High protein titers have been blamed, at least in part, for the mismatches of equipment, time, and facilities that result in downstream bottlenecks in chromatography and filtration operations. Less appreciated are upstream challenges due to an enabler of high-titer processing, namely the use of animal component-free media.
“High protein concentrations upstream require custom culture media that can be difficult to filter, or at best have different filter requirements from serum-supplemented media,” observes Anil Kumar, Ph.D., principal R&D engineer at Pall.
Moreover, high titers’ effect on filtration is multiplied throughout the process train, for example, in the huge quantities of buffer required by larger chromatography columns for high-titer processes, the difficulty filtering ever-more-viscous, valuable process fluids, and new concerns over contamination. “Filter manufacturers must consider upstream processing challenges, as well as develop solutions for downstream processing.”
Cell culture engineers believed that animal component-free media would eliminate the problem of mycoplasma contamination, but hope was illusory, as mycoplasma contaminate plant-derived raw materials and water, as well as animal-derived ingredients. In response, Pall developed a triple-layer culture media filter, Fluorodyne® EX EDT, purposely designed to provide greater than 10 log removal of mycoplasma from complex soy-based culture media.
Since time and cost remain critical elements, users have become cognizant of throughput and value. “When biotechnology was in its infancy, users worried about whether sterilizing filters could sterilize, then they worried about validation. Today, those concerns have been allayed. Now, the big concern is filter capacity and their impact on manufacturing costs.”
Nowhere are cost and throughput concerns more evident than in virus filtration. This is understandable, Dr. Kumar explains, because “users focus on the things that cost the most, and virus filters are indeed the second most costly downstream step, right after capture chromatography.”
One could argue that the situation tilts even more strongly against virus filters than chromatography resins since the latter are generally reusable, whereas validation of virus filter re-use is problematic. One-time use only amplifies the negative perception. Virus filter cost, says Dr. Kumar, is justified by their expensive materials of construction and manufacturing technology compared, say, with sterilizing filters.
Pall recently introduced the Ultipor VF UDV20, a filter cartridge the company says will process more fluid at lower overall processing cost. VF UDV20 employs Pall’s laid-over pleating and narrow core cartridge technologies to pack 2 m2 of membrane into each 10 inch element. “What might have previously required two 20 inch cartridges can now be done in one 20 inch cartridge, at lower cost per liter.”
All things being equal, one would expect serum-free media to be cleaner and filter more easily than traditional media, but all things are not equal. “Serum-free media does tend to filter more easily,” says Mandar Dixit, head of product management for filtration technologies, North America at Sartorius Stedim Biotech. “But biomanufacturers have many additives that make the media they use filter differently from something off-the-shelf.”
Another confounding factor is high-temperature, short-time (HTST) treatment, which is increasingly common during media preparation. In some instances, HTST-treated media is more difficult to filter than nontreated media, Dixit says, “and since more and more biomanufacturers are incorporating heat treatment as part of platform manufacturing process, vendors have to conduct filterability trials at small and intermediate scales to confirm filter sizing done during process development.”
Sartorius Stedim is taking media filtration seriously, recently entering a collaboration with SAFC Biosciences to develop cost-effective, optimum filter trains for critical cell culture media for the firms’ common clients.
On its own, Sartorius Stedim Biotech has introduced two additional multilayer PES membranes apart from its classical Sartopore 2 0.45/0.2 combination, to deal with evolving filtration needs. For example, the Sartopore 2 XLG, which was optimized for filtering soy-hydrolysate supplemented serum free media, features a 0.8 micron prefilter layer atop a 0.2 micron membrane; while the Sartopore XLI has 0.35 micron prefilter layer atop the same 0.2 micron final layer. Application-specific filter development represents the future of sterile filtration. Sartorius has also launched different double-layer PES prefilters, Sartoguard, to provide enhanced protection to its Sartopore 2 0.2 and 0.1 micron sterilizing-grade filters.