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Jun 1, 2005 (Vol. 25, No. 11)

Oligo Manufacturing Puts Emphasis on RNA

Improvements in Production are Keeping Pace with Advances in Drug Discovery

  • As more experimental DNA drugs move through the clinic and into large-scale trials, the demand for efficient and cost-effective manufacturing strategies is intensifying. Higher yields, greater purity, and lower production costs are the key drivers of new DNA synthesis technology. Similarly, as both therapeutic and research applications of RNA oligos increase dramatically, competition and price pressure are driving advances in RNA synthesis technology as well.

    Although there has "not been a lot of good news in the last 12 months or so from an antisense DNA perspective, there are still plenty of promising second-generation chemistry drugs that are in the clinic and are progressing," says Gaby Silver, marketing manager at Kinovate Life Sciences (www.kinovate.com), a new company solely held by Osaka, Japan-based Nitto Denko.

    At the recent "TIDES" meeting, in Boston, Kinovate launched NittoPhase, a novel solid support for oligo synthesis developed in collaboration with Isis Pharmaceuticals. The product is available in two grades: NittoPhaseTOS for high loading applications and NittoPhaseROS, composed of a bead with a lower loading capacity.

    "We have exploited Nitto Denko's polymer synthesis technology to engineer a bead with higher yield and purity than is currently available with a polystyrene bead, and at significantly less cost," says Silver. Significantly lower back pressure contributes to greater ease of use, Silver adds.

    Kinovate is in the final stages of optimizing its bead technology for RNA synthesis, and Silver anticipates a product launch within the next 12 to 18 months. RNA interference "is a technology with incredible promise," says Silver.

    The fact that the mechanism behind siRNA occurs in nature has led to much optimism that it will be effective in humans. Nitto Denko is committed to investing in engineering and optimizing a bead specifically for RNA synthesis conditions. The company hopes to "really raise the bar in terms of what's possible in the RNA synthesis world."

    The data to date suggest that the bead is "able to load at a higher capacity and, therefore, to produce a higher yield than existing products, whether controlled pore glass CpG or polysytrene.

    "The oligos market is trending toward RNA," Silver says. With the RNA market growing significantly faster than the antisense oligo market, he predicts that it will achieve parity with antisense market figures in about five years.

    Also launched at "TIDES" was Link Technologies' (www.linktech.co.uk) OligoPrep, a polyvinylacetate solid support for large-scale oligo synthesis using standard phosphoramidite chemistry. Link developed this product in collaboration with Merck KgaA.

    OligoPrep is a base-functionalized polyvinylacetate support designed for large-scale oligo synthesis using standard phosphoramidite chemistry. The support has a particle size of about 120 m and offers customized loadings up to 350 mol/g, according to the company.

  • Searching for Success

    The failure of Corgentech's (www.corgentech.com) edifoligide (E2F Decoy) in Phase III trials led to the company's decision to cease development of the drug, which had been in development with Bristol Myers Squibb to prevent vein graft failure following coronary artery bypass graft surgery.

    Corgentech will continue to pursue development of its other transcription factor decoys, oligo drugs focused initially on the treatment of inflammatory diseases and cancer.

    Producing oligos for Corgentech's E2F clinical trials was a major focus of the manufacturing activity at Avecia Biotechnology's (www.avecia.com) Grangemouth, Scotland facility. Following the release of the negative study results, Avecia announced the closing of its Grangemouth site. It is consolidating its oligo manufacturing activities at its facility in Milford, MA.

    Recently, Avecia entered a manufacturing agreement with Pfizer to produce clinical and commercial quantities of the active pharmaceutical ingredient for ProMune, an anticancer drug licensed to Pfizer by Coley Pharmaceutical Group. Avecia will focus on process development and optimization, scale-up, and process validation in applying its solid-phase amidite technology to produce ProMune.

    Driving efforts to make large-scale cGMP synthesis of oligos more efficient and cost effective is the continued hope that DNA drugs now in the development pipeline will establish themselves as effective therapeutics in the clinic and in the marketplace.

    Coley's lead compound is one such drug. ProMune is an agonist of Toll-like receptor (TLR) 9 and is in late Phase II clinical studies for the treatment of advanced non-small cell lung cancer, malignant melanoma, and cutaneous T-cell lymphoma.

