Researchers need to tweak their entire process to maximize the yield of difficult-to-express proteins. That’s the view of Martin Bertschinger, Ph.D., director of drug substance development at IGI (previously Ichnos Sciences), who last year published research showing yields up to 10 grams per liter of a multi-specific antibody.
According to Bertschinger, high-performance manufacturing of harder-to-express proteins requires bioprocessors to consciously choose which issues can be overcome and which need to be worked around. “During the molecule design, there are some things you need to accept because they’re part of the molecule’s characteristics and other things that you can productively tweak,” he explains.
For example, some features of a protein may be convenient for development but cause problems at the manufacturing stage.
“There were specific mutations that were helpful for multi-specific antibody assembly, but made the molecule harder to make,” he says. “But we were able to live with it by identifying a strong promoter to improve expression levels.”
IGI begins improving protein expression at the molecular design stage, he continues by, for example, changing signal peptides, codon usage, and the ratio of plasmids used to transfect cells to balance the subunits forming the multi-specific antibody.
Adjust cell-line development
Next, his team adjusted their cell-line development to be as cell-friendly as possible. This avoided the cell-line selection step being biased towards cells that can survive harsh conditions and away from maximally producing cells.
“We needed a smart screening process,” notes Bertschinger. “We screened our cells under different conditions and what worked for us, as well, was a temperature shift, as lowering the temperature also helped to improve expression.” Lower temperatures are known to improve the cell’s ability to secrete correctly folded proteins.
In combination with a high seeding density process, the team was able to increase titers of correctly folded multi-specific antibody to more than ten grams per litre, with downstream yields in the range of normal mAbs (70%).
Bertschinger, who will speak at Bioprocessing Summit Europe next week, told GEN that since the publication of the team’s paper, the company has now developed a proprietary semi-directed method for integrating DNA into genomic locations in manufacturing cell lines favourable for antibody expression. The PiggyBac transposon-based molecular platform leads to higher and more stable gene expression than their previous technique, he says, adding that IGI has used the new platform to yield more than fifteen grams per litre of trastuzumab.
“We were able to reach yields at the pool stage that we couldn’t achieve previously with a random integration approach. This is particularly valuable for overcoming expression limitations of difficult-to-express proteins,” points out Bertschinger.