Advertisement

Quality Aspects of Oligonucleotide Drug Development: Specifications for Active Pharmaceutical Ingredients

Abstract

This article, which is the first in a planned series intended to address chemistry, manufacturing, and control (CMC) aspects of therapeutic oligonucleotides, examines the topic of specifications for active pharmaceutical ingredients (APIs). The authors attempt to present basic scientific considerations for the broadest range of oligonucleotide APIs. Tests and analytical methods suitable for the control of single- and double-stranded oligonucleotide APIs and conjugated oligonucleotide APIs are discussed.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 189

This is the net price. Taxes to be calculated in checkout.

References

  1. 1.

    Sanger F, Coulson AR. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol. 1975;94:441–448.

  2. 2.

    Maxam AM, Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977;74:560–564.

  3. 3.

    Banoub JH, Newton RP, Esmans E, Ewing DF, Mackenzie G. Recent developments in mass spectrometry for the characterization of nucleosides, nucleotides, oligonucleotides, and nucleic acids. Chem Rev. 2005;105:1869–1915.

  4. 4.

    McLuckey SA, Van Berkel GJ, Glish GL. Tandem mass spectrometry of small, multiply charged oligonucleotides. J Am Soc Mass Spectrom. 1992;3:60–70.

  5. 5.

    Sannes-Lowery KA, Hofstadler SA. Sequence confirmation of modified oligonucleotides using IRMPD in the external ion reservoir of an electrospray ionization fourier transform ion cyclotron mass spectrometer. J Am Soc Mass Spectrom. 2003;14:825–833.

  6. 6.

    Capaldi DC. Development and validation of methods suitable for the analysis of oligonucleotide therapeutics. Paper presented at: TIDES—Oligonucleotide and Peptide Technology and Product Development; May 1–4, 2006; La Costa, CA.

  7. 7.

    Alazard D, Filipowsky M, Raeside J, et al. Sequencing of production-scale synthetic oligonucleotides by enriching for coupling failures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Biochem. 2002;301:57–64.

  8. 8.

    Keough T, Shaffer JD, Lacey MP, et al. Detailed characterization of antisense DNA oligonucleotides. Anal Chem. 1996;68:3405–3412.

  9. 9.

    Metz P. Opportunities for PAT in oligonucleotide development and manufacturing. Paper presented at: Eastern Analytical Symposium; November 17–20, 2008; Somerset, NJ.

  10. 10.

    US Food and Drug Administration. Process Analytical Technology (PAT): A Framework for Innovative Pharmaceutical Development, Manufacturing and Quality Assurance. FDA Guidance for Industry. Silver Spring, MD: US Health & Human Services; 2004.

  11. 11.

    Wyrzykiewicz TK, Cole DL. Sequencing of oligonucleotide phosphorothioates based on solid-supported desulfurization. Nucleic Acids Res. 1994;22(13):2667–2669.

  12. 12.

    Nielsen PE. Modulating gene function with peptide nucleic acids (PNA). In: Crooke ST, ed. Antisense Drug Technology. 2nd ed. Boca Raton, FL: CRC Press; 2006:507–518.

  13. 13.

    Iversen PL. Morpholinos. In: Crooke ST, ed. Antisense Drug Technology. 2nd ed. Boca Raton, FL: CRC Press; 2006:565–582.

  14. 14.

    Westheimer FH. Why nature chose phosphates. Science. 1987;235:1173–1178.

  15. 15.

    Beauchemin D. Inductively coupled plasma mass spectrometry. Anal Chem. 2010;82:4786–4810.

  16. 16.

    Bings NH, Bogaerts A, Broekaert JAC. Atomic spectroscopy: a review. Anal Chem. 2010;82:4653–4681.

  17. 17.

    Srivatsa GS, Batt M, Schuette J, et al. Quantitative capillary gel electrophoresis assay of phosphorothioate oligonucleotides in pharmaceutical formulations. J Chromatogr A. 1994;680:469–477.

