TrxR activity and NADPH concentration at different harvest conditions
TrxR and NADPH were reported to be the primary contributor for the disulfide bond reduction of a recombinant protein manufacturing (Koterba et al, 2012; Kao et al., 2010). To study them, TrxR activity and NADPH concentration were measured at different harvest conditions. Figure 1 shows the TrxR activities in the supernatant of the low-speed centrifugation (800 x g for 10min at room temperature), the filtrate of the low-pressure depth filtration (<0.8 bar), the filtrate of the high-pressure depth filtration (> 0.8 bar), and the post-filtration flush of CHO cell culture of molecule 1, respectively. As shown, the TrxR activities presented in all of them except the post-filtration flush. The TrxR activity was the lowest in the low-speed centrifugation supernatant, the middle in the filtrate of low-pressure depth filtration, and the highest in the filtrate of the high-pressure depth filtration. The differences between them were limited. The TrxR activity in the flush was below the quantification limit of the used test method. Since the low-speed centrifugation would not cause cell lysis, the TrxR in the low-speed centrifugation supernatant would attribute to the accumulation of TrxR released during the cell culture process instead of its harvest. The post-filtration flush didn’t cause a practical release of TrxR into the flush sample.
For the CCF of molecule 2, the depth filtration was conducted using Millipore’s D0HC at the feed flux of 50-60 LMH. The filtration pressure was monitored. The three fractions, 0.4-0.6 bar, >0.8bar, and flush, were collected. Additionally, the cell lysate was made by following the preparation method of cell lysate described above. Figure 2 and 3 show the TrxR activities and NADPH concentration in the different fractions of its depth filtration harvest process, respectively. As shown in Figure 2, the TrxR activities were 1100 mU/ml for the >0.8 bar fraction, 1050 mU/ml for the 0.4-0.6 bar fraction, 300 mU/ml for the flush, and 680 mU/ml for the cell lysate. The >0.8 bar fraction had the highest TrxR activities, followed by the 0.4-0.6bar fraction, the cell lysate, and the flush. The NADPH concentration shown in Figure 3 was 0.4 µM for the 0.4-0.6 bar fraction, 1.05 µM for the >0.8 bar fraction, 0.5 µM for the flush, and 2.0 µM for the cell lysate. The observation suggested that the NADPH accumulated during the cell culture process was limited, the high-pressure filtration could increase the release of NADPH largely, the flush did contain NADPH at a comparable level as the 0.4-0.6 bar fraction, but the intact cells contained NADPH at the highest level among them.