At the upcoming “TIDES” conference in Las Vegas later this month, oligo manufacturers will discuss a range of opportunities and challenges related to the growing market for DNA and RNA therapeutics. Topics will include the application of process analytical technologies (PAT) in oligonucleotide manufacturing, scale-up strategies in the synthesis of oligo-based therapeutics, quality issues related to primer and probe production for use in molecular diagnostics assays, and challenges in manufacturing RNA oligos.
In April, Eurogentec www.eurogentec.com) celebrated the opening of its new GMP oligonucleotide-manufacturing facility in Liège, Belgium for producing in vitro diagnostics (IVD). To meet the stringent requirements for IVD products, the new facility and quality-management systems are fully compliant with ISO 13485:2003 quality standards and the FDA’s 12 CFR 820.
Features include card-key access, airlock pass-through to minimize bioburden and cross-contamination risk, and gowning policies for all employees to ensure segregation of critical manufacturing steps that require the use of Class 100,000 and 10,000 cleanrooms and Class 100 working zones. The company anticipates receiving its ISO certification in the fourth quarter of this year.
Eurogentec also announced expansion plans for its oligonucleotide facilities in the U.S., with enhancements to match the capabilities of the new European site. The firm is also discussing with its Japanese partner, Nippon, an upgrade of the oligo manufacturing facility in Japan. Back-up capacity and redundancy are particularly important to serve the growing diagnostics market, according to Peter Haima, Ph.D., senior project manager, IVD, at Eurogentec.
“The regulatory aspect is complex, and IVD start-ups are struggling with the decision to use normal or more costly GMP oligos,” says Dr. Haima. “Both the FDA and the European Union’s IVD Directive state that CMOs and suppliers of critical components for IVDs are encouraged to comply with GMP guidelines.”
Integrated DNA Technologies (IDT; www.idtdna.com) is launching its European facility in Leuven, Belgium, next month.
“We have stayed close to home for more than 15 years, and it’s time to branch out,” says Trey Martin, III, COO and svp at IDT. “The European market is still more fragmented than in the U.S., with a lot of local service providers.” Although the oligo market is not quite as large in Europe as in the U.S., with the EU’s Framework Initiative trying to compete with the NIH in scope and size, “we’re putting a lot of emphasis on Europe because we think they will be successful.”
GE Healthcare (www.gehealthcare.com) recently installed an ÄKTA OligoPilot 400 DNA/RNA synthesizer at BioSpring (www.biospring.de). At the installation, Ann O’Hara, GM, bioprocess, at GE, remarked that “the European market has been behind the American market in clinical trials for oligonucleotides, so we are pleased to support BioSpring at the center of driving the region’s development of large-scale manufacturing.”
“The number of pipeline projects for therapeutic oligos is increasing substantially and several candidates are now in clinical trials, so the need for production of large quantities of highly pure oligos has increased,” explains Sylvia Wojczewski, Ph.D., CEO at BioSpring, commenting on the expansion of the company’s GMP production capabilities.
“We are seeing increasing demand in the market, with many new research projects for both DNA and RNA; RNA, in particular, is a very strong field at the moment,” says Dr. Wojczewski.
“The market is still in a relatively immature state, with many candidates in early phase trials,” observes Kevin Fettes, Ph.D. process development group leader at Avecia Biotechnology (www.aveciabiotech.com).
“With the rapid pace of discovery, we are confident that there will continue to be many organizations with compounds in development, and this will attract larger pharmaceutical organizations into collaborations and buyouts, as we have seen with, for example, Roche/Alnylam and Merck/Sirna and within-industry collaborations such as Regulus Therapeutics (Isis/Alnylam).”
Increasing competition within the CMO sector will accompany the continued maturation of the oligos sector as DNA compounds in development become candidates for process validation and commercial launch, notes Dr. Fettes.
Citing the continuing rapid growth in the therapeutic oligonucleotides market, Gary Carter, business development and market manager for Agilent’s (www.agilent.com) nucleic acid-solutions division, notes that “the number of publicly disclosed oligo therapeutic programs has almost doubled in five years, from 121 in 2002 to 213 in 2008, and the number of companies involved in the field either directly or via collaborations has increased from 40 to 89 during the same period.
