NADPH indicator application case
Generated the cell lysate for molecule 2 prior to its manufacturing, determined the NADPH concentration in the cell lysate. Diluted the cell lysate in a series based on NADPH concentration. Spiked the diluted cell lysates with purified molecule 2 to a final concentration of 1 mg/mL. Splited each diluted and spiked cell lysate into two portions. One series was overlaid with nitrogen and the another was sparged with air. Incubated the two series of the cell lysates at room temperature for 12h. The incubated samples were tested on reduction occurrence with non-reduced SDS PAGE. The results were given in Table 3 and Table 4. The reduction under nitrogen overlay occurred when the NADPH concentration was ≥0.5 µM while the reduction under air sparging only occurred when the NADPH concentration was ≥1.25 µM. 0.5 µM and 1.25 µM of NADPH are corresponding to 25% and 100% of cell lysis, respectively. In the GMP manufacturing of molecule 2, the HCCF was sampled and tested. Its NADPH concentration was 0.4 µM, which was close to 0.5 µM. The reduction risk was considered to be moderate. The air sparging was applied as a prevention measure. The manufacturing went successfully without reduction.