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Apr 15, 2006 (Vol. 26, No. 8)

Market Increases Oligonucleotide Demands

Driven by Promise of miRNA and siRNA for Therapeutic and Research Applications

  • Even as new players join the competitive market for research- and pharmaceutical-grade oligonucleotides, acquisitions and partnerships continue to change the face of the supplier landscape. Demand is driven by the established market for primers and probes and the growing market for RNAs for research and therapeutic applications. With immunostimulatory DNA drugs now in Phase III trials and antisense oligos striving for greater recognition in the therapeutics arena, manufacturers of cGMP DNA eagerly await approval of the next oligo-based drug.

  • Acquisitions and Alliances

    To strengthen its position in the DNA medicines market, Avecia Biotechnology (www.aveciabiotechnology.com) entered into a strategic alliance with TriLink BioTechnology (www.trilinkbiotech.com) based on the adoption of common technology platforms that will enable the companies to provide integrated manufacturing scale-up capabilities.

    In a move to increase its range of molecules, materials, and solutions for novel applications, Solvay (www.solvay.com) last year acquired Girindus (www.girindus.com), a manufacturer of cGMP oligonucleotides for therapeutic use.

    “We are seeing a greater diversity of therapeutic strategy and chemical approaches,“ says Charles Shields, vp of global business development at Avecia. “To be specific the relative proportion of RNAi and immunostimulants are increasing markedly,“ whereas the field had been dominated by phosphorothioate DNA compounds operating through an antisense mechanism.

    With regard to advances in synthesis and purification technology, Shields notes, “In our hands we have seen improvements in crude quality and overall yield with some new-generation solid supports.“ Avecia has scaled up production in the area of siRNA to 50 mmol.

    In terms of price pressure in the therapeutic oligos market, “there are no clear trends emerging yet,“ adds Shields. “However, we still hope that successful approval of a large volume product will introduce economy-of-scale dynamics within the starting material supply chain.“

    Applied Biosystems (ABI; www.appliedbiosystems.com) recently completed its acquisition of the research products division of Ambion (www.ambion.com), which provides products for RNA synthesis, stabilization, detection, amplification, and analysis, including reagents for miRNA and RNA interference research.

    The $35 million in proceeds from the December sale is helping to fund a new company called Asuragen (www.assuragen.com), which is comprised of three business units: Molecular Diagnostics, Molecular Biology Services, and Discovery, a new R&D-based unit.

  • Producing cGMP Oligos

    Dowpharma´s (pharma.dow.com) addition of the OligoPilot 400 synthesizer (GE Healthcare; www.gehealthcare.com) to its commercial-scale cGMP manufacturing facility has given the company greater flexibility in oligo synthesis and increased production speeds. Sufficient manufacturing capacity is in place to meet current demand, says Noel Irizarry, business director of nucleic acid medicines at Dowpharma, as most oligo therapeutics are still in relatively early stages of development. The need for future scale-up is as yet unclear.

    “Oligos continue to be a highly speculative field,“ says Irizarry. In 2005, some players had setbacks in product development, “but we are fully committed to this market and have the analytical expertise and manufacturing unit in place.“

    OligoPilot 400 was designed to synthesize intermediate quantities of therapeutic-grade oligonucleotides. Nominal synthesis scales range from 4󈞊 mmol using 70- or 100-mm diameter columns. This translates into more than 150 grams of crude 20-mer oligos per run.

    With the additions of AKTA OligoPilot 10 Plus and AKTA OligoPilot 100 Plus, GE Healthcare has a full range of systems for manufacturing of oligonucleotides going from 1 µmol to 1 mol. The AKTA OligoPilot 10 Plus is an upgraded version of AKTA oligopilot 10 in which hardware as well as methods have been optimized for 1 µmol scale synthesis of both DNA and RNA.

    The AKTA OligoPilot 10 Plus operates in the 1󈞞 µmol range and the AKTA OligoPilot 100 Plus operates in the 100 µmol to 9 mmol range. OligoProcess systems from GE Healthcare have been designed for manufacturing of kg quantities of clinical and commercial material. Two models are currently available, operating in the 50� and 100𔂿,000 mmol scales respectively

    Lonza (www.lonza.com), another supplier of cGMP oligos—including DNA, RNA, and siRNA—manufactures oligos at small to industrial scale.

