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MS approach for absolute quantification and determination of phosphodiester to phosphorothioate ratio in therapeutic oligonucleotides
Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Development of an ICP-MS/MS approach for absolute quantification and determination of phosphodiester to phosphorothioate ratio in therapeutic oligonucleotides Juliusz Bianga, Magali Perez, Damien Mouvet, Caroline Cajot, Philippe De Raeve, Arnaud Delobel ∗ Quality Assistance sa, Technoparc de Thudinie 2, B-6536 Donstiennes, Belgium
a r t i c l e
i n f o
Article history: Received 15 December 2019 Received in revised form 13 February 2020 Accepted 14 February 2020 Available online 15 February 2020 Keywords: Absolute quantification ICP-MS/MS Mass spectrometry Oligonucleotide Phosphorothioate
a b s t r a c t A new analytical method based on ICP-MS/MS is proposed for the characterization of synthetic phosphorothioate oligonucleotides. Absolute quantification of oligonucleotides is challenging, as well as the determination of phosphodiester to phosphorothioate ratio for phosphorothioate oligonucleotides. Both are considered as critical quality attributes and should be determined using robust validated methods. The method we developed was designed to be easy to apply, fast, and robust. It allows simultaneous absolute quantification of an oligonucleotide (based on the quantification of phosphorus), determination of the phosphodiester to phosphorothioate ratio (based on the quantification of phosphorus and sulfur) and optionally determination of sodium (or any other metal) as a counter ion. The performance of the method was demonstrated on O,O-diethyl thiophosphate potassium salt, a well characterized model substance that possesses similar composition to phosphorothioate oligonucleotides. Method was also tested on different synthetic phophorothioate oligonucleotides, showing excellent accuracy and precision. © 2020 Elsevier B.V. All rights reserved.
1. Introduction Oligonucleotides are a class of therapeutics that has been under clinical development for the past 30 years. Different categories of oligonucleotides have been developed, among which anti-sense oligonucleotides (ASO) and aptamers, and more recently small interfering RNA (siRNA) [1]. As of November 2019, 9 oligonucleotides were approved for therapeutic use in Europe and/or in the US. They have a potential to be used in a wide range of diseases, including cancer, cardiovascular and metabolic conditions, neurological disorders, and ophthalmic diseases [2–7]. One of the challenges for oligonucleotides used as therapeutics is their pharmacokinetics. In order to make oligonucleotides amenable to their use as medicines, many chemical modifications designed to increase resistance against enzymatic digestion have been developed. Among them, phosphorothioate oligonucleotides [8], which include a modification on the phosphate backbone by the substitution of sulfur for a non-bridging oxygen (see Fig. 1), have shown promise, especially for antisense applications [2].
∗ Corresponding author. E-mail address: [email protected] (A. Delobel). https://doi.org/10.1016/j.jpba.2020.113179 0731-7085/© 2020 Elsevier B.V. All rights reserved.
As for all therapeutic molecules, the determination of their quantity is critical for both safety and efficacy reasons. Quantification can be either absolute or relative. Absolute quantification means that no well-characterized reference standard is required. The most commonly used technique for the routine quantification of oligonucleotides in drug substances or drug products is UV spectroscopy [9], but it requires the prior knowledge of the extinction coefficient [10]. This coefficient can be estimated by calculation using the base composition of the oligonucleotide, but such an estimation has been shown to produce a bias of around 10 %. It can also be determined experimentally by measuring the absorbance at 260 nm of solutions of known concentrations, but the problem remains the measurement of this concentration. Another possibility is to hydrolyze the oligonucleotide with an enzyme and measure the concentration of released nucleotides by liquid chromatography with UV or MS detection [11]. But this methodology also shows limited precision and accuracy. Quantification can also be done using a relative method, such as LC/MS, LC/UV or PCR, but these methods require a well-characterized reference standard. In phosphorothioate oligonucleotides, the molar ratio of phosphodiester to phosphorothioate linkages is also considered as a critical quality attribute and should be determined experimentally during characterization studies. Due to the sequential process, even a highly efficient sulfurization process may result in high amounts
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J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179
Fig. 1. Structure of RNA and phosphorothioate RNA.
of impurities. A missing substitution may play a role in the potency and the in vivo stability of the oligonucleotide versus nucleases activity. 31 P-NMR (Nuclear Magnetic Resonance) is the common way to determine this ratio [12]. This technique requires expensive equipment and skilled operators, is relatively slow, sequential and lacks sensitivity [13]. Moreover, it cannot be easily implemented in a GMP (Good Manufacturing Practices) environment if this method has to be used for batch release. Another possibility is to use strong-anion exchange chromatography (SAX) with UV detection [14,15]. This technique is more sensitive than NMR but shows strong limitations in the presence of other process- or product-related impurities. ICP-MS is mainly used to analyze metals in a wide range of applications, from environmental analysis to quality control of pharmaceuticals and biopharmaceuticals. However, it can also be used in biotechnology and proteomics [16] thanks to its ability to quantify elements such as phosphorus (for phosphoproteins) or sulfur [17,18] (for virtually all proteins). Sulfur determination by ICP-MS has long been a challenge because of the high ionization potential of this element (10.4 eV) and the interferences from polyatomic ions for all sulfur isotopes. The same issue is observed for the determination of phosphorus. Triple-quadrupole ICP-MS [19] can easily circumvent these issues, with the formation of sulfur and phosphorus oxide cations in the reaction cell, as shown previously by Diez-Fernandez et al. [20]. Different groups had already mentioned the use of ICP/MS for the quantification of oligonucleotides, but in other contexts [21,22]. The ability of triple quadrupole ICP-MS to quantify both phosphorus and sulfur appeared to us as an opportunity for the absolute quantification and the efficient determination of phosphodiester to phosphorothioate ratio in phosphorothioate oligonucleotides, if possible in the same analytical run. Provided the structure of the oligonucleotide is known (which is always the case for oligonucleotide drug substances and drug products), the phosphorus content can be easily calculated. Therefore, measuring the phosphorus content by ICP-MS/MS can give access the oligonucleotide concentration, without the need for a standard. Only a certified solution of phosphoric acid is needed. Moreover, by measuring both the phosphorus and the sulfur content, the phosphodiester to phosphorothioate ratio can also be readily determined. In an ICP-MS/MS instrument, samples are introduced through a nebulization device, but nebulization and transport efficiency are not equivalent for small and large molecules. As the standardization is done using sulfuric acid and phosphoric acid, a very accurate method cannot be obtained by introducing directly the oligonucleotide solution in the nebulizer. Therefore, all samples were digested with nitric acid and hydrochloric acid in a microwave oven, after addition of yttrium as an internal standard.
