Investigation of low viability in sparged bioreactor CHO cell cultures points to variability in the Pluronic F-68 shear protecting component of cell culture media

• Drop in cell viability and titers mitigated by supplying additional Pluronic F-68.
• Cell damage not due to decreased Pluronic F-68 concentration in media.
• Pluronic F-68 lot variability in lowering surface tension in cell culture media.
• Surface tension measurement can predict cell culture performance.

An unexpected 6–9% drop of viability in suspension CHO cell cultures 24–48 h post-inoculation in sparged bioreactors, paired with reduced overall cell growth by an average of 43%, occurred upon switching to new serum and hydrolysate-free chemically defined media (CDM) lots. An investigation was undertaken to identify sources of variability. First, chemical/biological contaminants, including cleaning procedures, gas source/quality, compromised bioreactor parts and mycoplasma contamination, were ruled out. Second, we tested a gradient of sparging rates and observed that early post-inoculation cell death increased with the gas flow rate. While bubbles are known to cause cell damage, this effect was not previously observed at the high sparging rate used. Third, because hydrodynamic damage appeared to be responsible for cell death, we tested the addition of increased levels of surfactant Pluronic F-68 (PF-68), which reduces cell-to-bubble attachment and lowers dynamic surface tension, to the new CDM lots. This restored the viable cell density and viability profiles. Measurement of the dynamic surface tension of the CDM lots employed in this study indicated variability up to 8% between CDM lots supplemented with different PF-68 lots. In summary, our observations suggest that the raw material quality of PF-68 lots can alter the ability to lower the surface tension in chemically-defined cell culture media and significantly impact performance of CHO cell cultures in sparged bioreactors.