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Feature Articles : Mar 15, 2010 (Vol. 30, No. 6)

Oligo Therapeutics Enriching Pipelines

As Development Escalates, Delivery Strategies, Chemistries, and Modifications Also Improve
  • Vicki Glaser

Market research figures compiled by Agilent Technologies reveal that in 2009, $407 million in direct financing was targeted to the development of oligonucleotide therapeutics in the U.S. This figure represents a combination of publically announced venture capital, private placement, IPO, secondary financing, and up-front collaboration payments.

The total deal value for therapeutic oligos in 2009—including up-front payments and potential milestone royalties—approached $2.8 billion, about the same as in 2006 and less than half the total for 2008. This total covers the full spectrum of oligo-based drugs: miRNAs, siRNAs, aptamers, decoys, antisense, immunostimulatory oligos, and other related drug classes.

Continuing an upward trend begun in 2003, the number of oligo therapeutic programs has increased each year, growing to 231 in 2009, representing a more than 8% increase from 2008.

As the number of oligo therapeutics in development continues to increase, so too does their complexity, the scale of oligo manufacturing to support late-stage development and commercialization programs, and the range of chemistries, modifications, delivery strategies, and purification and analytical methods. This diversity will be evident in the scope of presentations at IBC’s upcoming “TIDES” conference.

At the meeting, Agilent will unveil a novel technology for large-scale RNA deprotection. The technology combines chemical and engineering solutions that overcome the problems caused by the exothermic nature of conventional deprotections.

Heat-induced degradation of RNA during deprotection has, to date, hindered the ability to scale RNA production above 200 grams. Agilent has coupled on-column cleavage of RNA from the solid support with removal of the deprotecting groups in a semi-continuous, scalable process. The cleavage step involves pumping reagents through the synthesis bed and into a holding vessel. For deprotection, the contents of this vessel are continuously combined with deprotection reagents in a temperature-controlled mixed stream.

Paul Metz, director of operations at Agilent, describes this technology as another step in the company’s goal of transforming discrete steps in the manufacturing process into more continuous, closed process steps that facilitate automation and large-scale production.

Scaling Up

Metz reports that beginning in late 2009 and continuing into this year, Agilent has seen a growing number of late-stage programs for oligo APIs. These include scale up and supply of Phase III trial materials through to full process validation and commercial launch. The company’s new large-scale, multipurpose GMP facility in Boulder, Colorado, was designed to accommodate kilogram-scale oligo production. The plant has capacity in the low hundreds of kilograms per year and is on-stream and fully qualified. Agilent anticipates several projects moving forward into large-scale production in the 2011–2013 timeframe.

Exemplifying some of the challenging aspects of producing larger quantities of atypical oligonucleotides is the experience of Noxxon Pharma, which has taken its Spiegelmer® oligos into clinical development. NOX-E36 is completing a Phase I trial in inflammation and will enter a Phase II study in 2010. NOX-A12, designed to stimulate autologous stem cell recruitment, is also finishing up a Phase I trial and will begin a proof-of-concept Phase II study this year.

In December, the company announced the identification of a Spiegelmer with picomolar affinity and high selectivity for a target being pursued in collaboration with Eli Lilly for the treatment of migraine headache.

Spiegelmers are mirror-image, L-configured oligos composed of the L-isomer of ribonucleic acid. They bind to a biological target in a manner similar to antibody-antigen recognition, explains Stefan Vonhoff, Ph.D., vp of chemistry, manufacturing and control at Noxxon. Because they are not degraded by naturally occurring nucleases, Spiegelmers offer an attractive in vivo stability profile. They are unmodified L-RNA oligonucleotides that are synthesized using standard production processes established for natural D-configured oligos.

Initially, “the development of a scalable and cost-efficient manufacturing process for the L-RNA monomers” was a challenge, says Dr. Vonhoff. Today, the monomers are synthesized at multikilogram scale and the key raw material, L-ribose, has turned into a chemical commodity produced at ton scale for the manufacturing of antiviral drugs such as clevudine, which is used to treat hepatitis B.