As sodium is the most common counter-ion for oligonucleotides, we also measured its concentration during the same analytical run, in order to be able to perform mass-balance calculations (vide infra). We present the preliminary results obtained for the development of an ICP-MS/MS method for the absolute quantification of therapeutic oligonucleotides in drug substances and drug products and for the determination of phosphodiester to phosphorothioate ratio in a single method. 2. Material 2.1. Reagents and material ICP/MS-grade standard solutions of sodium, phosphorus and sulfur were obtained from Merck Chemicals. ICP/MS-grade standard solution of yttrium was obtained from VWR International. Nitric and hydrochloric acid (TraceSELECTTM grade) were obtained from Honeywell Fluka. O,O-Diethyl thiophosphate potassium salt (DTP) and ammonium hydroxide solution (28.0–30.0%) were obtained from Merck Chemicals. Ultrapure deionized water (resistivity > 18 M.cm) from a Milli-Q system (Millipore) was used throughout the experiment. Oligonucleotide samples were obtained from Kaneka Eurogentec (Seraing, Belgium). 2.2. Sample and standard preparation Sample solutions were obtained by gravimetric reconstitution of the oligonucleotide lyophilizates or DPT in water. Then, the solutions were aliquoted into digestion vessels mixed with 0.1 mL of internal standard (50 ppm Yttrium solution), 2.5 mL of concentrated nitric acid and 0.5 mL of concentrated hydrochloric acid and covered with caps. Digestion was carried out using a single reaction chamber Ultrawave MW digestion system (Milestone Srl, Sorisole (BG), Italy), equipped with 15 position sample rack. The sample rack with the digestion vessels was installed in the reaction chamber containing outer bath (150 mL of water and 5 mL concentrated nitric acid). The reaction chamber was closed and pressurized to 40 bars with compressed nitrogen, heated to reach 220 ◦ C in 35 min and then the temperature was maintained at 220 ◦ C for 30 min. After digestion all the solutions for analysis were transferred to polypropylene metal free tubes and diluted to 50 mL with water. For each digestion cycle at least one procedure blank was included. Digested samples were analyzed versus an external five-point calibration with an internal standardization. Calibration was prepared by dilution of the reference standards of phosphorus, sulfur and optionally of sodium, in the matrix matched solvent (water solution of acids at the same level as for digests).
J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179 Table 1 ICP/MS acquisition parameters. Description
Acquisition mode 1
Acquisition mode 2
RF power (W) Spray chamber Nebulizer Sample flow (mL/min) Nebulizer gas (L/min) Dilution gas (L/min) Scan mode Reaction gas Integ. Time per ion (s) Acquisition mode Replicate Sweep per replicate
1550 Double pass (Scott type) Micromist 0.4 0.8 0.2 Mass-shift reaction mode O2 (45%) 0.45 Spectrum 3 100
1550 Double pass (Scott type) Micromist 0.4 0.8 0.2 Single quadrupole He 0.45 Spectrum 3 100
2.3. ICP-MS/MS analysis ICP-MS analysis was performed using an Agilent 8800 ICP-QQQ triple quadrupole ICP-MS (Agilent Technologies Inc., Santa Clara, CA, USA) in mass-shift mode using O2 as a reaction gas. For sulfur quantification, the first quadrupole was set to select 32 S+ (m/z 32) and 34 S+ (m/z 34) ions, mass shifted by the reaction with oxygen in the octapole reaction system and finally detected as 32 S16 O+ (m/z 48) and 34 S16 O+ (m/z 50) by the second quadrupole. For phosphorus quantification, the first quadrupole was set to select 31 P+ (m/z 31) ion, mass shifted by the reaction with oxygen in the octapole reaction system and finally detected as 31 P16 O+ (m/z 47) by the second quadrupole. Yttrium was used as internal standard for S and P quantification: the first quadrupole was set to select 89 Y+ (m/z 89) ion, mass shifted by the reaction with oxygen in the octapole reaction system and finally detected as 89 Y16 O+ (m/z 105) by the second quadrupole. Sodium determination was carried out in single-quadrupole mode; the second quadrupole was set to select 23 Na+ (m/z 23). Yttrium was used as internal standard: the second quadrupole was set to select 89 Y+ (m/z 89). The sample introduction system consisted of a quartz Micromist nebulizer, a quartz double path spray chamber and an integral quartz injector and torch (Agilent Technologies Inc.). The sample solutions were infused continuously into the spray chamber of ICP-MS by an integrated autosampler I-AS (Agilent Technologies Inc.) and a peristaltic pump. The operating conditions of the ICP-MS were optimized and are summarized in Table 1.
