Facility as a Purification Asset?
Inese Lowenstein, director for downstream processing at EMD Millipore, notes that as titers continue to rise, mAb producers face difficulties in maximizing facility utilization and avoiding high-capital investments. “Titers constrain the filtration area, buffer tanks, and hold-tank volumes—the entire downstream facility.”
Millipore has, therefore, focused on developing high-capacity products such as ProSep Ultra Plus, a protein A affinity medium that, like GE’s recently introduced MabSelectSure LX, achieves high throughput while reducing column sizes, buffer volumes, and number of purification cycles.
Lowenstein cautions against viewing high-productivity unit operations in isolation. Their greatest benefit, she says, is in their integration into a process that reduces buffer-related steps and the need for some or all holding tanks. Millipore has presented a three-step protein-purification process that achieves these goals: protein elutes directly from the protein A column to an Eshmuno S (cation-exchange) column, and finally through an anion-exchange membrane adsorber for polishing.
“The process reduces time and effort, minimizes precipitation, has a smaller footprint than conventional purifications, and better utilizes facility resources.”
Contract manufacturers are by nature even more risk-averse than innovator companies. The emphasis at CMOs is getting the job done as quickly and cost-effectively as possible. Therefore CMOs rarely stray from their clients’ idea of robust purification methodology.
Scott Kelly, manager for GMP manufacturing sciences at Paragon Bioservices, notes that disposables have made a significant difference in providing value for early downstream operations like recovery and clarification.
“The productivity of upstream operations means more protein, but also more cell debris and side products.” Removing those impurities before chromatography is a key element of success.
Utilization of space, equipment, and personnel become bottlenecks as processes grow in size. Processes that employ centrifugation at bench scale must eventually switch to tangential flow or depth filtration, which adds time and equipment. “You need staff to prepare those buffers, and footprint to prepare and store them. So the more efficient we can make recovery and clarification the better,” Kelly says.
Similarly, large-scale batches need to be split and held, with several capture cycles required per batch instead of one. Here, not only floor space, but resin capacities come into play. “You have to fit the project around what you’re capable of at any particular time.”
Since designing ease of purification into molecules is difficult, bioprocessors will increasingly turn to nonmammalian expression systems with a downstream advantage such as fewer steps or improved product quality.
For example Ajinomoto’s Corynebacterium-based expression system, Corynex, reportedly reduces costs and speeds time to market by expressing recombinant proteins directly into broth. Corynex works with cytoplasmic proteins, growth factors, enzymes, and mAb fragments, but not with membrane proteins or entire antibodies.
The elimination of lysis minimizes host-cell proteins, DNA, and other contaminants, with an additional bonus. “Corynex allows
purification of properly folded proteins directly from the broth, with no inclusion-body processing,” says Joel White, business manager for biotechnology. “In the case of IGF-1, a protein with three disulfide bonds, purification requires half as many steps as conventional bacterial fermentation.” By eliminating junk and folding proteins correctly, losses are reduced to half of what one would expect with E. coli.
Purification involves centrifuging the cells away, ion-exchange or reverse-phase chromatography, followed by gel filtration or ultrafiltration. Eliminated are harvest, disruption, removal of cell debris, and refolding.
Insect cells are viewed as a simpler alternative to mammalian cell culture, perhaps providing a purification advantage as well, but problems still need to be ironed out.
In many respects insect cell culture is at the stage at which mammalian cultures were about a dozen years ago. Cell densities barely reach 2.5 million cells/mL, limited by the necessity that cells be in their logarithmic growth phase during transfection. Titers are below average for many established protein processes, and quite low for mAb processes. According to Manon Cox, Ph.D., president of Protein Sciences, one can expect “a couple of hundred milligrams per liter” from insect systems.
“But a lot of productivity is still to be gained as soon as the first product produced in insect cells is approved,” says Dr. Cox. “At that point, media development and feed strategies will take off,” perhaps providing some of the dramatic improvements we’ve seen for CHO cells.
Advantages of insect culture include proper protein folding and rapid, transient expression. Baculovirus-based products are more immunogenic than those from more conventional expression systems, which may be advantageous for vaccines but not for other products. Another minus is the presence of substantial quantities of baculovirus, which—even though they are not pathogenic to humans—must be cleared.
But perhaps the greatest drawback is regulatory uncertainty. “We filed our license application for an influenza vaccine three years ago,” Dr. Cox says, “and it is still not approved. All the problems are substrate related.”
Where It All Starts
Downstream bottlenecks are not the exclusive domain of large-scale processors. Initial or proof-of-principle purifications, the training ground of sorts for production scale, must often start from scratch, particularly for contract R&D firms.
Outsourcing early-stage purification and expression is becoming popular due to the increasing number of competitive service providers, says Brett Modrell, director of process development at Monserate Biotechnology. Monserate, which specializes in batches of up to about 10 g, operates in the purification “trenches”—on molecules whose total purification portfolio may include little more than an HPLC trace.
Monserate first considers formulation and purification possibilities based on solubility and activity. At this stage, the major bottleneck is not too much protein straining facilities and equipment but the more prosaic lack of it. “The greater the expression levels, the easier the purification,” Modrell says. “Effort spent improving expression pays back many times over.”
Yet even here operations such as sample concentration and buffer exchange become as critical as they are for larger processes. One notable difference at this scale is the potential for using affinity tags based on transition metal chelates, which provides enough protein to characterize, and perhaps to experiment with. Affinity tags are impractical and undesirable at production scale.
Similarly, chromatography media chemistries, ligand density, capacity, and cleanability are characteristics as desirable at 5 g as 50 kg scales, as are the benefits of disposable mixing and fluid handling.
Eventually, if all goes well and the molecule progresses through development, the fruits of lessons learned at milligram and low-gram scales find their way to process development.