20 June 2005 00:01 [Source: ICB Americas]
High cost has always been the key shortcoming of protein drugs, but improvements in cell-culture expression, enabled by the discoveries of companies such as ICOS, Crucell, Selexis, ML Laboratories, Chromos and others will continue to lower prices, extending the penetration of biologics into ever smaller therapeutic niches.
Thirty years ago, cell-culture expression levels were a mere 10 milligrams per liter (mg/L), notes Howard Levine, president of Acton, Mass.-based BioProcess Technology Consultants Inc. By 1995, they were still only in the range of 100 to 500 mg/L. Expression levels now routinely exceed 1 gram per liter (g/L), he says, and by 2015, he projects, they may pass 10 g/L. The price implications of such an increase will be large.
“It is not unthinkable that expression levels could reach 10 to 15 g/L over the next ten years through the use of new vectors, further optimization of the medium and improvement in the production process,” comments Peter van Hoorn, executive vice president business development, Cambrex Biopharmaceuticals. “All the major players in monoclonal antibodies (such as Genentech, Roche etc.) are investing heavily to increase output per unit volume. At the moment the highest yielding strain process is Humira (Abbott) with yields of approximately 6 g/L.”
The problem would be much less severe if these products could be manufactured by microbial fermentation, which can be 50 to 60 times more productive by volume than cell culture. Unfortunately, most biopharmaceuticals in development require post-translational modifications that yeast and other microbial hosts cannot accomplish.
Instead, the task is entrusted mainly to the Chinese hamster ovary (CHO). According to Levine, CHO is the host cell line for as many as 80 to 90 percent of marketed or developmental products requiring post-translational modification, although there are alternatives, particularly human cell lines such as Per.C6, which has been developed and marketed by DSM in an alliance with Crucell. “The CHO dominance will certainly be challenged in the future as new cell lines with better properties will find their way into the market,” says Pieter de Geus, vice president, research and development, at DSM Biologics. “In general, cell line development is aiming for improved product quality attributes, e.g., with respect to glycosylation and product homogeneity, and for improved cell line generation times and yields. Although CHO has shown splendid performance in a number of cases, it might just not be the best choice of host cell in the long run.”
Human embryonic kidney cells are used by some companies. Another mammalian host that has found some application is the murine B cell myeloma cell line (NSO), and four products in clinical trials even use insect cells.
Still, the Chinese hamster ovary has much in its favor. Regulatory authorities are very comfortable with CHO cell lines, says Dan Allison, director, production development at ICOS Corp., and for now, the highest titers (or yield per unit volume) are being obtained using them. The engineering has already been done on CHO cell lines, he notes, and they offer the potential for more. “For the foreseeable future, I think CHO will continue to be the cell line of choice.”
Many tools have been developed to increase expression levels in mammalian cells. One of the earliest, developed in the early 1980s, couples the expression of the desired protein with the expression of dihydrofolate reductase (DHFR), an enzyme essential for growth. Cells in which these two products have been coupled are then exposed to methotrexate, an inhibitor of DHFR. Cells that survive do so by increasing the production of DHFR—and with it, the desired protein. Lonza developed a similar technique, the GS system, in which the coupled enzyme is glutamine synthetase, and the inhibitor used is methionine sulphoximine.
In addition to these so-called “gene expression” or “amplification” methods, there are newer technologies that enhance the insertion of the gene into the chromosome, says Levine. First, he explains, insertion of a new gene should not break up existing genes for essential proteins. Second, the gene should be located where the cell can easily reach it. “By putting certain sequences of DNA on either side of the gene for the protein of interest, these various technologies target where in the chromosome the gene gets inserted,” he says. “They also help to say, when the cell sees that particular piece of DNA, it should make a lot of that protein, rather than another.”
Levine points to UCOE, MARtech and STAR as examples of such technologies. STAR, for which Leiden, the Netherlands-based Crucell received a US patent in April, is now being evaluated by both Genentech and Medarex for use in its proprietary systems. MARtech (the name refers to Matrix Attachment Regions) was developed by Geneva-based Selexis, which began offering it for license in November and also recently teamed with Diosynth Biotechnology to market the technology. UCOE (Ubiquitous Chromatin Opening Elements) has been licensed to several companies, according to its developer, St. Albans, UK-based ML Laboratories PLC.