3
pound and digested as a sample. Accuracy and precision of the method were evaluated by digesting different amounts of DTP, resulting in solutions containing 0.2 to 0.6 mmol/L of phosphorus. Results are presented in Table 2 and Fig. 2. Precision was assessed by calculating the relative standard deviation (RSD) on three determinations at each concentration level. RSDs range between 0.2 and 0.4 %. Accuracy was assessed by calculating the recovery between the theoretical concentration and the one calculated from the standardization curve. The mean recoveries at each level range from 99.8–102.4%. The linearity of the method was assessed by plotting the experimental concentration versus the theoretical concentration. By linear regression, a slope of 0.989 was obtained, with a determination coefficient (R2 ) of 0.9999. The behavior of DTP during the sample preparation and the ICP-MS/MS process may be different compared to oligonucleotides, but no oligonucleotide with a certified concentration is commercially available. Therefore, to evaluate the accuracy of the method developed, we analyzed two purified oligonucleotide samples for which we measured the water content (by coulometric Karl-Fisher titration) and the phosphorus and sodium content by ICP-MS/MS. Based on the oligonucleotide content, we were able to calculate the expected amount of sodium present as a counter-ion of the oligonucleotide (1:1 molar ratio to phosphorus). The excess sodium content was easily calculated, and we assumed that this sodium was present in the form of sodium chloride (this assumption was done based information on the synthetic route provided by the supplier). The amount of chloride ions was therefore calculated. All these results are presented in Table 3. A mass balance close to 100 % was obtained for both samples, which demonstrates indirectly the excellent accuracy of the method. As the method is not intended for the quantification of impurities, sensitivity was not assessed, in accordance with ICH Q2(R1) guideline on the validation of analytical methods. In order to get reproducible and accurate results, the method was designed so as the signals measured are far from the detection limits of the instrument. The sulfur and phosphorus concentrations measured for oligonucleotide samples using the method described here are in the ppm range, while limits of quantification are in the low ppb range.
3.2. Determination of phosphodiester to phosphorothioate ratio 2.4. Determination of water content in oligonucleotide samples Water content determination was carried by coulometric Karl-Fisher titration, with 831 K F Coulometer equipped with a Thermoprep device (Metrohm AG, Herisau, Switzerland). An aliquot of a sample was heated at 180 ◦ C, under dry nitrogen flow (50 mL/min) which carried the desorbed water from the sample to the titration cell where after absorption in the electrolyte it was titrated electrochemically versus an indicator electrode. 3. Results and discussion 3.1. ICP-MS/MS for the analysis of phosphorus and sulfur 3.1.1. Absolute quantification of oligonucleotides Absolute quantification is the most straightforward application of ICP-MS/MS for oligonucleotides. By performing a digestion step in the presence of an internal standard before analysis, excellent precision and accuracy can be obtained. In order to evaluate the performance of the method, a certified reference standard is required. As no oligonucleotide with a certified accurate concentration is commercially available, a more characterized sample, O,O-diethyl thiophosphate potassium salt (DTP) was taken as a model com-
In order to evaluate the capabilities of the technique to measure accurately and precisely sulfur / phosphorus ratios, the instrument was calibrated with diluted NIST solutions of phosphoric and sulfuric acids, varying the sulfur/phosphorus molar ratios from 0 to 1 at constant phosphorus content. Results are presented in Table 4. Accuracy was close to 100 % at all levels tested (with a maximum bias of 3%, at low S/P ratio), with excellent precision (%RSD on 3 determinations below 0.6 %). The linearity of the method is presented in Fig. 3. The performance of the method was studied using a small molecule standard (O,O-diethyl thiophosphate potassium salt), containing one atom of sulfur and one atom of phosphorus, as well as samples of therapeutic oligonucleotides (information on sample characterization provided by the supplier allowed us to know accurately the phosphodiester to phosphorothioate ratio expected for each oligonucleotide sample). Results are presented in Table 5. Very good accuracies (between 99 and 101 %) were obtained for the P O/P = S ratio of all samples with a relative standard deviation on 3 determinations below 1%. Even for the most complex sample (oligonucleotide containing 23 bases with 6 phosphorothioate linkages and a cholesteryl group), an accuracy of 99 % is obtained, with a relative standard deviation on 3 determinations of 0.3 %.
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Table 2 Results obtained for the evaluation of absolute quantification method performance. Theoretical DTP concentration (mmol/L)
0.192
0.295
0.384
0.475
0.578
Replicate
Phosphorus concentration measured (mmol/L)
Accuracy (expressed as % recovery)
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
0.198 0.196 0.197 0.297 0.299 0.298 0.387 0.386 0.388 0.475 0.477 0.473 0.579 0.581 0.577
102.9 102.0 102.3 100.9 101.3 101.2 100.7 100.6 100.9 99.8 100.2 99.4 100.3 100.6 99.9
Average accuracy (%)
RSD% (n = 3)
102.4
0.4
101.2
0.2
100.7
0.2
99.8
0.4
100.3
0.3
Fig. 2. Evaluation of method linearity for DTP absolute quantification based on phosphorus content.
Table 3 Evaluation of method accuracy based on phosphorus, sodium, and water content on two purified oligonucleotide samples. % w/w in sample powder
Sample
Oligonucleotide 1 Oligonucleotide 2
Mass balance (%)
Oligonucleotide
Sodium (total)
Water
Excess sodium (calc.)