    ProMune is composed of unmethylated, synthetic CpG sequences that mimic a pattern of nucleotides commonly found in the DNA of bacteria and viruses, and it can bind to and activate TLR 9 to induce a T-cell and B-cell based immune response against malignant cells.

    Other oligo drugs in development include Hybridon's (www.hybridon.com) IMOxine, a second-generation synthetic oligonucleotide agonist of TLR 9.

    In April, Hybridon presented data from two preclinical studies in tumor models that demonstrated the drug's antitumor activity and its ability to enhance the effects of chemotherapy, radiation, and antibody therapy and to enhance the immunological response to and antitumor activity of peptide cancer vaccines. IMOxine is currently in a Phase II trial as a monotherapy to treat patients with renal cell carcinoma.

    Dynavax Technologies (www.dynavax. com) is developing short, immunostimulatory DNA sequences for three clinical indications: linked to allergens for the treatment of allergies and asthma; linked to antigens to enhance prophylactic and therapeutic vaccines and immunotherapy of cancer; and as treatments for infectious and inflammatory diseases.

    In March, Dynavax reported data from a Phase I trial of its AIC ragweed allergy immunotherapeutic showing a clinically significant improvement in symptoms and a reduction in medication usage compared to placebo. The company anticipates initiating a pivotal Phase III trial in early 2006 and hopes to begin a supportive Phase II trial in a pediatric indication in the first half of 2005.

    At its large-scale oligo manufacturing plant in Cincinnati, OH, Girindus (www.girindus.com) produces REP 9, Replicor's (www.replicor.com) lead antiviral drug candidate. REP 9 is an oligo drug designed to combat viral infections such as HIV, hepatitis B, herpes, respiratory syncytial virus, influenza, and Ebola virus. Girindus is applying its solution-phase synthesis technology to produce oligos at ton scale.

  • Enzymatic Synthesis of Circular DNA

    Circular, or plasmid DNAs, used in gene therapy and vaccine applications can be produced efficiently using enzymatic processes in place of traditional bacterial fermentation methodologies. CytoGenix (www.cytogenix.com) recently filed a patent application for its chemo-enzymatic synthesis technology, which can produce gram or greater quantities of circular DNA in less than a week at laboratory scale, according to the company. CytoGenix is applying its technology to the discovery and development of DNA-based therapeutics.

    With topical medicines in development to treat chronic herpes infections and inflammatory skin disorders, which require large quantities of DNA, "we looked at the cost to purchase clinical-grade DNA," says Frank Vazquez, executive vp and COO at CytoGenix, and found it too costly to enable the development of a viable product.

    "Bioprocessing is expensive," Vazquez says, and it is "inherently a dirty and risky process." Enzymatic synthesis eliminates the need to remove bacterial toxins and other impurities introduced by fermentation and allows for the design of a smaller plasmid, containing only a promoter and the transgene, without the need for a bacterial backbone or any antibiotic selective genes. The result, according to Vazquez, is a safer, more active, and more economical product.

    The cycle time of the synthesis process is about 26 hours, and all of the steps take place in one reaction vessel. CytoGenix is working with Alfa Laval Biokinetics (www.alfalaval.com) to design a manufacturing facility in the Houston area.

    Boehringer Ingleheim Austria (www.boehringer-ingleheim.com) and BIA Separations (www.biaseparations.com) have partnered to develop high cell density fermentation technology for phamaceutical-grade plasmid DNA manufacturing capable of producing titers of 1 g pDNA per liter.

    The automated bioprocessing technology relies on gentle lysis of the biomass combined with a novel purification process using BIA's Convection Interaction Media (CIM) short monolithic columns that increase productivity by 15-fold compared to traditional supports, according to the company.

    Despite the "slow-down of the clinical development" of gene therapy products since early 2000, in the past two years, those programs "are again being strongly pursued," says Monika Henninger, Ph.D., head of customer relations and projects at Boehringer Ingelheim Austria. She reports increased requests for contract manufacturing services for pDNA products.

    "For pDNA products for gene therapy applications or angiogenesis, our expectation would be that the first products could be approved in about five years." Henninger says.