    Scandinavian Gene Synthesis (SGS, www.sgsdna.com) produces cGMP DNA oligos primarily for the in vitro diagnostics market.

    With a focus on developing RNAi drugs, Sirna Therapeutics (www.sirnatherapeutics.com) produces its own cGMP oligos and recently received a U.S. patent covering the chemical synthesis and manufacturing of ribonucleic acids. Sirna´s process comprises six major steps—synthesis, deprotection, purification, annealing, ultrafiltration, and lyophilization—that encompass the 150 discrete chemical steps involved in manufacturing a siRNA.

    According to Sirna, the process achieves chemical yields in excess of 60%, with high-product purity. Sirna also produces oligos on a contract-manufacturing basis. Its processes are readily scaleable to hundreds of kilograms of siRNAs.

    “We have seen a major drop in the price of RNA-based raw materials,“ says Mark Egan, vp of operations at Sirna. “The DNA market has historically been a much bigger market,“ and DNA amidites “are cheap compared to the RNA amidites. But when you look at the actual cost of the RNA nucleosides versus the DNA nucleosides, which are the starting materials, RNA is actually cheaper than DNA.“

    The difference in price is due to economies of scale and the size of the DNA market, Egan observes. “With the evolution and development of the siRNA field, we are starting to see economies of scale and a reduction in the cost of those RNA amidites.“

  • Immunostimulatory Drugs

    Idera Pharmaceuticals (www.iderapharma.com) is capitalizing on the immunostimulatory effect induced by synthetic DNA compounds first recognized in the mid-1990s during clinical testing of early antisense drug candidates. This immune response is linked to unmethylated CpG oligonucleotides, which are present in bacterial DNA and recognized by human Toll-like receptor (TLR) 9, inducing a Th1-type of immune response.

    After years of trying to modify antisense compounds to minimize this response, Idera has changed focus and is now developing TLR agonists that target the TLR-9 receptor to modulate the immune response and treat disease.

    “Rather than working on CpG, we wanted to develop clear IP,“ says Sudhir Agrawal, D.Phil., CEO and CSO at Idera. The company developed alternative immunostimulatory oligonucleotide motifs called YpG and CpR, and a series of IMO synthetic DNA compounds.

    Compared to the typical half-gram doses of antisense DNA needed to achieve a therapeutic response, immunostimulatory DNA drugs induce a local effect, and a 10󈞀 milligram unit dose would typically be sufficient, allowing for smaller production lots.

    From a manufacturing perspective, compared to the production of antisense compounds, Dr. Agrawal explains, “The technology is the same. However, the raw materials [modified, unnatural nucleotides] and the DNA sequences are different.“ Overall, manufacturing, scale-up, and purification of immunostimulatory DNA compounds are similar to those for traditional antisense drugs.

  • Process Development

    According to Shields, customer demands include a wide range of volume requirements. “Clearly with more potent entities or niche indications, the volume requirement is low, but in other areas we are seeing large volume needs.“ The lower volume requirements for more potent products imply that “process validation has to be approached with limited batch history—this places a greater emphasis on the quality of process development and the depth of process understanding.“

    “Many of the oligonucleotide drugs currently in the clinic will only ever be required in low volumes, and this needs to drive a change in the paradigm for the suppliers,“ says Mick Mclean, CEO of Oligovax, a French company developing therapeutic oligos. “Rather than focusing on making high volumes in a dedicated plant, they need to become expert in making low volumes cost effectively in multi-product suites. Labor becomes the dominant cost driver in this scenario, so we´re looking to our suppliers to continuously reduce their costs by improving the changeover time, cleaning processes, analytical development, and project management.“

    The main change in emphasis among customers that are bringing their products into late-stage development is an increased focus on manufacturing and regulatory-related issues. They are looking more closely at the raw materials used to make their product, says Kami Beyzavi, Ph.D., a scientist in media applications development at Millipore´s (www.millipore.com) OEM Group, and asking questions such as, “where are they coming from, are they manufactured up to the specifications required, and is there sufficient capacity?“

    Millipore´s DNA Nucleoside Controlled Pore Glass (CPG) media provides a rigid support matrix for oligonucleotide synthesis using phosphoramidite chemistry. Available in pore sizes from 70 up to 3,000 , the company says that the media enables coupling efficiencies of >97.5% and provides a ligand loading range of 20󈞔 µmoles/g for 1000 CPG and 30󈞞 µmoles/g for 500 CPG, for example. Key features of Millipore´s CPG are its uniform pore size, high surface area, and broad solvent compatibility without swelling or shrinking, according to Dr. Beyzavi.