Chloride (calc.)
30.0 63.2
28.1 14.6
3.5 6.6
26.0 10.1
40.1 15.6
101.7 100.1
Table 4 Results obtained for the evaluation method performance for the determination of phosphodiester to phosphorothioate ratio. Test solution
T1
T2
T3
T4
Replicate
1 2 3 1 2 3 1 2 3 1 2 3
Measured conc. (mmol/L)
P/S molar ratio
P
S
Theoretical
Experimental
0.587 0.589 0.588 0.583 0.582 0.580 0.581 0.579 0.578 0.579 0.575 0.576
0.626 0.628 0.627 0.184 0.186 0.184 0.061 0.061 0.061 0.012 0.012 0.012
0.932 0.932 0.932 3.108 3.108 3.108 9.326 9.326 9.326 46.630 46.630 46.630
0.937 0.938 0.938 3.159 3.122 3.142 9.470 9.421 9.428 45.553 45.127 45.485
Accuracy %
100.5 100.6 100.6 101.6 100.4 101.1 101.5 101.0 101.1 97.7 96.8 97.5
Average Accuracy %
RSD %
100.6
0.06
101.1
0.60
101.3
0.26
97.3
0.49
J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179
5
Fig. 3. Evaluation of method linearity for phosphodiester to phosphorothioate ratio determination.
Table 5 Evaluation of the accuracy of phosphodiester to phosphorothioate ratio determination of DTP and oligonucleotide samples. Sample
Number of P/mol
Number of S/mol
Theoretical P/S ratio
Experimental P/S ratio
Accuracy %
RSD (n = 3)
O,O-diethyl thiophosphate Oligonucleotide 1 (20 mer, full 2 -OMe full PS) Oligonucleotide 2 (20 mer, full PS) Oligonucleotide 3 (20 mer, full 2 -OMe full PS) Oligonucleotide 4 (22 mer, full PS) Oligonucleotide 5 (22 mer, full PS) Oligonucleotide 6 (23 mer, full 2 -OMe, 6 PS, 5 -Cholesteryl)
1 19 19 19 21 21 23
1 19 19 19 21 21 6
1.000 1.0000 1.0000 1.0000 1.0000 1.0000 3.833
0.9913 1.010 0.9999 1.0032 0.9884 0.9935 3.876
99.1 101.0 100.0 99.7 101.2 100.7 101.1
0.1 0.4 0.4 0.3 0.3 0.8 0.3
4. Conclusion
CRediT authorship contribution statement
We developed an ICP-MS/MS method based on the quantification of phosphorus and sulfur after microwave digestion for the absolute quantification of oligonucleotides (based on phosphorus content), and for the determination of phosphodiester to phosphorothioate ratio in purified therapeutic oligonucleotide samples, in the same analytical run. Although the quantification of phosphorus and sulfur by ICP-MS/MS was previously described, this is to the best of our knowledge the first application of this technology to therapeutic phosphorothioate oligonucleotides for both absolute quantification and phosphodiester-to-phosphorothioate ratio determination. Around 10 mg of sample are required to perform the test, and results can be obtained within one day of analytical work, including sample preparation. Even if additional investigations may be needed, preliminary results are very encouraging, with excellent precision and accuracy obtained on reference standards and therapeutic oligonucleotide samples. As there is no chromatographic separation prior to ICP-MS/MS analysis, this method is intended to be applied to highly-purified therapeutic-grade oligonucleotides. The absolute quantification method will actually quantify the oligonucleotide and all the potential impurities, while another purity method (usually a chromatographic method with optical or MS detection) may be used to determine the purity profile. The method could be used in routine testing of oligonucleotides in a GMP environment, as an orthogonal method to 31 P-NMR.
Juliusz Bianga: Methodology, Investigation, Formal analysis, Writing - original draft. Magali Perez: Supervision. Damien Mouvet: Supervision. Caroline Cajot: Resources. Philippe De Raeve: Conceptualization, Methodology, Supervision. Arnaud Delobel: Project administration, Writing - review & editing.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments The authors would like to thank Kaneka Eurogentec for providing oligonucleotide samples.
References [1] C.A. Stein, D. Castanotto, FDA-approved oligonucleotide therapies in 2017, Mol. Ther. 25 (2017) 1069–1075, http://dx.doi.org/10.1016/j.ymthe.2017.03. 023, PMID - 28366767. [2] C.F. Bennett, Therapeutic antisense oligonucleotides are coming of age, Annu. Rev. Med. 70 (2019) 307–321, http://dx.doi.org/10.1146/annurev-med041217-010829, PMID - 30691367.
