April 15, 2012 (Vol. 32, No. 8)

Neil McKenna, Ph.D.

Oligonucleotide therapeutics make up a small but rapidly growing segment of the overall global drug pipeline, with 245 programs currently in development today. While only one approved oligo therapeutic is currently on the market (Pfizer’s Macugen®), there are currently nearly 250 programs under development. Direct investment in this space was nearly $400 million in 2011, and it is projected that this class of drugs could represent up to 10% of total approvals in the next two years in the U.S. and EU, according to Gary Carter, director of strategy and marketing at Agilent Technologies’ Nucleic Acid Solutions Division (NASD).

Oligo therapeutics include siRNAs, antisense oligos, miRNA inhibitors, and mimics, with cancer, infectious diseases, ocular conditions, and cardiovascular, CNS, and renal conditions being the primary targets among a diverse pipeline.

With increasing numbers of oligo therapeutics nearing the market, the manufacturing process for these drugs is assuming increased importance, with considerations of raw material availability, product yield and purity, cost effectiveness, reproducibility, validation, and product consistency during process scaling being critical parameters.

The forthcoming IBC Life Sciences “TIDES: Oligonucleotide and Peptide Research, Technology and Product Development” conference will feature several speakers from key players in the oligo therapeutic arena providing a variety of perspectives on the oligonucleotide manufacturing process.

Topics that will be addressed range from chemical strategies to improve oligonucleotide delivery, uptake, and targeting, to logistical issues associated with maintaining the worldwide oligonucleotide supply chain. The supply chain encompasses companies that specialize in sourcing raw materials required for the production of oligonucleotide synthesis reagents, as well as companies that focus on the logistics of commercial scale-up of the actual oligo production process.

Agilent is investing in its oligonucleotide manufacturing in anticipation of continued growth in this area. “NASD’s three-year plan includes further expansion of our state-of-the-art kilo manufacturing facilities to support a projected 10-fold increase in demand for a broad portfolio of oligo APIs,” says Skip Thune, general manger of Agilent’s NASD.

A critical hurdle for oligonucleotide therapeutics is efficient drug delivery. Selectively targeting the oligo to desired tissues and ensuring its efficient uptake at the cellular level and release within the cytosol requires specific design strategies for each oligo.

“One strategy for drug delivery is to chemically conjugate oligonucleotides to a delivery vehicle, which can include polyethylene glycol polymers, cholesterol, vitamins, sugars, and peptides,” says Kenneth Hill, Ph.D., principal investigator in Agilent’s NASD.

“An important consideration is whether or not the oligonucleotide can be modified while on the solid support,” he adds. If the modification group is not amenable or is unstable to the synthesis or deprotection conditions, then a post synthetic means of conjugation is required.

“Thiol-, amine-, or aldehyde-compatible chemistries originating from protein modification have been used for oligonucleotides,” says Dr. Hill. More recently, pericyclic reactions for conjugation have grown in popularity, since the reagents and conditions are very mild, with high yields and little to no side products.

At the opposite end of the drug pipeline to drug delivery, another key consideration in the manufacturing process is management of change and impact of raw material impurities on drug quality.

“A well-documented supply chain risk assessment is not only prudent but is expected by regulatory authorities as part of the development process,” observes Paul Metz, senior director of operations at Agilent.

Nucleoside phosphoramidites are highly reactive 3´-O-(N,N-diisopropyl phosphoramidite) derivatives of nucleosides that are the basic starting material in the oligo manufacturing process. DNA and RNA phosphoramidites are typically manufactured in reactors before purification by preparative column chromatography on silica gel with medium- to high-pressure chromatography equipment.

The presence of critical impurities in chemical preparations of phosphoramidites is an important regulatory and safety consideration during the manufacturing process. “Monitoring and testing for critical impurities in phosphoramidites starts as soon as we receive the raw material,” adds Metz. The industry is beginning to harmonize the testing methods and specifications for these key oligonucleotide building blocks.

Agilent is investing in oligonucleotide manufacturing in anticipation of continued growth in this area. Current facilities include a large-scale tangential flow filtration system in Boulder, CO.

