6 Conclusions and Future Directions


Figure 7. Non-denaturing SEC Analysis of a 21-mer siRNA double stranded oligonucleotide showing separation of the duplex from aggregates and excess single strand. See reference [49] for more details.


Oligonucleotide Analysis using Mass Spectrometric Detection


Mass spectrometry (MS) is routinely interfaced with HPLC separations to provide improved specificity, sensitivity, and structural characterisation capabilities. HPLC-MS or HPLC-MS/MS is routinely used for the confirmation of base sequences or to structurally characterise impurities. However, some authors are also reporting its use in release testing of API for the control of impurities co-eluting with the main peak [50]. This latter approach is important as it is directly related to the specificity achievable via the chromatographic separation, which in turn determines the necessity for high sensitivity impurity quantification via MS detection. Figure 8 demonstrates the collection of the MS and MS/MS spectra for the sense strand of a 21-mer siRNA from a IP-RP-UPLC separation [51]. Many chromatographic separations used for the analysis of oligonucleotides are not MS compatible, particularly non-denaturing techniques, due to the use of non-volatile buffers in the mobile phase. However, IP-RP HPLC is MS compatible and has become a standard separation platform for the analysis of oligonucleotides [52-58], [37]. The retention of oligonucleotide on the column is critically impacted by the concentration of the ion pair reagent. The more ion pairing agent used the greater the retention. However, high ion pair concentrations can significantly suppress the electrospray ionisation efficiency adversely impacting on mass spectrometric detection/analysis [53]. As a result, optimisation of the ion pairing agent concentration is commonly performed to enable suitable mass spectrometric detection [52-54]. In addition to TEA, other ion pairing agents have been explored based of their differences in structure and overall hydrophobicity [56-58], [37].


Despite the complexities associated with synthetic oligonucleotides, chromatography is still the analytical technique of choice for characterising these biopolymers. However, no one separation technique can fully characterise either the API, or the related impurities. Consequently, orthogonal and complementary separation techniques are routinely utilised, e.g. IP-RP-HPLC, AX-HPLC and SEC. The data from these separation techniques then need to be consolidated and integrated to provide an overall assessment of both purity and related impurities. Industry is working with the regulators to provide guidance in this evolving field. One of the biggest challenges to the regulatory mind-set is the need to re-define impurities. Classically, impurities have been regarded as related compounds that provide no beneficial attributes and thus need to be tightly controlled based on safety considerations, e.g. ICH Q3A and Q3B. In contrast, oligonucleotide derived related impurities (especially longmers /shortmers) may have similarly efficacy/safety to the parent API. Therefore, there are less safety concerns and thus greater levels can be tolerated in the final drug product.


Future progress is likely to be focussed on improving the selectivity and sensitivity of these existing chromatographic approaches. However, CE and related electrochromatographic techniques may play a greater role in the future. Recent advances with interfacing CE with ESI-MS (sheathless ESI) [59], have dramatically improved sensitivity to levels comparable with HPLC-ESI-MS, and hold much promise. This coupled with on-line pre-concentration mechanisms like ITP [60], electrokinetic supercharging [61] or immunoaffinity microreactors [62] has ensured that CE-ESI-MS is now one of the most selective and sensitive analytical separation techniques available.


Biography


Dr George Okafo is currently a Chemistry Science Director in Scinovo, a group in GSK dedicated to providing integrated pre-clinical drug development consultancy and technical diligence for GSK’s internal and external collaborators. Dr Okafo has been at GSK for over 20 years and has worked in all aspects of chemical development to support numerous projects. Dr Okafo has interests in all aspect of analytical chemistry and, more recently in oligonucleotides analysis.


Dr Daren Levin is currently an Investigator within Exploratory Development Sciences, a group at GSK focusing on pre-clinical drug product development. Dr Levin has spent the past four years at GSK developing internal capabilities/knowledge around the analysis and control of short interfering RNA (siRNA) therapeutic compounds. In addition, Dr Levin has been focused on the development of formulations used to enhance the cellular uptake of oligonucleotide therapeutics. Prior to joining GSK, Dr Levin spent seven years at Alkermes, Inc developing high efficiency inhalation products as well as sustained release microsphere injectable products.