6
J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179
[3] L. Echevarría, P. Aupy, A. Goyenvalle, Exon-skipping advances for Duchenne muscular dystrophy, Hum. Mol. Genet. 27 (2018) R163–R172, http://dx.doi. org/10.1093/hmg/ddy171, PMID - 29771317. [4] C.I.E. Smith, R. Zain, Therapeutic oligonucleotides: state of the art, Annu. Rev. Pharmacol. Toxicol. 59 (2018) 1–26, http://dx.doi.org/10.1146/annurevpharmtox-010818-021050, PMID - 30285540. [5] O. Khorkova, C. Wahlestedt, Oligonucleotide therapies for disorders of the nervous system, Nat. Biotechnol. 35 (2017) 249–263, http://dx.doi.org/10. 1038/nbt.3784, PMID - 28244991. [6] K.E. Lundin, O. Gissberg, C.I.E. Smith, Oligonucleotide therapies: the past and the present, Hum. Gene Ther. 26 (2015) 475–485, http://dx.doi.org/10.1089/ hum.2015.070, PMID - 26160334. [7] K. Takakura, A. Kawamura, Y. Torisu, S. Koido, N. Yahagi, M. Saruta, The clinical potential of oligonucleotide therapeutics against pancreatic cancer, Int. J. Mol. Sci. 20 (2019) 3331, http://dx.doi.org/10.3390/ijms20133331, PMID - 31284594. [8] F. Eckstein, Phosphorothioates, essential components of therapeutic oligonucleotides, Nucleic Acid Ther. 24 (2014) 374–387, http://dx.doi.org/10. 1089/nat.2014.0506, PMID - 25353652. [9] D. Michaud, Oligonucleotide assay and potency, in: Handb. Anal. Oligonucleotides Relat. Prod., CRC Press, 2011, pp. 285–305, http://dx.doi.org/ 10.1201/b10714-10. [10] V. Murugaiah, Determination of extinction coefficient, in: Handb. Anal. Oligonucleotides Relat. Prod., CRC Press, 2011, pp. 351–359, http://dx.doi.org/ 10.1201/b10714-13. [11] H. Aygün, Determination of Base composition, in: Handb. Anal. Oligonucleotides Relat. Prod, CRC Press, 2011, pp. 439–452, http://dx.doi.org/ 10.1201/b10714-18. [12] B.L. Hirschbein, K.L. Fearon, 31P NMR spectroscopy in oligonucleotide research and development, Antisense Nucleic Acid Drug Dev. 7 (1997) 55–61, http://dx.doi.org/10.1089/oli.1.1997.7.55, PMID - 9055040. [13] M. DeRider, D. Brooks, G. Burt, Structural Determination by NMR, Handb. Anal. Oligonucleotides Relat. Prod., 2011, pp. 361–384, http://dx.doi.org/10. 1201/b10714-14. [14] B.J. Bergot, W. Egan, Separation of synthetic phosphorothioate oligodeoxynucleotides from their oxygenated (phosphodiester) defect species by strong-anion-exchange high-performance liquid chromatography, J.
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
Chromatogr. A 599 (1992) 35–42, http://dx.doi.org/10.1016/00219673(92)85456-4. V. Metelev, S. Agrawal, Ion-exchange high-performance liquid chromatography analysis of oligodeoxyribonucleotide phosphorothioates, Anal. Biochem. 200 (1992) 342–346, http://dx.doi.org/10.1016/00032697(92)90476-n, PMID - 1632499. O. Chahrour, J. Malone, Inductively coupled plasma mass spectrometry (ICP-MS) applications in quantitative proteomics, Protein Pept. Lett. 23 (2016) 1, http://dx.doi.org/10.2174/0929866523666161213094936, PMID 27964701. H.-S. Lee, S.H. Kim, J.-S. Jeong, Y.-M. Lee, Y.-H. Yim, Sulfur-based absolute quantification of proteins using isotope dilution inductively coupled plasma mass spectrometry, Metrologia 52 (2015) 619–627, http://dx.doi.org/10. 1088/0026-1394/52/5/619. L. Cid-Barrio, F. Calderón-Celis, P. Abásolo-Linares, M.L. Fernández-Sánchez, J.M. Costa-Fernández, J.R. Encinar, A. Sanz-Medel, Advances in absolute protein quantification and quantitative protein mapping using ICP-MS, TrAC Trends Anal. Chem. 104 (2018) 148–159, http://dx.doi.org/10.1016/j.trac. 2017.09.024. L. Balcaen, E. Bolea-Fernandez, M. Resano, F. Vanhaecke, Inductively coupled plasma – tandem mass spectrometry (ICP-MS/MS): a powerful and universal tool for the interference-free determination of (ultra)trace elements – a tutorial review, Anal. Chim. Acta 894 (2015) 7–19, http://dx.doi.org/10.1016/j. aca.2015.08.053. S.D. Fernández, N. Sugishama, J.R. Encinar, A. Sanz-Medel, Triple quad ICPMS (ICPQQQ) as a new tool for absolute quantitative proteomics and phosphoproteomics, Anal. Chem. 84 (2012) 5851–5857, http://dx.doi.org/10. 1021/ac3009516. Q. Tu, E.N. Guidry, F. Meng, T. Wang, X. Gong, A high-throughput flow injection inductively coupled plasma mass spectrometry method for quantification of oligonucleotides, Microchem. J. 124 (2016) 668–674, http:// dx.doi.org/10.1016/j.microc.2015.10.011. ´ ´ S. Studzinska, S. Mounicou, J. Szpunar, R. Łobinski, B. Buszewski, New approach to the determination phosphorothioate oligonucleotides by ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry, Anal. Chim. Acta 855 (2015) 13–20, http://dx.doi. org/10.1016/j.aca.2014.12.010, PMID - 25542085.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Development of an ICP-MS/MS approach for absolute quantification and determination of phosphodiester to phosphorothioate ratio in therapeutic oligonucleotides Juliusz Bianga, Magali Perez, Damien Mouvet, Caroline Cajot, Philippe De Raeve, Arnaud Delobel ∗ Quality Assistance sa, Technoparc de Thudinie 2, B-6536 Donstiennes, Belgium
a r t i c l e
i n f o
Article history: Received 15 December 2019 Received in revised form 13 February 2020 Accepted 14 February 2020 Available online 15 February 2020 Keywords: Absolute quantification ICP-MS/MS Mass spectrometry Oligonucleotide Phosphorothioate
a b s t r a c t A new analytical method based on ICP-MS/MS is proposed for the characterization of synthetic phosphorothioate oligonucleotides. Absolute quantification of oligonucleotides is challenging, as well as the determination of phosphodiester to phosphorothioate ratio for phosphorothioate oligonucleotides. Both are considered as critical quality attributes and should be determined using robust validated methods. The method we developed was designed to be easy to apply, fast, and robust. It allows simultaneous absolute quantification of an oligonucleotide (based on the quantification of phosphorus), determination of the phosphodiester to phosphorothioate ratio (based on the quantification of phosphorus and sulfur) and optionally determination of sodium (or any other metal) as a counter ion. The performance of the method was demonstrated on O,O-diethyl thiophosphate potassium salt, a well characterized model substance that possesses similar composition to phosphorothioate oligonucleotides. Method was also tested on different synthetic phophorothioate oligonucleotides, showing excellent accuracy and precision. © 2020 Elsevier B.V. All rights reserved.
