New Technologies Improve Biotech's Downstream Processing

New Technologies Improve Biotech's Downstream Processing Author: Rebecca Andrews, p. 24, 25. Over the last few years, the number of recombinant pharmaceutical products that have entered the regulatory fray to gain approval for marketing has grown rapidly, and is now in the hundreds. As biotechnology companies seek regulatory approval for their products, however, they also face the problem of producing these proteins in quantity, at an affordable cost. Downstream processing is the isolation and purification of a desired protein from the soup of bacterial or animal cells that produced it. This stage, so termed because it takes place "downstream" from the production process, is "the rate-limiting step in the release of biotech products," says Bob Kennedy, applications lab manager at Membrex Inc., a Fairfield, N.J., company that specializes in membrane filtration systems. "Science and economics meet head to head." Kennedy points out that the actual production of recombinant proteins typically takes place in fermentation tanks (when bacterial cells are used) or bioreactors (if mammalian cells are used), and usually can be scaled up to batches in the tens of thousands of gallons. "But purification systems are just scaled-up--or attempted scale-up of--laboratory tools, so they are relatively inefficient." These systems were originally designed for analytical tasks, using comparatively small amounts of material. The companies that supply the biotechnology industry with the tools needed to purify their proteins are perpetually trying to increase the speed, lower the cost, and improve the productivity of their wares. Biotech companies develop unique, highly proprietary methods for purifying their pet protein, so that they can bring it to market faster and at a lower cost than can their competitors. "A lot of time and effort and cost goes into methods development work," says Kennedy. "People spend man-years of time optimizing just [one or two components]." As part of approving a product, the Food and Drug Administration must approve the processing behind it, as well. And changing even a small part--such as the type of filter or chromatography media used at a particular stage--may necessitate reapplying for approval. Downstream processing actually consists of many steps, each one increasing the purity of the final product. The number of separ- ations--from three to as many as seven--varies from protein to protein, as do the technologies used to effect the separations. The most widely used methods for processing proteins are chromatography and membrane filtration, both of which encompass a wide variety of techniques. Although downstream processing procedures vary, the first step is usually clarification--the removal of cell debris and insoluble matter from the cell-culture soup. Kennedy explains that with mammalian cell culture, the desired product is usually secreted into the growth media, so the cells can be removed intact, leaving behind a broth that is relatively easy to clarify. In bacterial cell culture, on the other hand, the product is inside the cells, and the cell membranes must be disrupted in order to remove it. The sticky nucleic acids released make for a highly viscous mixture that can be difficult and time-consuming to clarify. Clarification on a production scale is sometimes accomplished by the use of large centrifuges. But according to Kevin Smith, manager of bioprocessing systems at the Penn State University Bioprocessing Resource Center in State College, Pa., microfiltration is "the method of choice" for this first separation. In microfiltration, the mixture is passed through membranes with pores ranging from 0.1 to 0.8 microns in diameter. Smith notes that whereas in centrifugation, the high speeds at which the mixture is spun introduces the possibility of producing aerosols--airborne particles that could be infectious--this danger is eliminated with the use of ultrafiltration membranes. (He adds, however, that some centrifuges have been designed to contain any aerosols that are produced.) Once the flotsam and jetsam of the cell broth are removed, the solution must be concentrated and the desired protein isolated from extraneous proteins and other substances. Concentration, essentially the removal of water or other liquid, usually relies on ultrafiltration, with membrane pores several orders of magnitude smaller than those of microfiltration membranes. This step can take place a number of times. A mixture may be concentrated and the desired protein purified from the concentrate. If a solvent or buffer is used in the purification step, ultrafiltration may follow to remove these solutions. The number of suppliers of filtration equipment reflects the technique's universal application in downstream processing of biotherapeutics. Among the major manufacturers of process-scale filtration products are Millipore Corp. of Bedford, Mass., and the Amicon Division of W.R. Grace & Co., headquartered in Beverly, Mass., but dozens of smaller companies exist as well. These