July 1, 2009 (Vol. 29, No. 13)

Angelo DePalma Ph.D. Writer GEN

Technological Hoopla Aside, Simplification Is the One Trend on which Everyone Can Agree

Like all areas of bioprocessing, downstream purification has its own favored buzzwords—titers, bottlenecks, capacity, and others—meant to evoke a particular response depending on who is speaking or writing them. But, if we could just identify one leading, universal trend in downstream bioprocessing, it would be simplification.

Processes are becoming more streamlined with fewer steps, improved versatility, and rapid changeover between products. Disposable equipment plays a huge role in this evolution, particularly during steps that achieve several purification objectives simultaneously. For example, charged depth (lenticular) filters from several vendors now remove DNA and host-cell proteins as well as cellular debris.

According to Uwe Gottschalk, Ph.D., group vp for purification at Sartorius Stedim Biotech, process intensification has become the leading driver for large-scale chromatography. New processes for monoclonal antibodies, he says, are trending toward utilization of just two columns where three or four columns might have been the norm. Processors generally achieve capture and volume reduction through the initial column, which is followed by a flow-through polishing step typically composed of an anion exchange column or membrane adsorber.

The Real Hurdle

Rising protein titers have been blamed for numerous downstream bottlenecks in monoclonal antibody processes, but according to Dr. Gottschalk, capture is the real hurdle in terms of time and direct and indirect costs.

The titer question revolves around the fact that for a given process volume upstream productivity is driven by cell density, while downstream separations are mass-driven. Put another way: upstream volumetric productivity rises with cell density—the limit has apparently not yet been reached—whereas, capture resin binding capacity is constant. A doubling of cell density produces twice the protein in the same volume, but capturing it requires twice the column volume, twice the buffer, etc.

Protein A capture for mAbs works exquisitely well, but its cost is three to ten times that of nonaffinity resins, which amounts to hundreds of millions of dollars for resin over the lifetime of a successful mAb product. In addition, the protein A ligand leaches into the process and must be quantified at the end.

“Bioprocessors rely too much on chromatography,” explains Dr. Gottschalk. “We are beginning to face facility-fit problems with high-titer processes. It’s not so much the size of the columns but of peripheral activities such as buffer prep, buffer hold, and utilities for column washing.”

Dr. Gottschalk envisions emerging bioprocesses that rely more on precipitation, extraction, or crystallization for initial capture of monoclonal antibodies and other product types. Of the three, he prefers precipitation for its robustness, followed by resolubilization and polishing through a series of media—membranes, perfusion, monoliths, and columns—to clear contaminants sequentially in one operation.

“Processes of the future might have several cartridges linked together, operated by the same pump, that removes all contaminants down to specified limits for each.”

The individual pieces already exist for such a purification train but engineering work needs to be done in the areas of connecting sequential steps, synchronizing flows among them, and the measurement of conductivity and/or pH, all in disposable format.

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.

Titers, Protein A Issues Overblown?

Eric Grund, Ph.D., senior director, biopharma applications GE Healthcare believes that the buzz over rising protein titers is over-blown. Titers have been rising steadily for a decade, he argues, and bioprocessors have had more than sufficient opportunity to respond with larger, or in many cases, more-efficient separations. Moreover, higher titers have been accompanied, generally speaking, with raw product material that is more concentrated and of higher-quality, which has enabled simpler downstream processing, for example, by cutting the number of purification steps.

Dr. Grund, also a steadfast supporter of protein A capture, believes that to propose  precipitation or cation exchange capture is nothing but an attempt to fix something that already works phenomenally well.

“It’s naïve to say protein A is too expensive, and therefore, must be replaced. Protein A does most of the purification work for mAb processes, providing a product that is 99% pure. Precipitation cannot come close to that and provide a safe product.”

Dr. Grund notes that the plasma protein industry, which traditionally has used precipitation to produce IgG and albumin, is moving more and more toward chromatography-based purifications because “that is what makes these products highly pure, reproducible, and safe.”

