Incorporating Next-Generation Sequencing to Accelerate Cell Bank Release with Mitigated Risks
Biosafety of the cell banks is another important concern of the accelerated CMC strategy. Certain biosafety tests are required to control the risks before any cell banks are allowed to enter the GMP facilities. In a traditional CMC development, mycoplasma, typical viruses, retroviruses, and adventitious viruses are tested separately by more than ten different complicated assays. Even though not all the tests should be passed to release a cell bank into the GMP facility, mycoplasma, sterility, and 28-day in vitro virus testing are required to mitigate the risks of contamination, regardless. Although it takes only one or two weeks for relatively straightforward assays to conclude, some cellular assays, such as in vitro virus testing (IVV), could take several weeks to complete, which are too long to incorporate into an aggressively accelerated CMC strategy. On the other hand, given the current environmental control and extensive historical experience, the probability of biosafety failure is nearly negligible, enabling us to think about replacement tests for precautions.
To overcome this safety vs . time-efficiency dilemma, we performed an IVV test with the top 10 clone cell mixture and developed an NGS-based virus detection method (Khan et al., 2016 & 2018; Brussel et al., 2019). For the proof of concept, we mixed serial proportions of mAb-expressing cells with the host cells, wherein we measured the RNA levels of the mAb. As shown in Table 1, the expression of mAb could still be detected when the sample contains only 1% mAb expressing cells. Precedents are indicating that the virus will not grow at a composition of lower than 0.01 virus/cell (Richards et al., 2014; Wang, Feng and Duncan, 2014; Plavsic et al., 2016). Technically, we believe that the viral RNA can still be effectively detected even when the sample contains very low copies of the virus. For virus detection, the total RNA of the cell banks is analyzed by NGS and aligned to all available sequences in Reference Viral DataBase (RVDB). The virus contamination would be pronounced if the sequence coverage of any virus is higher than 50%. This test can be considered as a precautionary test for early warnings, granting preliminary but sufficient support to permit the cell banks to enter the GMP facilities given the extremely low risks. However, all of the biosafety tests should still be performed in parallel to authenticate the NGS-based results, which is necessitated by the generation of subsequent working cell banks (WCB) and cGMP manufacturing. In short, cell banks can enter GMP facilities with NGS supported warrants with a turnaround time as short as within 1 week, which is also accredited to our refined algorisms.