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.