What Experts Are Thinking
Several talks at Select Biosciences’ “Cell Culture” conference will shed light on current modes of thinking among upstream specialists.
Matthew Croughan, Ph.D., professor at the Keck Graduate Institute, will focus on new methods to control cell metabolism and advanced mass transfer strategies to achieve high titers and cell densities.
The need to control metabolism arises from the successes of modern mammalian cell culture: As cell densities increase, so do concentrations of waste products like lactic acid and ammonia. Dr. Croughan is looking at four techniques to reduce the waste burden in fed-batch cultures.
The first, reducing lactic acid production through feedback control of glucose, is based upon careful monitoring of “pH trajectories.” This approach, developed at Pfizer and known as HIPDOG (high-end pH-controlled delivery of glucose), is based on the fact that cells consume lactic acid when glucose levels fall below about 1 mM.
As acid levels fall, pH rises. Researchers devised a feeding method that delivers concentrated glucose solution, in response to rising pH, which reduces lactate accumulation during the cells’ growth phase.
The next technique, a patent-pending method invented by Dr. Croughan and Nate Freund, a former student, reduces lactic acid and ammonia production by adapting cells to high (40 mM) lactate.
“The high lactate concentration pushes the equilibrium toward pyruvate, away from lactate production,” he says. “In fact, if lactic acid concentration is high enough the cells will consume it.”
The other approaches involve reducing lactic acid production by transfecting cells with anti-apoptosis genes and controlling cell metabolism through “advanced flux modeling” and medium optimization. The material in this article related to Dr. Betenbaugh describes the anti-apoptosis approach.
The last technique was developed by Bernard Palsson, Ph.D., professor at the University of California, San Diego, and commercialized by GT Life Sciences, a division of Intrexon.
All well and good, but can these techniques be used together to create super-cells and super-cultures? Dr. Croughan is cautiously optimistic.
“They possess common components, but some overlap. It may be possible to combine flux modeling with anti-apoptosis strategies, lactate supplementation and adaptation, or HIPDOG.”
Interestingly, some of these techniques are being applied to transiently transfected cells, which currently produce at much lower levels than stably transfected lines. Transient transfections are normally conducted at research or investigational scale, but the extra productivity cannot hurt.