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May 1, 2013 (Vol. 33, No. 9)

Critical Tools for Oligo Characterization

  • But Is It Good Enough for QC?

    The high resolution of LC/MS makes it a powerful tool for analyzing oligonucleotide impurities during process development. The knock on this technique is its lack of robustness for quantitative QC determinations. Edward Huber, Ph.D., director, quality control and analytical development, Girindus America, hopes to demonstrate the suitability of LC/MS for both characterization and QC.

    Girindus (recently acquired by Nitto Denko Avecia) has a lot at stake in oligo characterization. Last year the company entered into a strategic alliance with Marina Biotech. The deal provides Girindus/Avecia with exclusive rights to Marina’s conformationally restricted nucleotide (CRN) synthetic process. In return Marina gets royalties from the sale of CRN-based oligonucleotide reagents, plus preclinical and clinical cGMP material for Marina and its drug development programs.

    CRNs are patented analogs in which a chemical bridge spans from the C2´ and C4´ carbons of ribose, the central component of a nucleotide. CRN locks the ribose within a fixed conformation, which restricts the molecule’s flexibility, thus allowing more specific reactivity and production of putative RNA medicines that were previously believed to be not “druggable.”

  • Orthogonal Methods

    Michele DeRider, Ph.D., lead scientist at Catalent Pharma Solutions, points out the strengths and weaknesses of nuclear magnetic resonance, circular dichroism, and fourier-transfer infrared spectroscopy (FTIR) in oligonucleotide analysis and characterization.

    Among NMR’s strengths: it is quantitative, has broad dynamic range, is nondestructive, and versatile. “There are so many levels of information available with NMR, depending on how you use it,” Dr. DeRider says. A big plus for oligos is that the typical formulation buffer is also the NMR solvent of choice. The most-studied nuclei are protons and 32P, the latter being diagnostic for specific base linkages.

    “NMR’s weaknesses include expensive equipment and operation,” Dr. DeRider says. NMR uses liquid helium to cool the probe to near absolute zero. The current helium shortage has made the material prohibitively expensive for many research groups.

    Circular dichroism, while extremely sensitive to subtle conformational variations, does not analyze molecular structure as deeply as NMR or IR. “You’re limited to looking for minima and maxima, but other than that it is not very feature-rich. It’s most useful for stability or thermodynamic features,” Dr. DeRider notes. “And it can be too sensitive at times to trace contaminants.”

    By contrast FTIR instrumentation, commonly used to identify compounds from the “fingerprint region” spectrum, is ubiquitous and easy to use. While some IR bands will be diagnostic for specific linkages, the spectra are extremely complex. This does not help with oligonucleotides, which are all composed of the same four building blocks plus sugars and phosphate.

    While Dr. DeRider is partial to NMR, she confirms that spectroscopic characterization of oligonucleotides is a puzzle-like exercise. “You have to piece information from different sources together. Each method yields more and more information.”

  • Product Specifications: Why We Analyze

    Click Image To Enlarge +
    Isis Pharmaceuticals has invested heavily in process and product knowledge, in-process controls, and process validation.

    The point of oligonucleotide analysis is to assure that products meet specifications. According to Daniel Capaldi, Ph.D., vp for analytic and process development at Isis Pharmaceuticals, “a specification is a critical quality standard, proposed by the manufacturer and approved by regulatory agencies. It is a contract among the manufacturer, the regulatory agencies, and the patient, which describes the quality criteria that the drug substance or drug product will meet consistently.”

    Interestingly, therapeutic oligonucleotides, as well as some peptides and radiopharmaceuticals, are excluded from the scope of International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guideline Q6A. Evidently the ICH expert working group lacked direct experience with oligos, and time-restrictions prevented their consideration within the guidance.

    “We believe that the spirit of Q6A, and that of the related impurities guidelines Q3A and Q3B, which also categorically exclude oligonucleotides, do, in fact, apply to oligonucleotide therapeutics, but that certain details of the guidelines require modification,” Dr. Capaldi explains.

    As with small molecule drugs, oligonucleotide specifications are not designed to establish full characterization, but rather focus on critical quality attributes to ensure safety and efficacy. This is achieved by confirming identity and strength and through impurity characterization.

    As one would expect from their structure, oligonucleotides present unique challenges. Impurity testing is relatively straightforward with small molecule drugs, but with oligos impurities can only be practically controlled as groups of related structures. “The size and complexity of oligonucleotide drugs often requires the application of sophisticated analytical techniques,” Dr. Capaldi adds.

    “The relatively limited data obtainable by analyzing an oligonucleotide drug also enhances the importance of other aspects of the control strategy. For example, at Isis we have invested heavily in process and product knowledge, in-process controls, and process validation, and we view these and other quality-by-design principles, such as risk assessment and adherence to current good manufacturing practices, as equally important components of any strategy designed to assure quality.”

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