Producing high yields of pure, high-quality proteins remains a goal of many academic and industrial labs. Their efforts can be thwarted by problems ranging from incomplete control over cells’ growth conditions to inadequate post-translational modifications. However, progress is being made on many fronts.
Bioreactors with rocker platforms, which literally rock the cell cultures back and forth, were introduced about a decade ago and have since become widely accepted for the development of seed cultures, says Loe Hubbard, applications manager of Pall’s cell culture systems. However, “because you are working with rocker shaking, the oxygen transfer that you’re able to achieve isn’t really high enough to do actual production for most processes.”
To address this limitation, “we’ve added an extra dimension to the shaking,” Hubbard notes. “Instead of just rocking back and forth, the XRS 20 Bioreactor also rocks side to side, so you almost have a hula motion to the platform.”
Two-dimensional rocking appears superior to one-dimensional rocking in several respects, Hubbard said. Thanks to the augmented oxygen transfer, higher cell densities can be supported. In addition, “We see less cell debris at the end of the run,” she says. “We think that the additional dimension gives a tumbling characteristic to the flow, so that the cells stay nice and round and are not getting pulled apart by extensional shear.”
Another innovation in bioreactor design—also exemplified by the XRS 20, made by Pall in conjunction with New Brunswick Scientific—is the transition to disposable plastic bioreactor vessels.
Though this trend might not sound environmentally friendly, “the people who are doing the calculations are showing that it’s more green overall,” says Hubbard. “The amount of energy that’s being used and the kinds of chemicals that are being used to sterilize and clean bioreactors after each use add up to much larger waste and energy usage than incinerating the bioreactors at the end.”
“It’s really counterintuitive, but it’s a really exciting thing,” Hubbard concludes. “You could get to where you could run a process in a conference room. You don’t need the infrastructure; you just plug it in and go.”
ArrayXpress’ general strategy is to study cell cultures by collecting samples at multiple time points, performing RNA-Seq to quantify gene expression, mapping the expression data onto biochemical pathways to see how the cells are responding to the task of making a recombinant protein, and analyzing this data at the pathway level to determine what is limiting the production of the protein.
As an illustration of this strategy, Len van Zyl, Ph.D., CEO, gives the example of a pharmaceutical company that was manufacturing a commercial therapeutic protein in E. coli, but was only achieving about half of the expected yield. To diagnose the problem, ArrayXpress grew the production strain alongside a control strain (which contained an expression vector lacking the open reading frame for the target protein) and compared their expression patterns after 11, 18, 24, and 28 hours of growth.
“Based on this, we could very quickly pinpoint at which hour there was a real issue, and what the issue was,” says Dr. van Zyl. “What we could see from this experiment was that there was a very significant shutdown of purine metabolism in the production strain.”
Mining of the peer-reviewed literature then led to the hypothesis that excess adenine in the medium might be causing the formation of PurR-hypoxanthine complexes, which sensitize the cells to growth arrest by adenosine and inosine, thus reducing protein yields.
This hypothesis, in turn, led ArrayXpress to propose several chemical and genetic means of correcting the imbalance in nucleotide metabolism. One possible solution, adding guanosine to the media, improved the yield by over 150%.