In May, Chromos Molecular Systems of Burnaby, British Columbia, Canada, licensed its ACE System for the first time, to Cambridge Antibody Technology.
“One of the advantages of the newer technologies is that while the DHFR amplification system takes a long time to develop high-expressing clones through multiple rounds of selection and amplification, some of these other newer technologies enable companies to obtain a higher level of expression right off the bat,” says Levine. DHFR amplification can take anywhere from 18 to 30 weeks, he points out, whereas some of the other technologies claim to achieve the same expression levels in a few months.
Another approach to higher expression levels focuses on “promoters.” Simply put, promoters are pieces of code that tell the cell which genes to read for instructions. The CMV promoter, named after the cytomegalovirus from which it is derived, is used to manufacture most mammalian cell-culture products on the market, says Levine. ICOS, however, has developed an alternative, the CHEF1 system, and made it available for license. Based on the CHO EF-1a promoter, CHEF1 provides expression levels over six to 25 times greater than those obtained with other promoters such as CMV, without amplification, says ICOS’s Allison. “ICOS is focused on using off-the-shelf technologies for screening large numbers of clones derived from CHO cells transfected with CHEF1 vectors. We’re also looking at using CHEF1 vectors to express cellular proteins that may be rate-limiting for expression/folding/secretion/modification of the protein of interest.”
An old technology—the screening of clones—updated with high throughput, is proving key to obtaining high-titer cell lines, Allison adds. “This is being done largely through the use of robotics to assay cell-culture media from thousands of clones, and using improved assay systems such as homogeneous assays that don’t require dilution (e.g., Europium based assays).” He points to several examples. BioProcessors, of Woburn, Mass., does high-throughput screening of clones in miniaturized bioreactors to look for good clones. Zeiss reportedly has an imaging system that facilitates clonal screening. And Genetix, a company based in New Milton, UK, has a method for automated screening and picking clones from semi-solid agar using an imaging system coupled with the use of fluorescently-labeled anti-IgG (for antibody-producing cells).
All told, the toolbox is broad and deep. “I believe there is a great potential for these technologies to improve cell productivity,” Allison comments.
Nonetheless, intrinsic methods such as these, which engineer the cell to improve expression levels, are not the whole story, Levine points out: “The second piece is optimization of the cell culture process itself.” In particular, the industry has become very sophisticated about the cell culture media, which contain the nutrients necessary for growth. “Now that we understand the cell’s metabolism better, we know that too much of a good thing is not always good,” he explains. There are major benefits. “By playing with the media, you can optimize the cell density, the number of cells per unit volume,” he says. “You can also optimize the cell density and viability.” The more viable cells are in the reactor at any given time, the more productive the reactor will be. The result has been a trend away from traditional “undefined” media, so-called because they rely on variable natural extracts for a complete range of nutrients, and toward “defined” media—in which, beginning with water, each constituent is added individually by design to serve each particular culture system.
“Two things are allowing companies to do better,” comments Levine. “One is that we understand the fundamental metabolism of cells better, we understand better what makes the cell live and what makes it die. And somewhat related to that, we have more and more experience. The industry in general, and specific companies in particular, have been growing CHO cells making antibodies or recombinant proteins for quite some time now, so they have some institutional knowledge of what helps the cell grow and what keeps it from growing.”
The window closes early on yield improvement through cell modification, but the cell culture process can be adjusted indefinitely. “Since it is rare that clones are changed during clinical trials, the only other alternatives to increase yield are to try to improve the cell culture medium or the process,” notes Roel Gordjin, global director of sales and marketing for biotherapeutic media, Cambrex Bio Sciences. Cambrex, a major supplier of media, collaborates closely with companies doing process development and designing feed strategies by providing spend-medium analysis, optimization solutions, and trial batches that customers can evaluate in-house, he says. “We also have the option to do a complete medium optimization program. Cambrex is able to design and optimize a whole process from the gene of interest to manufacturing with medium optimization, cell line development, process development, manufacturing, fill/finish, and testing services on intermediates and the final product.”
