Where Rising Titers Are Welcome
Dr. Gottschalk acknowledges that many manufacturers scoff at the idea that precipitation can produce biopharmaceuticals safely and at high yield. Yet a number of companies that have tried precipitation, including Genentech, Pfizer, Biogen Idec, and Amgen, have presented data on processes that are “competitive with protein A capture.”
Precipitation is one area where rising protein titers can actually help. Years ago, when proteins were expressed at 100 mg/L, precipitation with polyethylene glycol required near-saturation conditions in the polyol (19%), which generated tons of waste for a 30,000 L bioreactor. Today’s cell cultures produce a hundred times more protein on a volumetric basis, and therefore, require much less of whatever reagent is used to precipitate the protein.
Implementing precipitation will require smarter processes and approaches, e.g., polyelectrolytes that efficiently induce precipitation of either product or impurities. “Precipitation techniques are much more advanced than when I joined the industry, when we were looking to replace precipitation with chromatography,” Dr. Gottschalk remarks.
Georges Belfort, Ph.D., professor at Rensselaer Polytechnic Institute, confirms bioprocessors’ love-hate relationship with protein A capture for monoclonal antibodies. Dr. Belfort, who cofounded the North American Membrane Society, reports that you can’t go to a bioprocessing meeting without hearing a litany of complaints about protein A—that it leaches into solution, is too expensive, and so on. But everyone recognizes that it works really well.”
In a recent paper in Biotechnology and Bioengineering, Dr. Belfort described a technique for selectively precipitating immunoglobulin G from an albumin-rich bovine serum using ammonium sulfate. Selectivity of this technique for IgG over BSA was 36, resulting in removal of 97% of the albumin and 93% purity for the antibody. The BSA solution was eliminated by filtration through a large-pore membrane.
Dr. Belfort’s demonstration solves the classic problem of removing IgGs from BSA in solution. Even though the two molecules differ significantly in molecular weight (155 kD for IgG vs. 69 kD for BSA), interactions between the two molecules in the filtration medium prevent separation by membranes alone. Dr. Belfort believes that the technique would need to be modified for separations downstream of cell culture, but it could work. He cites several recent publications and presentations by leading biotechnology companies as proof that the industry is seriously considering alternatives to protein A capture.
Alahari Arunakumari, Ph.D., senior director for process development at Medarex, is equally wary of protein A, but takes a different tack from Drs. Gottschalk and Belfort. Protein A’s dominance in mAb manufacture, according to Dr. Arunakumari, is based more on custom than science. “Low antibody titers of less than one gram per liter once justified the use of protein A capture. Today, with titers commonly greater than 10 g/L, we need a higher-binding, cheaper resin to reduce the cost of goods, keep column sizes manageable, make purification processes more efficient, and reduce—but not eliminate—downstream bottlenecks.
Dr. Arunakumari’s capture resin of choice, cation exchange, provides both efficiency and economy. For example, antibodies bind to protein A at between 30 and 50 mg/mL of resin, and to cation exchangers at up to 160 mg/mL, for a fivefold improvement. Factor in the cost differential—Dr. Arunakumari estimated this at three- to fivefold—and cation exchange provides as much as 25 times the benefit per volume per dollar cost.
“It’s a matter of mathematics. We can reduce the number of cycles and process larger masses at significantly less cost.” The savings in time, buffer, and human resources are substantial.
Medarex has developed more than a dozen antibodies at clinical and preclinical stages using cation exchange capture. Three of these products have been transferred to contract manufacturers in protein A-less format.