1. Introduction Oligonucleotides are a class of therapeutics that has been under clinical development for the past 30 years. Different categories of oligonucleotides have been developed, among which anti-sense oligonucleotides (ASO) and aptamers, and more recently small interfering RNA (siRNA) [1]. As of November 2019, 9 oligonucleotides were approved for therapeutic use in Europe and/or in the US. They have a potential to be used in a wide range of diseases, including cancer, cardiovascular and metabolic conditions, neurological disorders, and ophthalmic diseases [2–7]. One of the challenges for oligonucleotides used as therapeutics is their pharmacokinetics. In order to make oligonucleotides amenable to their use as medicines, many chemical modifications designed to increase resistance against enzymatic digestion have been developed. Among them, phosphorothioate oligonucleotides [8], which include a modification on the phosphate backbone by the substitution of sulfur for a non-bridging oxygen (see Fig. 1), have shown promise, especially for antisense applications [2].
∗ Corresponding author. E-mail address: [email protected] (A. Delobel). https://doi.org/10.1016/j.jpba.2020.113179 0731-7085/© 2020 Elsevier B.V. All rights reserved.
As for all therapeutic molecules, the determination of their quantity is critical for both safety and efficacy reasons. Quantification can be either absolute or relative. Absolute quantification means that no well-characterized reference standard is required. The most commonly used technique for the routine quantification of oligonucleotides in drug substances or drug products is UV spectroscopy [9], but it requires the prior knowledge of the extinction coefficient [10]. This coefficient can be estimated by calculation using the base composition of the oligonucleotide, but such an estimation has been shown to produce a bias of around 10 %. It can also be determined experimentally by measuring the absorbance at 260 nm of solutions of known concentrations, but the problem remains the measurement of this concentration. Another possibility is to hydrolyze the oligonucleotide with an enzyme and measure the concentration of released nucleotides by liquid chromatography with UV or MS detection [11]. But this methodology also shows limited precision and accuracy. Quantification can also be done using a relative method, such as LC/MS, LC/UV or PCR, but these methods require a well-characterized reference standard. In phosphorothioate oligonucleotides, the molar ratio of phosphodiester to phosphorothioate linkages is also considered as a critical quality attribute and should be determined experimentally during characterization studies. Due to the sequential process, even a highly efficient sulfurization process may result in high amounts
2
J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179
Fig. 1. Structure of RNA and phosphorothioate RNA.
of impurities. A missing substitution may play a role in the potency and the in vivo stability of the oligonucleotide versus nucleases activity. 31 P-NMR (Nuclear Magnetic Resonance) is the common way to determine this ratio [12]. This technique requires expensive equipment and skilled operators, is relatively slow, sequential and lacks sensitivity [13]. Moreover, it cannot be easily implemented in a GMP (Good Manufacturing Practices) environment if this method has to be used for batch release. Another possibility is to use strong-anion exchange chromatography (SAX) with UV detection [14,15]. This technique is more sensitive than NMR but shows strong limitations in the presence of other process- or product-related impurities. ICP-MS is mainly used to analyze metals in a wide range of applications, from environmental analysis to quality control of pharmaceuticals and biopharmaceuticals. However, it can also be used in biotechnology and proteomics [16] thanks to its ability to quantify elements such as phosphorus (for phosphoproteins) or sulfur [17,18] (for virtually all proteins). Sulfur determination by ICP-MS has long been a challenge because of the high ionization potential of this element (10.4 eV) and the interferences from polyatomic ions for all sulfur isotopes. The same issue is observed for the determination of phosphorus. Triple-quadrupole ICP-MS [19] can easily circumvent these issues, with the formation of sulfur and phosphorus oxide cations in the reaction cell, as shown previously by Diez-Fernandez et al. [20]. Different groups had already mentioned the use of ICP/MS for the quantification of oligonucleotides, but in other contexts [21,22]. The ability of triple quadrupole ICP-MS to quantify both phosphorus and sulfur appeared to us as an opportunity for the absolute quantification and the efficient determination of phosphodiester to phosphorothioate ratio in phosphorothioate oligonucleotides, if possible in the same analytical run. Provided the structure of the oligonucleotide is known (which is always the case for oligonucleotide drug substances and drug products), the phosphorus content can be easily calculated. Therefore, measuring the phosphorus content by ICP-MS/MS can give access the oligonucleotide concentration, without the need for a standard. Only a certified solution of phosphoric acid is needed. Moreover, by measuring both the phosphorus and the sulfur content, the phosphodiester to phosphorothioate ratio can also be readily determined. In an ICP-MS/MS instrument, samples are introduced through a nebulization device, but nebulization and transport efficiency are not equivalent for small and large molecules. As the standardization is done using sulfuric acid and phosphoric acid, a very accurate method cannot be obtained by introducing directly the oligonucleotide solution in the nebulizer. Therefore, all samples were digested with nitric acid and hydrochloric acid in a microwave oven, after addition of yttrium as an internal standard.