companies sell a variety of products such as polymeric filters with low protein-binding qualities, so that a minimum of protein gets stuck to the filter; sturdy ceramic or stainless steel filters that can be cleaned, sterilized, and reused indefinitely; and filter systems that use novel configurations to increase efficiency. "There's so much competition out there, it's unbelievable," says Joseph Kutzko, a research scientist in the process development division of Boston-based Genzyme Corp. "Everybody's got a ceramic or a zirconium oxide they want you to try, and some of them are wonderful and some are garbage." A significant problem in scaling up filtration technologies, says Kennedy, is the clogging and fouling of the filter. "Dead-end" filter cartridges are designed to be used once and discarded when they clog. "Tangential flow" systems extend the life of the filter by forcing the liquid to flow across the filter and creating turbulence, slowing fouling. Kennedy's company, Membrex, takes this idea one step further with "vortex flow filtration." The membrane, which can be either a micro- or an ultrafiltration membrane, depending on the application, is wrapped around a cylinder that rotates within another cylinder. Little vortices are created by the spinning motion of the filter membrane, and these whirlpools keep particulates and proteins from settling on it. Kennedy says that since Membrex's system takes longer to clog than those using tangential flow, it is especially useful in the separation of viscous materials. Membrex markets two systems: the laboratory-scale Benchmark, at $7,500, which can process batches of about 20 liters, and Pacesetter, which can go up to 100 liters. A Pacesetter system with one filtration module runs from $17,000 to $25,000, depending on the filters used. Kennedy notes that at present, scale-up to large quantities can become prohibitively expensive. When scaling up tangential flow systems, "you just undo some bolts and snap in some more filters," he says. But with Membrex's units, "you have to add more hardware that will make more vortices." Within the next year, however, Membrex plans to offer a system that allows the filter area within a module to be expanded, bringing the cost down substantially. Kennedy also points out that "scaled-up" in biotech doesn't necessarily mean huge batches. "Some scaled-up biotech processes are only 100-liter production sizes," he says. "If the product the cell is making ... is active in microgram or nanogram quantities, a cell culture of 100 liters would produce enough to last a lifetime." Of the 200 or so Benchmark systems that have been sold, he adds, several are being used in production. The "nit and the grit" of downstream processing is the final purification, says Smith--getting a substance down to the desired product and nothing else. While membrane filtration suffices for broad separations, Genzyme's Kutzko says that chromatographic techniques are necessary for precise separations: "There's really no other way to achieve purity." In chromatography, the clarified and, possibly, concentrated material, often diluted in a solvent, is passed through a column containing tiny particles that catch hold of the protein of interest while the rest of the solution passes through. The captured protein is then dislodged from the solid media and collected in its now-purified form. A variety of methods can be used to hook the protein. Ion-exchange chromatography uses electrical charge, while size-exclusion chromatography catches the proteins on the basis of size. As in filtration, the chromatography market is immense, but many in the industry point to Pharmacia Inc. of Piscataway, N.J., as the leader in process-scale chromatographic supplies. One of the most interesting techniques now available is affinity chromatography, in which the medium is coated with a ligand that attaches to the protein of interest. Affinity chromatography can be highly specific, reducing or eliminating the need to repeat chromatographic separations to reach a final purity. A ligand could be an antigen that binds to a specific antibody, such as protein A that attaches to immunoglobulin G proteins. Some biotech companies have designed proprietary monoclonal antibodies just for this purpose. Other types of affinity chromatography include metal affinity, in which a metal such as zinc attaches to certain types of protein, and dye affinity, based on the tendency of some dyes, such as Cibacron Blue (a textile dye), to bind to certain proteins. But while chromatography, developed nearly a century ago as an analytical technique, may be unsurpassed in achieving very fine separations, it can be difficult and expensive to scale up. Larger chromatography columns are often taller, and as height increases, so does the amount of pressure created (termed "pressure drop") in the column. The solid phase particles, often made of soft gels, compact under these conditions, drastically slowing--or even halting--the throughput of the column. Pressure drop can be ameliorated to some degree by using more rigid media and