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.