Now, notes Dr. Vonhoff, Noxxon’s main process-development focus is on GMP manufacturing of Spiegelmers, in which efficient purification strategies are key to producing the drug substance at a consistently high quality. Noxxon has initiated a program with a CMO partner “to improve loading efficiency while maintaining the quality of the product,” says Dr. Vonhoff. The main impurities produced during the manufacture of Spiegelmers are “failure sequences,” he adds. “Due to their unnatural L-configuration, those N(+)-mers and N(-)-mers” are unlikely to exert off-target effects.

As therapeutic oligos move through preclinical testing into human studies and on to commercialization there is an increasing need for higher throughput analytical methods and strategies for assessing the composition and purity of larger amounts of compound isolated from biological samples such as blood and tissue. Analytical processes focus on identifying potentially active metabolites, quantifying and identifying impurities, and characterizing the pharmacokinetic properties of the drug as part of required ADME studies.

Analyzing Oligos

Michael McGinley, bioseparations product manager at Phenomenex, describes the pharmacokinetic analysis of oligonucleotide drugs as “a brand new field” and “a growing area of concern.”

Oligo therapeutics are essentially small polymers. “They are highly polar and most have been massively modified,” he says. This makes even traditional reverse-phase liquid chromatography a challenge. For pharmacokinetic studies that require analysis of oligo drugs isolated from biological samples, the difficulties include purifying the oligos from serum or plasma and getting rid of the lipids, organelles, and other large molecules present in tissue samples.

Oligos do not adhere well to reverse-phase chromatographic media because of their high polarity. “You have to add ion-pairing reagents, which tend to suppress the sensitivity of mass spectrometry.” 

Adding yet another level of complexity are the modifications to the oligo backbone intended to enhance the stability, bioavailability, and activity of oligo therapeutics. Many of the drugs in development “are more RNA than DNA and the modifications are getting even more esoteric these days,” McGinley says. Further complicating the picture is the attachment of peptide or lipid leaders to facilitate entry of the oligos into cells.

With recent improvements enabling the synthesis of larger quantities of therapeutic oligos, many of the past manufacturing challenges are being overcome. However to bring down costs, some manufacturers are looking to overseas amidite vendors, primarily in India and China, and “we are starting to see some impurities in these amidites” making the need for LC/MS analysis even more critical for monitoring the impurity profiles of oligo APIs.

Phenomenex’ Clarity® BioSolutions portfolio of products for synthetic oligonucleotide purification and analysis has grown and evolved to include Clarity Oligo-RP™, reverse-phase HPLC columns launched in 2006 for achieving greater than 90% purity at both small and large synthesis scales, to Clarity QSP™ for high-throughput trityl-on purification in 96-well plates formats. Clarity Oligo-WAX™ ion-exchange columns and bulk media are designed for preparative purification on HPLC or FPLC systems.

At “TIDES” in 2009, the company introduced the Clarity OTX™ solid-phase extraction system for cost-efficient isolation of oligo therapeutics from biological fluids and tissues in 15 minutes. And most recently, it unveiled Clarity Oligo-MS™, which McGinley describes as an ultra-high resolution reverse-phase column for rapid and efficient LC/MS analysis on both HPLC and UHPLC systems.

State-of-the-art oligo synthesizers can produce oligos at millimolar scale, putting pressure on purification technology and strategies to be able to handle gram quantities of oligo drugs. McGinley sees a need for “better ion-exchange preparatory and analytical solutions” and continued development of HPLC solutions. “People started out treating oligos like proteins, using low pressure LC for purification. Now they are taking advantage of the benefits of HPLC.” He also notes a shift toward the use of stainless steel dynamic axial compression columns in place of glass columns.

Leveraging Chemistry

Chemistry-based solutions can contribute to the design of more efficient and cost-effective synthetic protocols for complex oligo therapeutics. Geron developed a chemo-enzymatic strategy for producing the 3´-aminonucleosides that comprise its telomerase inhibitor currently in several clinical trials against cancer. Imetelstat (GRN163L) is an oligo (N3´-P5´-thio-phosphoramidite)-lipid conjugate. The drug competes with telomeres for the active site of telomerase, inhibiting its enzymatic activity.