short, fat columns. However, as Kutzko points out, greater diameter means less support to the solid phase, and the shrinking and swelling of the media under these conditions can make separations difficult. Another problem with scaled-up chromatography is the amount of time it takes to achieve a proper separation. If a liquid is pushed through a column too fast, the protein may not have time to bind to the matrix, resulting in an incomplete separation and low recovery. But several companies have come up with ways of getting around chromatography's inherent limitations. One such company is PerSeptive Biosystems Inc. of Cambridge, Mass., which has developed "perfusion chromatography," a method that it claims achieves separations 10 to 100 times faster than standard liquid chromatography. The company's POROS media contains pores of two sizes: "throughpores" that transect the solid-phase particles, allowing convective flow through them, and "diffusive pores," which provide a high adsorption surface area. The vast increase in speed means that biotech companies involved in scale-up could potentially process bigger batches of material in smaller columns, obviating some of the aforementioned shortcomings. Several scientists interviewed for this story expect perfusion chromatography to revolutionize the field within the next few years, although the relatively high price tag on POROS products at present may rule out their use for many bioprocessing applications. Sepracor Inc. of Marborough, Mass., has taken a different tack in speeding process-scale protein purification. The company's Trio Bioprocessing System uses affinity technologies developed for chromatography, but puts the affinity ligands on a hollow-fiber membrane, instead of a particle. The material to be separated flows down the inside of the fiber, and as it passes through the membrane--the wall of the fiber--the ligands in the membrane capture the target proteins. Because the fiber wall--analogous to a bed of chromatographic media--is so thin, the separation takes place extremely rapidly (up to 100 times faster than conventional chromatography), and uses the ligand quite efficiently, according to the company. The Trio system can also be configured to do clarification or concentration, simply by substituting the appropriate hollow-fiber ultrafiltration or microfiltration membrane cartridge. A user can even network three systems for a continuous processing system. According to John Chickosky, vice president of business development at Sepracor, one of the system's greatest advantages is the fact that it is automated. "The multistep process of loading the protein on the matrix, washing it, and eluting it, can take hours on a chromatography column," explains Chickosky. "With a hollow-fiber membrane, it can take minutes. It's physically difficult to achieve the possible speed unless you automate it." The Trio system now available can achieve affinity separations at a rate of up to 40 liters per hour, says Chickosky. Next year, the company plans to introduce a system with up to twice that capacity. Even after several separation and purification steps, it is virtually impossible to get a completely pure product. Genzyme's Kutzko explains that manufacturers aim for target purities of less than 10 nanograms contaminating protein per milligram, and DNA contamination at or below 1 picogram per milligram (the limit of detectability). However, these standards can be difficult to achieve. One contamination threat that exists even at high levels of purity is that of viral contamination. "There's always the potential for products made from human or animal cells to be contaminated with virus," says Paul Sekhri, manager of Millipore's virus removal program. To address this problem, Millipore recently introduced Viresolve, a filtration membrane--employed at the end of the downstream process--which is specifically designed to remove viruses. Sekhri says that standard ultrafiltration membranes have a fairly wide range of pore sizes, and sometimes pass even the largest virus particles. Viresolve, on the other hand, has a very narrow pore size distribution, and the removal of viruses using it can be predicted based on their size. This means that a user doesn't need to know what, if any, viruses are present--if a virus falls within a certain size range, the likelihood that it will be removed can be predicted mathematically. "We wanted to design this as a `black box,' " says Sekhri. Viresolve was introduced in June, and it is currently being tested by biotech companies across the country, says Sekhri. At $400 per disposable membrane--capable of processing about two liters of product--Viresolve isn't cheap. But as Sekhri points out, "these are for recombinant proteins, where a one-liter batch can be worth hundreds of thousands of dollars." And the high-stakes nature of the industry is bound to drive the search for more products to optimize the large-scale processing of these pricey molecules.

New Technologies Improve Biotech's Downstream Processing

Interested in reading more?

Become a Member of