“Furthermore, oligo therapeutic companies have received over $3.8 billion in direct investment funding (upfront collaboration payments, venture funding, and private, IPO, and secondary placement funding) since 2002,” notes Carter.
Compared to 2006, the total potential deal value including all upfront payments and potential milestone and royalty payments nearly doubled for the 16 therapeutic oligo collaborations signed in 2007, rising from $2.7 billion to $5.1 billion. “Big pharma is rapidly increasing its presence in the oligo therapeutic field,” observes Carter, with all of the top 12 pharma companies involved in at least one therapeutic oligo collaboration in 2008, compared to only three companies in 2002.
Agilent Technologies recently purchased the nucleic acids-medicine assets of DowPharma, giving it ownership of DowPharma’s lab-to-commercial-scale GMP oligo-manufacturing capabilities.
The acquisition “gave us a significant addition of scale and capacity,” says James Powell, GM of Agilent’s nucleic acid-solutions division. The DowPharma deal is just one component of Agilent’s ongoing expansion initiative. The company has initiated two expansion projects during the past 18 months at its nucleic acid-solutions site in Boulder, CO.
The first expansion added two ÄKTA OligoPilot 400 synthesizers and tripled downstream-purification capacity. The second project, now under way, will integrate a 1 mol synthesizer, a 30 L/min chromatography skid, two lyophilizers, each with 5 kg/batch capacity, and supporting utilities and infrastructure for commercial-scale GMP manufacturing.
In addition to expanding capacity and expertise, Agilent is focused on innovations aimed at reducing cost, increasing manufacturing throughput, and improving product quality. For example, Agilent entered into an exclusive licensing agreement with Aktiv-Dry (www.aktiv-dry.com) that provides access to CAN-BD (carbon dioxide assisted nebulization with a Bubble Dryer®) for nucleic acid spray-drying as an alternative to traditional, batch freeze-drying techniques. This low-temperature, closed, continuous drying process is milder, faster, and substantially less costly than lyophilization, according to Paul Metz, senior director, manufacturing operations, at Agilent.
“Spray-drying yields a uniform, fine, free-flowing powder with low static properties,” says Powell. Aktiv-Dry’s spray-drying technology involves high-pressure, continuous mixing of an oligo API feed stream with supercritical fluid CO2. The combined stream passes through a flow restrictor, after which the CO2 is evaporated, producing a micronized API powder. Agilent has completed proof-of-principle studies with CAN-BD spray-drying of DNA and siRNA APIs. Process optimization and scale-up are under way, and Agilent plans to offer spray-drying to its customers later this year.
Agilent will preview at “TIDES” a new oligo-synthesis chemistry designed to reduce the cost of siRNA therapeutics, which it plans to launch later this year. The technology aims to reduce the cost of monomers with the introduction of a 2´-hydroxy protection scheme.
In parallel with the construction of several kilo-scale manufacturing laboratories at its site in Bellshill, Scotland, Link Technologies (www.linktech.co.uk) recently announced receipt of ISO 9001:2000 certification. Link will use the new facilities for both routine production to support its established catalog business and to produce custom products for diagnostic and therapeutic applications.
John Bremner, Ph.D., business development director at Link, cites continued strong market demand for a variety of oligo modifiers such as linkers, dyes, and amino modifiers for postsynthetic modifications. The advantages of introducing modifications may include greater stability, reduced dosing, and better product performance.
In particular, he notes growing interest in the company’s new 5´-Cholesterol-CE Phosphoramidite, which has a hydrophobic cholesterol group that is intended to improve penetration into cells. In contrast to a postsynthetic method of conjugating an amino-modified oligo to cholesterol chloroformate, the Link process achieves direct 5´ attachment during oligo synthesis using the modified phosphoramidite, he says.
Compared to other cholesterol amidites and pegylated oligos, this product offers several advantages for therapeutic oligo development. According to the company, it is not susceptible to 1,2-diol elimination; it does not contain a trityl group, which can interfere with purification; it has a greater than 90% coupling efficiency for high yield of modified product; and it dissolves in dichloromethane for use in automated synthesis.
Link also recently commercialized the sulphur-transfer reagent EDITH, an acronym for 3-ethoxy-1,2,4-dithiazoline-5-one. Sulphurization modifies the DNA or RNA backbone by replacing one nonbridging oxygen atom in the phosphodiester to create a phosphorothioate linkage.