  18. 18.

    Capaldi DC, Scozzari AN. Manufacturing and analytical processes for 2′-O-(2-methoxyethyl)-modified oligonucleotides. In: Crooke ST, ed. Antisense Drug Technology. 2nd ed. Boca Raton, FL: CRC Press; 2006:401–434.

  19. 19.

    Zhou X, Hines P, Borer MW. Moisture determination in hygroscopic drug substances by near infrared spectroscopy. J Pharm Biomed Anal. 1998; 17:219–225.

  20. 20.

    Chen D, Yan Z, Cole DL, Srivatsa GS. Analysis of internal (n-1)mer deletion sequences in synthetic oligodeoxyribonucleotides by hybridization to an immobilized probe array. Nucleic Acids Res. 1999;27:389–395.

  21. 21.

    Krotz AH, Klopchin PG, Walker KL, Srivatsa SG, Cole DL, Ravikumar VT. On the formation of longmers in phosphorothioate oligodeoxyribonucleotide synthesis. Tetrahedron Lett. 1997;38:3875–3878.

  22. 22.

    Tamm C, Hodes ME, Chargaff E. The formation of apurinic acid from the desoxyribonucleic acid of calf thymus. J Biol Chem. 1952;195:49–63.

  23. 23.

    Capaldi DC, Gaus H, Krotz AH, et al. Synthesis of high-quality antisense drugs. Addition of acrylonitrile to phosphorothioate oligonucleotides: adduct characterization and avoidance. Org Proc Res Dev. 2003;7:832–838.

  24. 24.

    Cohen AS, Vilenchik M, Dudley JL, Gemborys MW, Bourque AJ. High-performance liquid chromatography and capillary gel electrophoresis as applied to antisense DNA. J Chromatogr A. 1993;638:293–301.

  25. 25.

    DeDionisio L. Capillary gel electrophoresis and the analysis of DNA phosphorothioates. J Chromatogr A. 1993;652:101–108.

  26. 26.

    Bergot BJ, Egan W. Separation of synthetic phosphorothioate oligodeoxynucleotides from their oxygenated (phosphodiester) defect species by strong-anion-exchange-high-performance liquid chromatography. J Chromatogr A. 1992;599:35–42.

  27. 27.

    Metelev V, Agrawal S. Ion-exchange high-performance liquid chromatography analysis of oligodeoxyribonucleotide phosphorothioates. Anal Biochem. 1992;200:342.

  28. 28.

    Srivatsa GS, Klopchin P, Batt M, Feldman M, Carlson RH, Cole DL. Selectivity of anion exchange chromatography and capillary gel electrophoresis for the analysis of phosphorothioate oligonucleotides. J Pharm Biomed Anal. 1997;16:619–630.

  29. 29.

    Huber CG, Krajete A. Analysis of nucleic acids by capillary ion-pair reversed-phase HPLC coupled to negative-ion electrospray ionization mass spectrometry. Anal Chem. 1999;71:3730–3739.

  30. 30.

    Huber CG, Oberacher H. Analysis of nucleic acids by on-line liquid chromatography-mass spectrometry. Mass Spectrom Rev. 2001;20:310–343.

  31. 31.

    Apffel A, Chakel JA, Fischer S, Lichtenwalter K, Hancock WS. Analysis of oligonucleotides by HPLC-electrospray ionization mass spectrometry. Anal Chem. 1997;69:1320–1325.

  32. 32.

    Gilar M. Analysis and purification of synthetic oligonucleotides by reversed-phase high-performance liquid chromatography with photodiode array and mass spectrometry detection. Anal Biochem. 2001;298:196–206.

  33. 33.

    Bothner B, Chatman K, Sarkisian M, Siuzdak G. Liquid chromatography mass spectrometry of antisense oligonucleotides. Bioorg Med Chem Lett. 1995;5(23):2863–2868.

  34. 34.