  • Price War for Custom Oligos

    SAFC Supply Solutions (www.safcsupplysolutions.com), a division of Sigma-Aldrich, manufactures the raw materials for oligo synthesis, including DNA and RNA phosphoramidites, controlled pore glass solid supports, and liquid reagents. In its SAFC Proligo Reagents production facility in Hamburg, Germany, the company says it can produce five metric tons of high-purity amidite products each year.

    New in 2006 is SAFC´s Pharmadite product group, which provides phosphoramidites for pharmaceutical applications. Among their advantages, according to SAFC, are the use of synthetic starting materials, traceability of raw materials, impurity profiles, and method validation, which help users satisfy regulatory requirements. SAFC will introduce the Pharmadite line next month.

    The price war brewing among primer and probe manufacturers has trickled down to exert pressure on raw materials suppliers, observes Mirjam Collegeman, sales manager at SAFC Supply Solutions. She points to the expiring patents on phosphoramidites as another factor driving increased competition, with “a new crop of competitors“ coming into the market and using price to gain a foothold.

    “We believe the next growth area for the phoshophoramidites will be on the pharmaceutical side,“ Collegeman says, “with excitement centering on the developments in RNA applications, futuristic inquiries for thousands of kilos of RNA phosphoramidites have started to emerge.“

  • Quality Control

    In the speciality oligonucleotides business, Trey Martin, COO of Integrated DNA Technologies (IDT; www.idtdna.com), points to the positive and negative aspects of the aggressive push by some oligo suppliers to gain market share by driving down prices. While the availability of high-throughput, lower-priced oligos enabled research that could not have been done years ago, quality control of manufacturing processes comes at a cost. Customers may have to pay a higher price for a high-quality product.

    Pursuing a strategy of expansion via acquisition of companies with complementary technologies, IDT recently acquired Synthegen, a company that specialized in the synthesis and purification of modified oligos. Early this year, IDT also began operations at its new West Coast oligo production center—a development made possible by IDT´s acquisition of San Diego-based GenBase in December. Establishment of a local manufacturing base allowed IDT to offer next-day oligo synthesis and delivery to the California biotech industry.

    The company has set its sights on expansion into Europe, according to Martin, a market that is “much more fragmented and much less defined, with no clear dominant player.“ Similar to the challenges European and Asian suppliers have faced trying to break into the U.S. market, “you have to be very sensitive to the needs of the local market,“ says Martin.

    IDT, along with Eurogentec (www.eurogentec.com), a supplier of oligos with a range of modifications for research use and a contract manufacturer of cGMP oligos, and MWG (www.mwgbiotech.com), a producer of modified and unmodified oligos, PCR probes, and siRNAs, acquired a non-exclusive license from Alnylam Pharmaceuticals (www.alnylam.com) to provide research products and services for RNAi under the company´s Kreutzer-Limmer patent family.

  • Large-Scale Production

    In late 2005, Invitrogen (www.invitrogen.com) made available tube-based oligos at 25 nmole starting synthesis scale. The company attributes its ability to provide this quantity of research-grade oligos for the same price as it had previously offered 10 nmole tube-based oligos to its collaboration with Illumina (www.illumina.com) and access to Illumina´s DNA synthesis technology.

    Mary Buchanan, business area manager for oligos at Invitrogen, says, “In collaboration with Illumina, we have rolled out a new synthesis platform that is capable of manufacturing over 13,000 unique oligos simultaneously.“ The robotic nature of the manufacturing procedure maximizes product consistency.

    She points to the genomics market as one sector showing continued growth. With the completion of the Human Genome Project, a lot of large project biology on some of the more esoteric genomes is now under way, generating demand for sequencing primers and probes for microarray analysis.

    Bioneer (www.bioneer.com) developed a synthesizer that it says is capable of producing 384 oligos in one cycle. Bioneer´s large-scale oligonucleotide manufacturing facility can generate more than 23,000 custom oligos per day, according to the company.

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