Since designing ease of purification into molecules is difficult, bioprocessors will increasingly turn to nonmammalian expression systems with a downstream advantage such as fewer steps or improved product quality.
For example Ajinomoto’s Corynebacterium-based expression system, Corynex, reportedly reduces costs and speeds time to market by expressing recombinant proteins directly into broth. Corynex works with cytoplasmic proteins, growth factors, enzymes, and mAb fragments, but not with membrane proteins or entire antibodies.
The elimination of lysis minimizes host-cell proteins, DNA, and other contaminants, with an additional bonus. “Corynex allows
purification of properly folded proteins directly from the broth, with no inclusion-body processing,” says Joel White, business manager for biotechnology. “In the case of IGF-1, a protein with three disulfide bonds, purification requires half as many steps as conventional bacterial fermentation.” By eliminating junk and folding proteins correctly, losses are reduced to half of what one would expect with E. coli.
Purification involves centrifuging the cells away, ion-exchange or reverse-phase chromatography, followed by gel filtration or ultrafiltration. Eliminated are harvest, disruption, removal of cell debris, and refolding.
Insect cells are viewed as a simpler alternative to mammalian cell culture, perhaps providing a purification advantage as well, but problems still need to be ironed out.
In many respects insect cell culture is at the stage at which mammalian cultures were about a dozen years ago. Cell densities barely reach 2.5 million cells/mL, limited by the necessity that cells be in their logarithmic growth phase during transfection. Titers are below average for many established protein processes, and quite low for mAb processes. According to Manon Cox, Ph.D., president of Protein Sciences, one can expect “a couple of hundred milligrams per liter” from insect systems.
“But a lot of productivity is still to be gained as soon as the first product produced in insect cells is approved,” says Dr. Cox. “At that point, media development and feed strategies will take off,” perhaps providing some of the dramatic improvements we’ve seen for CHO cells.
Advantages of insect culture include proper protein folding and rapid, transient expression. Baculovirus-based products are more immunogenic than those from more conventional expression systems, which may be advantageous for vaccines but not for other products. Another minus is the presence of substantial quantities of baculovirus, which—even though they are not pathogenic to humans—must be cleared.
But perhaps the greatest drawback is regulatory uncertainty. “We filed our license application for an influenza vaccine three years ago,” Dr. Cox says, “and it is still not approved. All the problems are substrate related.”