The Band-Aid Approach
Companies have thus far used the band-aid approach to overcome these difficulties, solving one problem only to have others crop up, says Dr. Romero. Capture/affinity resins with higher capacity are typical. A complete re-evaluation of the capture step would be better—for example, moving to expanded bed absorption. “At some point, bioprocessors must deal at a high level with the inevitable rise of protein titers to 10 g/L.”
Anything that favorably alters the protein/impurity ratio is helpful. Cells may be engineered to accomplish this, or processors can harvest earlier, with the goal of sacrificing yield of raw protein for final product. “At the end of the day, overall recoverable yield might be higher,” Romero says.
Other suggestions include investigating flocculation for cell harvesting, a technique that might reduce the burden on chromatography columns. Another strategy is to design secondary chromatography steps to operate in flow-through mode—binding impurities, not product—to take advantage of the relative fractions of these two components. Dr. Romero also likes membrane absorption, especially in flow-through mode early in the process, to remove impurities. Finally, eliminating buffer tanks by reconstituting chromatography buffer at the point of use, and eliminating steps between columns (elute, collect, load, repeat) would “save a lot of time and eliminate validation and tankage needed between columns.”
Other experts propose similar measures. Bruno Marques, Ph.D., senior research chemical engineer at Merck & Co. (www.merck.com), believes that reducing reliance on chromatographic separations with nonchromatographic techniques would be valuable as process streams become larger and more concentrated.
“Chromatography offers high resolution but low throughput, especially at larger scales,” he explains. “In the near future, companies will routinely manufacture 100 kg of a mAb per batch. Chromatography is not well-suited for such scales, especially when you consider the buffer requirements of two- or three-chromatography processes.”
Dr. Marques would like to see the development of alternative, simpler, technologies, such as impurity precipitation immediately after harvest. For replacing resin chromatography in flow-through, as well as bind-and-elute, mode, Dr. Marques likes disposable technologies but notes that selectivity and capacity “need further development, especially as far as adsorptive properties and fluid distribution, in the next several years.”
Mimetic replacements for expensive chromatography media are also of great interest. “Whenever we hear about mimetic resins Merck evaluates them, but so far we haven’t found any that meet all our requirements.” Drawbacks of newer mimetic resins include capacity, selectivity, packing, and linear-velocity capabilities.
Single-use equipment now serves nearly every upstream unit operation in one way or another, with single-use bioreactors in the 500–1,000-L range quite common. Even larger processes can benefit from small-volume single-use seed bioreactors and multiple disposable buffer and storage bags of up to approximately 1,000-L working volume each.
Fully disposable, large-scale downstream processing is probably years away, primarily due to the impracticality of single-use chromatography media, equipment (e.g., centrifuges), and certain filtration systems. While single-use direct flow, sterilizing, and certain types of depth filtration are available in disposable format up to the largest scale, disposable tangential flow filtration has only recently been introduced. Pall’s (www.pall.com) Kleenpak™ TFF MF capsule, which debuted at “Interphex 2007” along with disposable fluid management and containers, combines microfiltration with disposability.
According to Jerold Martin, Pall’s senior vp for scientific affairs, Kleenpak TFF resembles a dead-end filter capsule, but is used for cell removal or harvesting. “Kleenpak TFF is much simpler and easier than a cassette system,” Martin says.
Between clarification and polishing, filtration is mostly fully disposable. Pall has developed one fully assembled system whereby harvest fluid passes through depth filter, sterilizing filter, and virus filter, all presterilized and linked through sterile tubing and connectors. This filtration train, which handles process fluids of up to about 500 liters, was custom-designed using off-the-shelf single-use components.
Pall has been working with several larger customers on cassette-based disposable ultrafiltration systems. In these systems all buffer mixing takes place in disposable tank liners and is fed into cassettes, with all the fluid contact components being disposable and supported within stainless steel holders. “This is moving away from where the whole system is disposable, to where there is a fixed platform containing a disposable fluid path,” says Martin. “What people really want is a clean fluid path.”
Polishing and virus filtration can be carried out at almost any scale using presterilized filters connected to collection bags. Pall has created several integrated systems, for example a Mustang Q membrane chromatography module upstream of Ultipor® VF virus filters.
While disposables are rapidly maturing, gaps remain compared to traditional stainless steel manufacturing. “End users and vendors need to work together to identify these gaps and find solutions,” says Royce. One approach is to eschew completely disposable systems for disposable components that work harmoniously with stainless steel. Several vendors, including Pall and Millipore, offer such products.
Chromatography remains an expanding frontier for disposables. The high cost of resins precludes disposable chromatography for high-value products in the near term.