“Our customers tell us that competing sulphurization agents are less suitable in a production arena,” says Dr. Bremner. “EDITH offers an all-around capacity. It is soluble in the standard solvent for phosphoramidite dilution, acetonitrile (other Beaucage alternatives require either pyridine or picoline) is stable in solution for several months and exhibits high sulphurization efficiency with both DNA and RNA.”
Link offers a range of SynBase™ controlled-pore glass solid supports including unfunctionalized and universal supports and for automated solid-phase oligo synthesis. Universal supports offer the advantage of low inventory costs. “The support does not determine the first base or modifier,” notes Dr. Bremner. “It also leaves nothing behind on the oligo.”
Overall, Dr. Bremner describes a larger and more diverse customer base for the company’s catalog business and growing demand from oligo manufacturers for more projects and larger scale.
BioSpring has been focusing a great deal of its efforts on analysis and characterization of products, from the raw materials to the finished oligos. Identifying impurities and changes in RNA preparations is more difficult than for DNA, according to Hüseyin Aygün, Ph.D., CSO at BioSpring, who will take part in a “TIDES” presentation on the issues related to RNA-oligo manufacturing.
“My talk will focus on the impact of raw materials and batch-to-batch changes in serial batch production of the same oligo,” says Dr. Aygün. “For example, how does the fingerprint of the product change when you change the raw materials? Parts of the final product do not change, and these are quite clear to characterize; the parts that do change are more difficult to characterize.”
A lot of characterization work has been done with DNA oligos, but RNA synthesis involves different protecting groups and different chemistries, “which have an impact on the final product and the side products you can generate,” says Dr. Aygün.
“We have purchased some new instruments to fulfill the requirements of our customers, who are not only asking for larger-scale quantities” but are also demanding better characterization of the impurities of RNAs. BioSpring is implementing LC-MS analysis to characterize its products. LC-MS analysis has become increasingly common for DNA oligo-product characterization, and RNA analysis by LC-MS is becoming increasingly important. It enables a detailed analysis of impurities and side products, Dr. Aygün asserts.
“Interestingly, about a year ago, customers developing diagnostic applications have started asking for impurity characterization of their products,” according to Dr. Aygün. Additionally, whereas impurity analysis has typically been part of the validation process implemented in later-stage clinical development, he notes that customers developing therapeutic RNAs “are asking very early for validated processes,” moving up the pipeline into toxicology testing and the early clinical trial phase.
This more stringent analytical effort “is helping us optimize our processes and check for batch-to-batch variation in oligonucleotide purity,” comments Dr. Aygün.
With the integration of its new drying technology, Agilent takes a step closer to fulfilling its vision “of a continuous, closed process, from synthesis, all the way through to drying,” says Metz, rather than a series of discrete process steps with isolated intermediates.
The company plans to support this vision with PAT, which “can allow for feedback control of critical process steps such as chromatography fractionation in real time or near-real time,” says Metz, who will give a “TIDES” presentation titled “Opportunities for Process Analytical Technologies in Oligonucleotide Development and Manufacturing.”
Metz will discuss the implementation of PAT to support a risk-based approach to drug development, API manufacturing, and process control. “PAT technologies are already incorporated in automated oligo-synthesis equipment, and we are evaluating additional opportunities to implement PAT in synthesis and downstream processing.”
Initially, the company is developing approaches to online sampling and continuous dilution of the synthesis process stream. This is being applied to nucleotide sequencing. “Rather than waiting until the end of the process and sending the completed molecule to the lab for analysis, we want to be able to confirm the sequence chemically online in real time or at-line in near-real time,” says Metz. Agilent is exploring UV and direct MS approaches to PAT.
“What we learn in this investigation should be applicable to downstream-process sampling and analysis,” notes Metz. “We are just at the beginning stages of identifying best practices for continuous oligo process sampling and applying our analytical expertise to online process monitoring and control.”
A variety of factors will contribute to reducing the costs of oligo manufacturing, including lower raw-materials costs as demand increases, improvements in synthesis chemistry, enhanced synthetic efficiency and yield, refined purification strategies, and process optimization across the production stream.