Large-Scale Manufacturing

SAFC has more than 25 years experience in the manufacturing of DNA and RNA synthesis reagents. Its main oligonucleotide reagent manufacturing facility is located in Hamburg, Germany. Annual amidite production capacity at the Hamburg plant is in excess of five tons, making it the largest such facility in the world, according to the company. Large-scale production facilities at the plant include 1,000 L glass-lined reactors in addition to mid-size reactors (20–200 L).

“The supply chain for phosphoramidites starts with plant-derived materials like corn starch, and encompasses both unprotected and protected nucleosides,” says Andreas Wolter, site manager at SAFC. “Whereas many unprotected nucleosides are manufactured in large scale for the production of nucleoside-based APIs like zidovudine or as food additives, other nucleosides can be made from these major raw material streams through simple chemical or biocatalytic transformations,” he adds.

SAFC taps into this established supply chain to obtain unprotected and protected nucleosides, which are converted to phosphoramidites in the Hamburg plant. “In contrast to other manufacturers, we employ large-scale chromatographic purification as the key step for the purification of our amidites,” says Wolter.

“This results in greatly increased consistency, and reduced lot-to-lot variations,” he adds. SAFC utilizes a multivendor strategy to ensure a stable supply of raw materials into the manufacturing pipeline. “While some vendors have dropped out of the market over time and others have entered it, we have not experienced any shortage in these materials over the past decade,” says Wolter.

According to SAFC, annual amidite production capacity at its Hamburg plant is in excess of five tons.

Liquid Reagents

In solid-phase synthesis of oligonucleotides, liquid reagents are used in each step of the synthesis cycle. Overall synthesis performance, and therefore total product yield and purity of the crude oligonucleotide, is highly dependent on the chemical purity of the monomers and the supporting liquid reagents.

Over the past decade or so EMD Millipore has been involved in liquid reagent supply for oligonucleotide synthesis. John Koterba, product manager solvents, highlights the commercial-scale logistics associated with scale-up from clinical volumes to commercial scale and how this impacts the supply chain and the impact to the customer/vendor relationship.

“Manufacturing gram to kilogram to multiple kilograms of APIs on an annual basis has an impact on the critical liquid reagents supply,” says Koterba. “EMD Millipore has made recent capacity enhancements to account for these commercial-scale requirements,” he adds.

Deblocking is a critical step in oligonucleotide synthesis, in which the protecting group from the 5´ hydroxyl moiety of nucleotides already incorporated into the growing nucleic acid are removed prior to the addition of the next phosphoramidite. Removal of the blocking group allows the unprotected 5´ hydroxyl moiety to react with a new phosphoramidite in a subsequent extension reaction.

“We can produce halogenated acid deblocking solutions in the range of 400–7,400 liters,” says Koterba. During the oligonucleotide synthesis cycle, typically 1–2% of oligonucleotide chains will contain unreacted 5´-hydroxyl groups that did not react with the phosphoramidite moiety.

These unreacted groups must be blocked from further elongation in order to prevent the introduction of errors into the final oligo product. Capping with an acetyl group renders these 5´ hydroxyl groups unreactive for subsequent synthesis steps.

Blockmer Technology

Other companies represented at the meeting are developing chemical strategies to streamline the oligonucleotide synthesis process in order to reduce costs. Kyeong Eun Jung, Ph.D., senior director of oligonucleotide research and development at ST Pharm Co. (formerly Samchully Pharmaceutical) cites blockmer technology as a promising approach to more efficient oligonucleotide synthesis.

Conventional oligonucleotide synthesis involves the sequential coupling of monomeric nucleoside phosphoramidites. In the blockmer synthetic strategy, an oligonucleotide analog is generated by sequential coupling of short protected oligomers or blocks (for example, a dinucleotide) on a solid support. Advantages of this approach include a smaller number of synthesis cycles required to prepare an oligonucleotide, saving time and reducing the amount of starting reagent required.

“During synthesis of a 21 mer anticancer siRNA targeting noxin-like UV-induced anti-apoptotic protein, we reduced the number of coupling steps from 20 to 10,” says Dr. Jung. Another advantage is increased purity. “We have found that incorporation of a single dimer into a 21 mer siRNA can result in a 10% to 20% increase in purity,” he adds.

STPharm is also investigating the feasibility of more efficient recovery of phosphoramidite dimers as a means of reducing the costs of oligonucleotide synthesis.

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