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.
SangerFCoulsonAR. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol. 1975;94:441–448.
2.
MaxamAMGilbertW. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977;74:560–564.
3.
BanoubJHNewtonRPEsmansEEwingDFMackenzieG. Recent developments in mass spectrometry for the characterization of nucleosides, nucleotides, oligonucleotides, and nucleic acids. Chem Rev. 2005;105:1869–1915.
4.
McLuckeySAVan BerkelGJGlishGL. Tandem mass spectrometry of small, multiply charged oligonucleotides. J Am Soc Mass Spectrom. 1992;3:60–70.
5.
Sannes-LoweryKAHofstadlerSA. 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.
CapaldiDC.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.
AlazardDFilipowskyMRaesideJ. 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.
KeoughTShafferJDLaceyMP. Detailed characterization of antisense DNA oligonucleotides. Anal Chem. 1996;68:3405–3412.
9.
MetzP. Opportunities for PAT in oligonucleotide development and manufacturing. Paper presented at: Eastern Analytical Symposium; November 17-20, 2008; Somerset, NJ.
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.
WyrzykiewiczTKColeDL. Sequencing of oligonucleotide phosphorothioates based on solid-supported desulfurization. Nucleic Acids Res. 1994;22(13):2667–2669.
12.
NielsenPE. Modulating gene function with peptide nucleic acids (PNA). In: CrookeST, ed. Antisense Drug Technology. 2nd ed. Boca Raton, FL: CRC Press; 2006:507–518.
BeaucheminD. Inductively coupled plasma mass spectrometry. Anal Chem. 2010;82:4786–4810.
16.
BingsNHBogaertsABroekaertJAC. Atomic spectroscopy: a review. Anal Chem. 2010;82:4653–4681.
17.
SrivatsaGSBattMSchuetteJ. Quantitative capillary gel electrophoresis assay of phosphorothioate oligonucleotides in pharmaceutical formulations. J Chromatogr A. 1994;680:469–477.
18.
CapaldiDCScozzariAN. Manufacturing and analytical processes for 2′-O-(2-methoxyethyl)-modified oligonucleotides. In: CrookeST, ed. Antisense Drug Technology. 2nd ed. Boca Raton, FL: CRC Press; 2006:401–434.
19.
ZhouXHinesPBorerMW. Moisture determination in hygroscopic drug substances by near infrared spectroscopy. J Pharm Biomed Anal. 1998;17:219–225.
20.
ChenDYanZColeDLSrivatsaGS. 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.
KrotzAHKlopchinPGWalkerKLSrivatsaSGColeDLRavikumarVT. On the formation of longmers in phosphorothioate oligodeoxyribonucleotide synthesis. Tetrahedron Lett. 1997;38:3875–3878.
22.
TammCHodesMEChargaffE. The formation of apurinic acid from the desoxyribonucleic acid of calf thymus. J Biol Chem. 1952;195:49–63.
23.
CapaldiDCGausHKrotzAH. 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.
CohenASVilenchikMDudleyJLGemborysMWBourqueAJ. High-performance liquid chromatography and capillary gel electrophoresis as applied to antisense DNA. J Chromatogr A. 1993;638:293–301.
25.
DeDionisioL. Capillary gel electrophoresis and the analysis of DNA phosphorothioates. J Chromatogr A. 1993;652:101–108.
26.
BergotBJEganW. 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.
SrivatsaGSKlopchinPBattMFeldmanMCarlsonRHColeDL. Selectivity of anion exchange chromatography and capillary gel electrophoresis for the analysis of phosphorothioate oligonucleotides. J Pharm Biomed Anal. 1997;16:619–630.
29.
HuberCGKrajeteA. 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.
HuberCGOberacherH. Analysis of nucleic acids by on-line liquid chromatography-mass spectrometry. Mass Spectrom Rev. 2001;20:310–343.
31.
ApffelAChakelJAFischerSLichtenwalterKHancockWS. Analysis of oligonucleotides by HPLC-electrospray ionization mass spectrometry. Anal Chem. 1997;69:1320–1325.
32.
GilarM. 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.
BothnerBChatmanKSarkisianMSiuzdakG. Liquid chromatography mass spectrometry of antisense oligonucleotides. Bioorg Med Chem Lett. 1995;5(23):2863–2868.
34.
LecchiPPannellLK. The detection of intact double-stranded DNA by MALDI. J Am Soc Mass Spectrom. 1995;6:972–975.
35.
BahrUAygünHKarasM. Detection and relative quantification of siRNA double strands by MALDI mass spectrometry. Anal Chem. 2008;80:6280–6285.
36.
BayerEBauerTSchmeerKBelicherKMaierMGausH. Analysis of double-stranded oligonucleotides by electrospray mass spectrometry. Anal Chem. 1994;66:3858–3863.
37.
DoktyczMJHabibi-GoudarziSMckluckeyS. Accumulation and storage of ionized duplex DNA molecules in a quadrupole ion trap. Anal Chem. 1994;66:3416–3422.
38.
WuY.Characterization of PEGylated aptamers. Paper presented at: DIA 2nd Oligonucleotide-Based Therapeutics Conference; September 22-24, 2008; Bethesda, MD.
39.
LevinD. 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.
McCarthySMGilarMGeblerJ. Reversed-phase ion-pair liquid chromatography analysis and purification of small interfering RNA. Anal Biochem. 2009;390:181–188.
41.
BeverlyMHartsoughKMachemerLPavcoPLockridgeJ. 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.
SzékelyLKiessigSSchwartzMAKálmánF. Capillary gel electrophoresis of therapeutic oligonucleotides: analysis of single- and double-stranded forms. Electrophoresis. 2009;30:1579–1586.
43.
HuberCG. Micropellicular stationary phases for high-performance liquid chromatography of double-stranded DNA. J Chromatogr A. 1998;806:3–30.
44.
HerbertBSGellertGCHochreiterH. Lipid modification of GRN163, an N3′→P5′ thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition. Oncogene. 2005;24:5262–5268.
45.
LonnbergH. Solid-phase synthesis of oligonucleotide conjugates useful for delivery and targeting of potential nucleic acid therapeutics. Bioconjugate Chem. 2009;20:1065–1094.
46.
WuY. 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.
ManoharanMKallanthottathilGR. Utilizing chemistry to harness RNA interference pathways for therapeutics: chemically modified siRNAs and antagomirs. In: CrookeST, ed. Antisense Drug Technology. 2nd ed. Boca Raton, FL: CRC Press; 2006:437–464.
48.
LevinD.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.
