DCPIP indicator application case
Generated the cell lysate for molecule 6 prior to its manufacturing. Diluted the cell lysate in a series of 100%, 75%, 50%, 30%, 25%, 20%. Putted 199.2 µL of each diluted cell lysate on 96-well plate. Spiked 0.8 µL of DCPIP stock solution of 10 mg/mL into each diluted cell lysate to a final concentration of 0.04 mg/mL. After incubated at room temperature for 10 min, the OD at 600 nm of both the series and the control were measured. The results were given in Table 5. As seen, the OD at 600nm increased with the dilution factor. The less the dilution was, the lower the OD at 600 nm was.
Putted each diluted cell lysate into a vial. Spiked the purified molecule 6 into each diluted cell lysate to a final concentration of 1 mg/mL. Displaced air in the head space of each vial with nitrogen. After incubated at room temperature for 12h, the samples were tested on reduction occurrence with non-reduced SDS PAGE. The results were given in Table 6. For molecule 6, the reduction occurred when the cell lysis level reached 75% and above. According to Table 5, the OD of 0.381 was correlated with 75% cell lysis. In the GMP manufacturing of molecule 6, the HCCF was sampled and tested using DCPIP. Its OD at 600 nm was 1.14 which was much higher than 0.381, the reduction sensitivity of molecule 6. The reduction risk was considered to be low. No prevention measure was taken. The manufacturing went successfully without reduction.
In the above two cases, the concentration of the purified recombinant molecules used was > 10 mg/mL. The target concentration of recombinant molecules in a spiked series of cell lysates was 1 mg/mL. The spiking of recombinant molecules should dilute the corresponding cell lysates somewhat. For simplicity, such a dilution effect was neglected as long as the dilution factor was less than 10% or the purified recombinant molecules was more than 10 times concentrated than the target one.