As sodium is the most common counter-ion for oligonucleotides, we also measured its concentration during the same analytical run, in order to be able to perform mass-balance calculations (vide infra). We present the preliminary results obtained for the development of an ICP-MS/MS method for the absolute quantification of therapeutic oligonucleotides in drug substances and drug products and for the determination of phosphodiester to phosphorothioate ratio in a single method. 2. Material 2.1. Reagents and material ICP/MS-grade standard solutions of sodium, phosphorus and sulfur were obtained from Merck Chemicals. ICP/MS-grade standard solution of yttrium was obtained from VWR International. Nitric and hydrochloric acid (TraceSELECTTM grade) were obtained from Honeywell Fluka. O,O-Diethyl thiophosphate potassium salt (DTP) and ammonium hydroxide solution (28.0–30.0%) were obtained from Merck Chemicals. Ultrapure deionized water (resistivity > 18 M.cm) from a Milli-Q system (Millipore) was used throughout the experiment. Oligonucleotide samples were obtained from Kaneka Eurogentec (Seraing, Belgium). 2.2. Sample and standard preparation Sample solutions were obtained by gravimetric reconstitution of the oligonucleotide lyophilizates or DPT in water. Then, the solutions were aliquoted into digestion vessels mixed with 0.1 mL of internal standard (50 ppm Yttrium solution), 2.5 mL of concentrated nitric acid and 0.5 mL of concentrated hydrochloric acid and covered with caps. Digestion was carried out using a single reaction chamber Ultrawave MW digestion system (Milestone Srl, Sorisole (BG), Italy), equipped with 15 position sample rack. The sample rack with the digestion vessels was installed in the reaction chamber containing outer bath (150 mL of water and 5 mL concentrated nitric acid). The reaction chamber was closed and pressurized to 40 bars with compressed nitrogen, heated to reach 220 ◦ C in 35 min and then the temperature was maintained at 220 ◦ C for 30 min. After digestion all the solutions for analysis were transferred to polypropylene metal free tubes and diluted to 50 mL with water. For each digestion cycle at least one procedure blank was included. Digested samples were analyzed versus an external five-point calibration with an internal standardization. Calibration was prepared by dilution of the reference standards of phosphorus, sulfur and optionally of sodium, in the matrix matched solvent (water solution of acids at the same level as for digests).
J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179 Table 1 ICP/MS acquisition parameters. Description
Acquisition mode 1
Acquisition mode 2
RF power (W) Spray chamber Nebulizer Sample flow (mL/min) Nebulizer gas (L/min) Dilution gas (L/min) Scan mode Reaction gas Integ. Time per ion (s) Acquisition mode Replicate Sweep per replicate
1550 Double pass (Scott type) Micromist 0.4 0.8 0.2 Mass-shift reaction mode O2 (45%) 0.45 Spectrum 3 100
1550 Double pass (Scott type) Micromist 0.4 0.8 0.2 Single quadrupole He 0.45 Spectrum 3 100
2.3. ICP-MS/MS analysis ICP-MS analysis was performed using an Agilent 8800 ICP-QQQ triple quadrupole ICP-MS (Agilent Technologies Inc., Santa Clara, CA, USA) in mass-shift mode using O2 as a reaction gas. For sulfur quantification, the first quadrupole was set to select 32 S+ (m/z 32) and 34 S+ (m/z 34) ions, mass shifted by the reaction with oxygen in the octapole reaction system and finally detected as 32 S16 O+ (m/z 48) and 34 S16 O+ (m/z 50) by the second quadrupole. For phosphorus quantification, the first quadrupole was set to select 31 P+ (m/z 31) ion, mass shifted by the reaction with oxygen in the octapole reaction system and finally detected as 31 P16 O+ (m/z 47) by the second quadrupole. Yttrium was used as internal standard for S and P quantification: the first quadrupole was set to select 89 Y+ (m/z 89) ion, mass shifted by the reaction with oxygen in the octapole reaction system and finally detected as 89 Y16 O+ (m/z 105) by the second quadrupole. Sodium determination was carried out in single-quadrupole mode; the second quadrupole was set to select 23 Na+ (m/z 23). Yttrium was used as internal standard: the second quadrupole was set to select 89 Y+ (m/z 89). The sample introduction system consisted of a quartz Micromist nebulizer, a quartz double path spray chamber and an integral quartz injector and torch (Agilent Technologies Inc.). The sample solutions were infused continuously into the spray chamber of ICP-MS by an integrated autosampler I-AS (Agilent Technologies Inc.) and a peristaltic pump. The operating conditions of the ICP-MS were optimized and are summarized in Table 1.