Dr David Elder is currently a pharmaceutical development Science Director in Scinovo. Dr Elder has spent 33-years in the Pharmaceutical industry, with over half this period with GSK. He has a specific interest in drug impurities and is a member of the EfPIA, PhRMA and PQRI sub-groups on genotoxic impurities. He is a committee member of Joint Pharmaceutical Analysis Group and a member of the BP-Committee PCY: Pharmacy.


References


[1] S. Agrawal: Trends Biotechnol. Sci. 10 (1992) 3499-3507. [2] K. Sobczak, N. Bangel-Ruland, J. Semmler, H. Lindemann, R. Heermann und W.-M. Weber , HNO (2009), 1106 – 1112.


[3] G. Degols, J.P. Leonitti, N. Mechti, B. Lebleu. Nucl. Acids. Res. 19 (1991) 945-948.


[4] A. Aartmus-rus, W.E.Kaman, J.T. den Dunnen, G.J. van Ommen, J.C.van Deutekim, Mol. Ther.14(3) (2006);401-407. [5] E.J. Sontheimer, RW. Carthew, Cell 122(1) (2005) 9–12.


[6] US FDA: Drug approval package:vitravene (fomivirisen sodium intraveal injectable) injection; www.fda.gov/cder/foi/nda/98/20961_vitravene.html.


[7] US FDA:FDA approves new drug treatment for age-rlated macular degeneration; www.fda.gov/bbs/topics/news/2004/new01146.html.


[8] R. Bhindi, R.G. Fahmy, H.C. Lowe . Am J. Pathol. 171(4), (2007), 1079-1088. [9] S. Sel, W.Henke, A.Dietrich, U.Herz, H.Renz, Curr. Pharm. Des. 12(25) (2006);3293-3304.


[10] J.Kurreck, E.Wyszko, C.Gillen, V.A.Erdmann, Nucleic Acid Research, 30(9), (2002), 1911- 1918.


[11] L.Gold, B.Polisky, O.C.Uhlenbeck, M.Yarus, Ann. Rev. Biochem 64 (1995);763-797. [12] G.Purschke, F.Radtke, F.Kleinjung, S.Klussmann,Nucleic Acids Res.31 (2003); 3027-3032.


[13] M.E. Zubin, E.A. Romanova, E.M. Volkov, V.N. Tashlitsky, G.A. Korshunova, Z.A. Shabarova, T.S. Oretskaya, Febs Letters, 456 (1) (1999) 59-62. [14] A. Dorn, S. Kippenberger. Curr. Opin. Mol. Ther. 10(1) (2008) 10-20. [15] N.K. Sahu,G. Shilakari, A. Nayak, D.V. Kohli, Curr. Pharm. Biotechnol. 8(5) (2007) 291-304.


[16] J.S. Cohen. Oligodeoxynucleotides:. Antisense inhibitors of gene expression, 1989; CRC Press, Boca Raton FL, p.255.


[17] ICH Q6A, Specifications:Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances, 1999.


[18] ICH Q6B, Specifications:Test Procedures and Acceptance Criteria for Biotechnological/ Biological Products, 1999.


Figure 8. RP-IP-UPLC-MS data showing A) siRNA UV chromatogram under denaturing conditions (sense strand has been highlighted); b) An MS spectrum of the sense strand peak (6860 MW) showing the multiple charge states of this oligonucleotide (-4 through - 7) formed as a result of the electrospray ionisation; and C) The MS/MS spectrum of the 1713.9 m/z ion (-4 charge state) showing the various fragment ions associated with the specific nucleotide sequence. See reference [51] for more details.


[19] ICH Q3A (R2), Impurities in Drug Substance, 26th October 2006. [20] ICH Q3B (R2); Impurities in New Drug Products (Revised Guideline), 2006.


[21] B.L. Gaffney, R.A. Jones, Tetrahedron Letters, 29 (22) (1988) 2619-2622.[22] S.L. Beaucage, M.H. Caruthers. Tetrahedron Letters 22 (1982) 90461-7.[23] H.Koster, J.Biernat, J.McManus, A.Wolter, A. Stumpe, Ch.K.Narang, N.D.Sinha, Tetrahedron, 40(1), (1984), 103-112.


[24] J.F.Labadie, Current Opinion in Chemical Biology, 2(3), (1998), 346-352.


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