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.