Imetelstat is a 13-mer uniformly modified oligonucleotide molecule. During the earliest stages of development, Geron experimented with many chemical synthetic processes to yield a molecule with optimal potency and bioavailability. The company developed a phosphoramidate and thio-phosphoramidate chemistry that resulted in an estimated 10-fold increase in telomerase inhibition.

With the addition of a 5´ palmitoyl lipid group the molecule demonstrated higher bioavailability and prolonged half-life in plasma. The lipid domain may allow the molecule to self-formulate into pseudomycellular structures under certain conditions.

Following selection of the most potent molecule and the optimal chemistry for producing the conjugated compound, Geron focused on improving the efficiency and economic viability of the synthetic manufacturing process, ultimately reducing the number of steps required to produce the nucleoside monomer building blocks from 36 to 11. It accomplished this by switching from an entirely chemical process to a chemo-enzymatic synthesis involving trans-glycosylation.

Geron relies on 3´-amino-thymidine as the starting material. It is derived from a component of the readily available and affordable anti-HIV agent 3´-azidothymidine (AZT) and serves as the common precursor for making three of the four monomer building blocks.

This switch “reduced the cost for producing our amidites by 95%,” says Sergei Gryaznov, Ph.D., director and senior research fellow at Geron, adding that the current synthetic process is readily scalable by at least 10- to 100-fold.

Sourcing Raw Materials

Agilent is continuing to develop Process Analytical Technology for monitoring process steps in real-time, as well as LC/MS and MS-MS technology to enhance the analysis of amidite purity.

“Identification of degradation products and process-related impurities that couple during oligo synthesis is key to setting meaningful raw material specifications,” says Metz. As more products move into later stage development, “there could be pressure on the amidite supply by 2013, and security of supply will be an important commercialization factor to consider.”

“While there are currently stable Western sources, multisourcing options from Asia are also being developed. Agilent has built analytical centers of excellence in both India and China and plans to use these resources to work with new amidite supplies to “help them better understand their starting raw materials and processes and to control amidite impurities.”

Agilent’s goal is to ensure a more efficient and reliable supply chain by creating “supplier partnerships” that will allow for in-process testing and prerelease of amidites to Agilent in-country. After processing, the amidites would be analyzed for purity and impurity levels before they are sent on to Agilent’s manufacturing facility in Boulder, where they would then undergo regular GMP testing prior to use in production.

Agilent has completed proof-of-concept for its thin-film evaporation and spray-drying technologies, and these are now available as processing options in early-stage development for products expected to require large-scale manufacturing as they progress toward commercialization.

The company is building a commercial-scale thin film evaporator to support one of its late-stage programs. Both the thin-film evaporation and the spray-drying technologies reduce “product time-at-temperature,” which translates to less chance for thermal degradation, explains Metz. They also provide for higher-throughput compared to current rotary evaporation and lyophilization technologies.

Quantifying Oligos in Clinical Samples

Hybridization assays for quantifying oligo drug levels in biological samples have advantages over conventional chromatographic techniques such as capillary gel electrophoresis or HPLC. The main advantage is the high sensitivity of ELISA assays, the small sample volumes required, and the ease of use, with little or no sample clean-up required prior to assay processing.

In fact, “oligo levels can be measured in homogenized tissue samples without any prior extraction,” says Helen Legakis, research scientist in immunochemistry laboratory services at Charles River Laboratories. In contrast, tissue samples are subjected to sonication and enzyme digestion to break down the tissue membranes.

As oligo-based therapeutics are mainly administered parenterally, a high assay sensitivity is essential to characterize their pharmacokinetic and toxicokinetic properties following systemic administration. This is particularly important at “the terminal elimination phase, characterized after near-complete distribution of the drug has occurred,” notes Legakis.

To achieve optimal sensitivity, Charles River designs its probes to include Locked Nucleic Acid inserts, which contain a rigid bicyclic structure in the ribofuranose ring. This reduces the background noise of the assay and increases the thermal melting temperature of the oligo analyte and the complementary probe. The increased binding affinity to the probe prevents competitive re-annealing of double-stranded analytes to their complementary strand, Legakis explains.

Looking to the future, Legakis sees a growing demand for multiplexing technologies coupled to an ELISA-based assay format. With the emergence of multiple therapeutic targets, these methods will be able to measure more than one analyte in a multianalyte drug cocktail in a single well.