Moreover, he feels that protein A’s reliability as a platform technology is a huge advantage. “If you have 20 mAbs in your pipeline, you can purify them all without too much trouble.”

Manufacturers of blockbuster products, says Dr. Grund, will always look for cost advantages through lean and six sigma approaches, and through optimizing utilization of plant resources. But that is not justification for abandoning tried-and-true methodology that generates safe products. “Of course some monoclonals can probably be prepared using methods developed in the plasma products industry years ago, but I’m not certain you’ll get the same level of purity or yield as you will with protein A capture.”

According to GE Healthcare, the plasma protein industry is moving more toward chromatography-based purifications, because it helps ensure product purity, reproducibility, and safety.

Single-Use Still News

Disposable processing continues to provide versatility and ease-of-use for downstream operations. “Turning around existing purification equipment faster is critical to eliminating capacity constraints and bottlenecks,” says Paul Chapman, vp for downstream processing at Millipore. “Increasing the facility’s total throughput means not spending an inordinate amount of time on cleaning and validation steps.”

Recent advances extending the reach of disposables from upstream operations to clarification and chromatography, and even to viral filtration and final formulation, put upstream “on the road to realizing a fully disposable purification process, at least for the 2–3 kg scale.”

Millipore’s single-use product line reflects the versatility and scale of which Chapman speaks. The company’s Mobius FlexReady line combines single-use filters and assemblies with process-ready hardware platforms for media/buffer prep, clarification, tangential flow filtration (TFF), and virus filtration. FlexReady products allow users to install equipment, configure applications, and validate processes quickly from development-scale through small-scale commercial manufacturing, he adds.

Watching disposables make their way into relatively complex downstream unit operations is encouraging. Tangential flow (also called crossflow) filtration (TFF) is a preferred method for concentrating protein solutions, buffer exchanges, and fractionation by molecular weight. Where normal flow filtration directs fluids into the membrane under pressure, TFF works by pumping the process fluid across the membrane surface. Lower fouling in TFF comes at the cost of a recirculation loop.

Earlier this year Pall obtained the rights to a new type of TFF, single-pass TFF (SPTFF), from SPF Innovations (spfinnovations.com). Pall claims the technique provides high concentration factors and high recovery. And, while it is not yet available in fully disposable format, SPTFF caused considerable stir at its debut at “Interphex 2009”, according to the company.

SPTFF operates similarly to TFF, but instead of concentrating through one membrane, product is transferred to sequential membranes where it becomes progressively more concentrated. Thus SPTFF eliminates the recirculation loop, reduces mixing or foaming issues, and has lower hold-up volumes for easier recovery.

SPTFF allows for in-line processing and easier integration with other process steps, for example, chromatography and in-line concentration. Residence times are on the order of minutes vs. several hours for conventional TFF, which generates lower shear exposure and enables operation at elevated temperature to reduce viscosity, reports marketing director Ian Sellick. “SPTFF is a derivation of a technology that everybody uses, but in a more functionally simple format that saves significant costs.”

Dr. Grund goes so far as to call the deployment and integration of disposables the big downstream processing story over the past 12 months. “There has been a good deal of discussion recently on when fully disposable downstream equipment makes sense, and when stainless steel might be more appropriate.”

GE’s ReadyToProcess™ line of disposable equipment includes the Wave disposable bioprocess Cellbag™, packed ready to use chromatography columns, filters, membranes, tubing and connectors. Also in this product line are ÄKTA™ready preparative chromatography systems built for process scale-up and production for early clinical phases. These systems operate with ready-to-use, disposable flow paths.

ReadyToProcess is based on the emerging need among bioprocessors, at least at small and mid-sized scales, for agility and flexibility—plug and play is the term GE applies. Not all the equipment needs to be disposable. “You probably would not want to use all the components only once, but even where you don’t you can at least plug them in rather quickly,” Dr. Grund says.

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