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Mar 15, 2010 (Vol. 30, No. 6)

Oligo Therapeutics Enriching Pipelines

As Development Escalates, Delivery Strategies, Chemistries, and Modifications Also Improve

  • 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.


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