    Lecchi P, Pannell LK. The detection of intact double-stranded DNA by MALDI. J Am Soc Mass Spectrom. 1995;6:972–975.

  35. 35.

    Bahr U, Aygün H, Karas M. Detection and relative quantification of siRNA double strands by MALDI mass spectrometry. Anal Chem. 2008;80:6280–6285.

  36. 36.

    Bayer E, Bauer T, Schmeer K, Belicher K, Maier M, Gaus H. Analysis of double-stranded oligonucleotides by electrospray mass spectrometry. Anal Chem. 1994;66:3858–3863.

  37. 37.

    Doktycz MJ, Habibi-Goudarzi S, Mckluckey S. Accumulation and storage of ionized duplex DNA molecules in a quadrupole ion trap. Anal Chem. 1994;66:3416–3422.

  38. 38.

    Wu Y. Characterization of PEGylated aptamers. Paper presented at: DIA 2nd Oligonucleotide-Based Therapeutics Conference; September 22–24, 2008; Bethesda, MD.

  39. 39.

    Levin D. Challenges to validation of methods for double stranded oligonucleotides. Paper presented at: DIA 3rd Oligonucleotide-Based Therapeutics Conference; March 23–25, 2010; Bethesda, MD.

  40. 40.

    McCarthy SM, Gilar M, Gebler J. Reversed-phase ion-pair liquid chromatography analysis and purification of small interfering RNA. Anal Biochem. 2009;390:181–188.

  41. 41.

    Beverly M, Hartsough K, Machemer L, Pavco P, Lockridge J. Liquid chromatography electrospray ionization mass spectrometry analysis of the ocular metabolites from a short interfering RNA duplex. J Chromatogr B Analyt Technol Biomed Life Sci. 2006;835:62–70.

  42. 42.

    Székely L, Kiessig S, Schwartz MA, Kálmán F. Capillary gel electrophoresis of therapeutic oligonucleotides: analysis of single- and double-stranded forms. Electrophoresis. 2009;30:1579–1586.

  43. 43.

    Huber CG. Micropellicular stationary phases for high-performance liquid chromatography of double-stranded DNA. J Chromatogr A. 1998;806:3–30.

  44. 44.

    Herbert BS, Gellert GC, Hochreiter H, et al. Lipid modification of GRN163, an N3′→P5′ thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition. Oncogene. 2005;24:5262–5268.

  45. 45.

    Lonnberg H. Solid-phase synthesis of oligonucleotide conjugates useful for delivery and targeting of potential nucleic acid therapeutics. Bioconjugate Chem. 2009;20:1065–1094.

  46. 46.

    Wu Y. The sequencing of a 37-mer aptamer via failure sequence analysis has been reported. Paper presented at: DIA 2nd Oligonucleotide-Based Therapeutics Conference; September 22–24, 2008; Bethesda, MD.

  47. 47.

    Manoharan M, Kallanthottathil GR. Utilizing chemistry to harness RNA interference pathways for therapeutics: chemically modified siRNAs and antagomirs. In: Crooke ST, ed. Antisense Drug Technology. 2nd ed. Boca Raton, FL: CRC Press; 2006:437–464.

  48. 48.

    Levin D. A comprehensive look at siRNA impurity formation as a function of melting temperature, and is water content a critical attribute for siRNA stability? A Paper presented at: TIDES—Oligonucleotide and Peptide Technology and Product Development; April 25 to 28, 2010; Boston, MA.

Download references

Author information

Correspondence to Daniel Capaldi PhD.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Capaldi, D., Ackley, K., Brooks, D. et al. Quality Aspects of Oligonucleotide Drug Development: Specifications for Active Pharmaceutical Ingredients. Ther Innov Regul Sci 46, 611–626 (2012) doi:10.1177/0092861512445311

Download citation

Keywords

  • therapeutic oligonucleotides
  • active pharmaceutical ingredients
  • specifications
  • specification tests
  • analytical methods