiked samples with nitrogen and incubate them for 12h at room temperature, and analyze the obtained samples with non-reduced SDS PAGE. The results will tell whether the reduction has occurred and if occurred, what is the minimum concentration of NADPH and the maximum OD of decolored DCPIP for the reduction occurrence of the tested molecule. The reduction sensitivity of the molecule can be quantified either by the minimum concentration of NADPH or by the maximum OD of decolored DCPIP for its reduction occurrence.
For a given manufacturing, the risk level of reduction occurrence is correlated with the difference between the reduction power of its HCCF and the reduction sensitivity of its recombinant protein. The difference can be quantified by using anyone of those two indicators (the concentration of NADPH and the OD of decolored DCPIP) as long as the same indicator is used for the quantifications. If the reduction power of the HCCF was lower than the reduction sensitivity of the recombinant protein, the reduction unlikely will occur. If the reduction power of the HCCF was higher than the reduction sensitivity of the recombinant protein, the reduction likely will occur without the implementation of a reduction prevention measure. The larger the difference is, the stronger the reduction measure would be needed for its reduction prevention.