NADPH concentration in different stage of cell culture
The CHO cell fed-batch cultures of three molecules, molecule 8, molecule
9, molecule 10, were sampled on Day 9, Day 11 and Day 14. The cell
pellets of the low-speed centrifugation (800 x g for 10 min at room
temperature) were collected and re-suspended to their initial cell
density. Similar to the above, the cell lysates were generated. Their
NADPH concentrations were measured and the results were shown in Figure
6. The NADPH concentration in all the three different cells varied with
cell culture stage largely. The concentration was the highest on Day 11.
The sharp increase in the intracellular ratio GSH/GSSG from day 7
onwards of CHO cultures was reported by Ruaudel et al., 2015. The both
observations suggested the intracellular redox potential was not
constant but changed with cell culture stages. Figure 7 showed the NADPH
concentration in the cell lysate of the seed, the cell lysate at
harvest, and the supernatant of the CCF for molecules 1. The NADPH
concentration in the cell lysate of the seed was about five times of the
cell lysate at harvest. The NADPH concentration in the supernatant of
the CCF was below the quantification limit of the test method. Both
Figure 6 and Figure 7 suggested that the NADPH concentration in the
cells varied largely with cell culture stages. The minimization of cell
lysis during its harvest process could minimize the release of NADPH
into its HCCF so that the risk of disulfide bond reduction could be
reduced at least if it can’t be prevented completely.
The summarization of the above results leads to the following
conclusions. The reducing agents such as NADPH varied largely not only
with cell clones but also with growth stages. NADPH presented both in
intact cells and in the cell culture supernatants. But the quantities of
NADPH in the intact cells were far more than in the cell culture
supernatants. Besides cell clones, the growth stage and harvest process
could affect the NADPH concentration in HCCF largely. The reduction
power of HCCF is correlated with its NADPH concentration better than its
TrxR activity. Another key factor for a reduction occurrence is
recombinant molecule itself. It has been reported in the literature
(Hutterer et al., 2013) and observed in this study (data not shown here)
that the reduction sensitivities of recombinant molecules varied
largely. The reduction sensitivity of a given molecule shall not vary
with cell culture conditions, growth stages, and harvest processes and
can be quantified prior to its manufacturing run. However, the level of
the reducing agents such as NADPH in HCCF varies largely not only with
cell clones but also might vary with cell culture conditions, growth
stages, and harvest processes. For each given HCCF, its reduction power
needs to be measured individually.