This case-study describes a sudden, unexpected loss of early post-inoculation viability paired with reduced cell growth in suspension CHO cell cultures inside 3-L glass sparged bioreactors, which occurred when switching to new serum and hydrolysate-free chemically defined media (CDM) lots. While in historical runs, performed with the same cell bank and older CDM lots, the viability stayed within 10% viability drop within 24 hours post-inoculation. The poor viability partially recovered 3-4 days later, however viable cell density (VCD) profiles remained 50-60% lower than historical runs. An investigation was undertaken to identify the sources of variability. First, we tested for chemical/biological contaminants, such as cleaning procedures, gas source/quality, compromised bioreactor parts and mycoplasma contamination, which were all ruled out. Second, we tested a gradient of gas sparging rates and observed that early post-inoculation cell death increased with the gas flow rate. While that was not surprising since bubbles are known to cause cell damage, this effect was never observed at the high sparging rate that was used historically. Third, because hydrodynamic damage appeared to be responsible for cell death, we tested the addition of increased levels of the surfactant pluronic F-68 (PF68) to the new CDM lot, which reduces cell-to-bubble attachment and lowers dynamic surface tension. This restored the VCD and viability profiles. However, measurement of the dynamic surface tension drop in CDM lots used historically and in the problematic runs did not reveal any differences, suggesting that the reduced viability could not be attributed to surface tension differences. Fourth, upon PF68 supplementation to the new CDM lot, we observed a metabolic shift resulting in decreased lactate consumption. In summary, our observations indicate that differences between lots of the same CDM can lead to undesirable variability.

Investigation of an early loss of cell culture viability in sparged bioreactors points to chemically defined media lot variability

247th National Meeting (2014)

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