Cambrex offers twenty-three serum-free CHO media, most of which are non-animal origin, says Gordjin, and this year launched the fully chemical-defined PowerCHO series. “Cambrex serum-free media like the ProCHO series and the PowerCHO series are non-animal origin, regulatory-friendly media that give excellent expression levels and are used in a number of processes that are currently in clinical trials.”
When the cell culture process is complete, the protein must be separated from the rest of the biomass. This phase, downstream processing, is rapidly becoming the predominant time- and cost-bottleneck in bioprocessing, according to Cambrex’s van Hoorn. “There are limitations to scaling columns. [C]olumns, gels and the validation of column re-use are very expensive. Engineers continue to look for ways to limit the number of column steps in favor of more conventional and readily scalable technologies like filtration, centrifugation and precipitation. One cannot continue to add larger chromatography resins, as the column material will be prohibitively expensive.”
For most processes producing monoclonal antibodies, the isolated yield is around 50 or 60 percent, estimates Levine. “I think the best processes out there are probably around 70 percent. 80 percent is probably the limit.”
The bottleneck has created an opportunity for companies like Cambrex, Gordjin notes. “Since many companies need to devote significant time and resources to focus on [downstream processing], they want to minimize time spent on non-core activities like validations, cleaning and buffer preparation. Disposable technologies for medium, buffer bags and prefab buffers can reduce downtime spent on validation and cleaning and reduce manufacturing costs.” Cambrex’s contribution includes fully validated and non-animal origin Platinum UltraPAK bags used not only for storing buffers or cell culture media but also for sampling and harvesting. “We have added tank liners and tubing sets to our disposable system product offerings,” van Hoorn notes. “We also recently invested in a 10,000 liter liquid medium/buffer facility in Verviers, Belgium, which is completely animal origin-free and can produce cell culture medium or buffers in bags up to 1000 liters.”
ICOS’s Allison also laments the cost of resins. Higher capacity resins and more rapid recycling would reduce raw material costs and plant time for each purification step, he says. “Automation of purification steps would reduce labor costs. Another area for improvement is in QC/QA—more automated tests, more efficient procedures (e.g., barcodes, electronic data storage), and fewer discrepancies would reduce costs in these areas.”
More productive cells, better optimized cell culture processes, and more efficient product isolation together have the potential to dramatically improve reactor output, and yet there is little likelihood that biopharmaceutical manufacturers will find themselves with excess capacity on their hands. “The market is growing tremendously,” Levine observes. “And what’s interesting is that for the products that have been approved most recently, the doses are getting higher and higher. The amount of product required per patient per year is higher, so we have to make more product. That’s one of the things that has been driving the development of new technology for expression. For antibodies, the compound annual growth rate for the last four to five years has been 20 plus percent, and you’ve got products like Avastin, which was just approved, that are going to take huge quantities of protein. So even though the expression level and the yields and so forth are higher, the amount of capacity required to make all these products is pretty large.”
Van Hoorn is not worried either. “Mammalian bioreactor cell culture capacity has doubled in the last two years, but this capacity could be completely utilized if a significant number of biopharmaceutical products that are currently in clinical trials make it to market.”
Indeed, higher expression levels are likely to create a dynamic encouraging a broader pipeline of biologics. “With higher expression levels in the future, it is expected that there will be increased demand for smaller bioreactors in the range of 1000 to 2000 liters,” van Hoorn says. “There will also be opportunities for fully disposable facilities with disposable bioreactors, purification units etc. The combination of better expression levels and the lower-cost disposables will enable the industry to construct facilities faster, reduce validation and cleaning time, resulting in the overall cost reduction of biopharmaceuticals. With this in mind, the outlook for transgenics as a lower cost alternative might be less promising then it was a couple of years ago. More importantly, lower manufacturing costs can make it more economically viable to launch non-block buster drugs for rare diseases in the future. The overall lower costs could enable the development of drugs for diseases that we cannot treat today because it is too expensive to make the product.”
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