  • A Difference in Scale

    An emerging trend in oligo manufacturing overall is the shift away from larger-scale production and focusing instead on improved efficiency and reducing the cost of oligo synthesis. This is relevant in particular for the burgeoning RNA market.

    Based on the products currently in development, it appears that the scale of synthesis for RNA drugs will be significantly less than for DNA medicinestens of grams rather than hundreds of kilograms. "The hope is that these compounds will be more potent," says Micheal Mclean, president of Avecia Biotechnology.

    Avecia recently sub-licensed from GE Healthcare the Duplaa RNA synthesis technologyreagent technology based on triethylamine-HF deprotection chemistry to produce siRNA molecules.

    Although the core phosphoramidite solid support-based chemistry used to produce synthetic DNA is the same basic technology used for RNA synthesis, the need to remove a protective hydroxyl group in the RNA molecules represents a fundamental difference between the two processes.

    With RNAi medicines gaining momentum in development, "it is important to keep the manufacturing technology moving on as drug discovery moves on," says Mclean.

    The Duplaa technology is fast, notes Mark Douglas, IP manager at Avecia Biotechnology, which "is important because you are dealing with a sensitive molecule at that point" in the synthesis. He points to the relatively low cost of the technology and the ease of salt removal during purification as other advantages.

    Avecia is manufacturing siRNAs in clinical development for companies such as Acuity Pharmaceuticals (www. acuitypharma.com). Earlier this year Acuity filed an IND for its lead siRNA product, CAND5, for the treatment of age-related macular degeneration. The drug targets vascular endothelial growth factor (VEGF) to inhibit blood vessel overgrowth and vascular leakage.

    As the cost of producing RNA molecules is higher than for DNA due to the more complex chemistry required and the higher cost of raw materials, Douglas points to the predictions of 10-fold greater efficacy of siRNA compared to DNA antisense as a key factor in keeping down the ultimate cost of oligo therapeutics.

  • Moving siRNAs Forward

    Boulder, CO-based Sirna Therapeutics (www.sirna.com) is also moving forward with its first clinical trial of a chemically modified short interfering RNA (siRNA). The trial is evaluating Sirna-027 in age-related macular degeneration; the drug targets VEGF receptor-1 (VEGFR-1) to inhibit angiogenesis.

    In May, Sirna announced the results of an interim analysis of the Phase I trial, reporting that the drug appeared to be safe and well tolerated, with no dose-limiting toxicities.

    Of the 14 patients who each received a single intravitreal dose of Sirna-027, ranging from 100 to 800 micrograms, all of the patients experienced stabilization in visual acuity, and half the patients had improved visual acuity, which appeared to be dose dependent.

    "We have the capacity to manufacture 20 to 30 kilos of siRNAs, or other oligos, per year, and can purify more than 80 kilos per year," says Howard Robin, Sirna president and CEO.

    Sirna makes all of the siRNAs in its own pipeline and has an ongoing agreement with Archemix (www.archemix.com) to produce all of the aptamers it will need through Phase IIa trials.

    "We are in the range of being able to manufacture oligos at prices that would make them commercially reasonable today," says Robin. And he expects costs to continue to drop as the manufacturing process becomes even more efficient.

    Sirna's other development projects include a partnership with Eli Lilly to assess the effectiveness of siRNAs against various oncology targets, interfering RNAs to treat hepatitis C, asthma, diabetes, and Huntington's disease, and the establishment of Sirna Dermatology, which will initially apply siRNAs for permanent hair removal.

    Atugen (www.atugen.com) is also focused on the development of siRNA-based drugs and announced a collaborative discovery program with Asinex (www.asinex.com) to identify novel protein kinase drug targets.

    Atugen plans to take its first siRNA therapeutic into the clinic later this year.

    The company's proprietary technology yields chemically stabilized siRNA molecules (atuRNAi's) intended to be less susceptible to degradation by nucleases present in body fluids.

    These siRNA molecules are 19-21 nucleotides in length, blunt-ended, and composed solely of RNA. Atugen is focusing on two disease indications for its intravenous and intramuscular siRNA products (metabolic diseases and epithelial cancers) and two indications for its topical formulations (ocular diseases and skin diseases).



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