Biological Basis for Media Development
Optimizing cell culture processes is a balancing act, where the more that is known and controllable, the greater the opportunity to create a more predictable process. Among the critical variables are gas entrance kinetic energy and vessel liquid height.
Gas entrance kinetic energy is determined by the velocity of gas entering the bioreactor. All things being equal, it would seem that sparging at a higher gas volume per process volume over time would result in higher gas kinetic energy. Not so.
“Higher flow is only part of the issue,” says Christopher Brau, associate engineer for bioprocess production at Thermo Fisher Scientific. While kinetic energy may be directly increased and decreased in existing systems by raising or lowering the volume of gas delivered, in new systems one has the option of changing the size and quantity of the pores through which gas passes. Pore size, notes Brau, directly affects not only the velocity at which a given gas flows, but also how fast the velocity will increase or decrease relative to pore size.
Brau studied systems in which the velocity or kinetic energy of the gas entering the vessel was maintained below thresholds determined to create more uniform behavior. “Excessive kinetic energy in a sparge system risks damage to cells in addition to generating a wide bell curve of bubble sizes which has its own host of potential issues,” he explains.
Similarly, vessel liquid column height and mixer flow pattern determine the total area available for mass transfer between liquid bulk and sparged gas. When bioprocessors do not account for these factors and the same sparge design is used despite increasing liquid columns, the total area available for mass transfer skews the behavior of a sparge system, potentially quite unfavorably. “This often manifests as a combination of CO2 buildup, a gas gradient in the column, excess foam, high holdup volume, and higher cell entrainment in the foam layer, while reducing the uniformity of scale up behavior between vessels,” Brau elaborates.
Brau’s work holds special significance for kLa, the mass transfer coefficient (kL) multiplied by the area (a) available for mass transfer based on a simplified gas liquid film theory equation. Here, kLa is a combined term describing relative mass transfer efficiency for a given set of operating conditions.
Brau measured kLa for both O2 delivery and CO2 stripping. “The significance here compared to most of industry is that we look at and consider the tradeoff between the two processes, as well as the limitations that govern and drive the behavior in the first place,” Brau says.
An excessively efficient sparge will achieve the maximum possible oxygen delivery, but is likely to generate excessive foam and ensure CO2 buildup. “That is why you have to account for the ratio of performance a sparge delivers under given operating conditions, specifically the ratio of O2 delivery and CO2 stripping,” Brau explains. Going too far toward CO2 stripping causes the overall sparging efficiency to suffer.
“You have to strike a perfect balance to achieve an O2 delivery and CO2 stripping kLa that is harmonious for your process conditions,” concludes Brau. “In other words, [this is about] managing the quantity of O2 consumed and CO2 produced. Striking this balance means you are maintaining your cell culture in ideal conditions with [the smallest possible] gas flow rates, relatively speaking.”