3
pound and digested as a sample. Accuracy and precision of the method were evaluated by digesting different amounts of DTP, resulting in solutions containing 0.2 to 0.6 mmol/L of phosphorus. Results are presented in Table 2 and Fig. 2. Precision was assessed by calculating the relative standard deviation (RSD) on three determinations at each concentration level. RSDs range between 0.2 and 0.4 %. Accuracy was assessed by calculating the recovery between the theoretical concentration and the one calculated from the standardization curve. The mean recoveries at each level range from 99.8–102.4%. The linearity of the method was assessed by plotting the experimental concentration versus the theoretical concentration. By linear regression, a slope of 0.989 was obtained, with a determination coefficient (R2 ) of 0.9999. The behavior of DTP during the sample preparation and the ICP-MS/MS process may be different compared to oligonucleotides, but no oligonucleotide with a certified concentration is commercially available. Therefore, to evaluate the accuracy of the method developed, we analyzed two purified oligonucleotide samples for which we measured the water content (by coulometric Karl-Fisher titration) and the phosphorus and sodium content by ICP-MS/MS. Based on the oligonucleotide content, we were able to calculate the expected amount of sodium present as a counter-ion of the oligonucleotide (1:1 molar ratio to phosphorus). The excess sodium content was easily calculated, and we assumed that this sodium was present in the form of sodium chloride (this assumption was done based information on the synthetic route provided by the supplier). The amount of chloride ions was therefore calculated. All these results are presented in Table 3. A mass balance close to 100 % was obtained for both samples, which demonstrates indirectly the excellent accuracy of the method. As the method is not intended for the quantification of impurities, sensitivity was not assessed, in accordance with ICH Q2(R1) guideline on the validation of analytical methods. In order to get reproducible and accurate results, the method was designed so as the signals measured are far from the detection limits of the instrument. The sulfur and phosphorus concentrations measured for oligonucleotide samples using the method described here are in the ppm range, while limits of quantification are in the low ppb range.
3.2. Determination of phosphodiester to phosphorothioate ratio 2.4. Determination of water content in oligonucleotide samples Water content determination was carried by coulometric Karl-Fisher titration, with 831 K F Coulometer equipped with a Thermoprep device (Metrohm AG, Herisau, Switzerland). An aliquot of a sample was heated at 180 ◦ C, under dry nitrogen flow (50 mL/min) which carried the desorbed water from the sample to the titration cell where after absorption in the electrolyte it was titrated electrochemically versus an indicator electrode. 3. Results and discussion 3.1. ICP-MS/MS for the analysis of phosphorus and sulfur 3.1.1. Absolute quantification of oligonucleotides Absolute quantification is the most straightforward application of ICP-MS/MS for oligonucleotides. By performing a digestion step in the presence of an internal standard before analysis, excellent precision and accuracy can be obtained. In order to evaluate the performance of the method, a certified reference standard is required. As no oligonucleotide with a certified accurate concentration is commercially available, a more characterized sample, O,O-diethyl thiophosphate potassium salt (DTP) was taken as a model com-
In order to evaluate the capabilities of the technique to measure accurately and precisely sulfur / phosphorus ratios, the instrument was calibrated with diluted NIST solutions of phosphoric and sulfuric acids, varying the sulfur/phosphorus molar ratios from 0 to 1 at constant phosphorus content. Results are presented in Table 4. Accuracy was close to 100 % at all levels tested (with a maximum bias of 3%, at low S/P ratio), with excellent precision (%RSD on 3 determinations below 0.6 %). The linearity of the method is presented in Fig. 3. The performance of the method was studied using a small molecule standard (O,O-diethyl thiophosphate potassium salt), containing one atom of sulfur and one atom of phosphorus, as well as samples of therapeutic oligonucleotides (information on sample characterization provided by the supplier allowed us to know accurately the phosphodiester to phosphorothioate ratio expected for each oligonucleotide sample). Results are presented in Table 5. Very good accuracies (between 99 and 101 %) were obtained for the P O/P = S ratio of all samples with a relative standard deviation on 3 determinations below 1%. Even for the most complex sample (oligonucleotide containing 23 bases with 6 phosphorothioate linkages and a cholesteryl group), an accuracy of 99 % is obtained, with a relative standard deviation on 3 determinations of 0.3 %.
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J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179
Table 2 Results obtained for the evaluation of absolute quantification method performance. Theoretical DTP concentration (mmol/L)
0.192
0.295
0.384
0.475
0.578
Replicate
Phosphorus concentration measured (mmol/L)
Accuracy (expressed as % recovery)
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
0.198 0.196 0.197 0.297 0.299 0.298 0.387 0.386 0.388 0.475 0.477 0.473 0.579 0.581 0.577
102.9 102.0 102.3 100.9 101.3 101.2 100.7 100.6 100.9 99.8 100.2 99.4 100.3 100.6 99.9
Average accuracy (%)
RSD% (n = 3)
102.4
0.4
101.2
0.2
100.7
0.2
99.8
0.4
100.3
0.3
Fig. 2. Evaluation of method linearity for DTP absolute quantification based on phosphorus content.
Table 3 Evaluation of method accuracy based on phosphorus, sodium, and water content on two purified oligonucleotide samples. % w/w in sample powder
Sample
Oligonucleotide 1 Oligonucleotide 2
Mass balance (%)
Oligonucleotide
Sodium (total)
Water
Excess sodium (calc.)
Chloride (calc.)
30.0 63.2
28.1 14.6
3.5 6.6
26.0 10.1
40.1 15.6
101.7 100.1
Table 4 Results obtained for the evaluation method performance for the determination of phosphodiester to phosphorothioate ratio. Test solution
T1
T2
T3
T4
Replicate
1 2 3 1 2 3 1 2 3 1 2 3
Measured conc. (mmol/L)
P/S molar ratio
P
S
Theoretical
Experimental
0.587 0.589 0.588 0.583 0.582 0.580 0.581 0.579 0.578 0.579 0.575 0.576
0.626 0.628 0.627 0.184 0.186 0.184 0.061 0.061 0.061 0.012 0.012 0.012
0.932 0.932 0.932 3.108 3.108 3.108 9.326 9.326 9.326 46.630 46.630 46.630
0.937 0.938 0.938 3.159 3.122 3.142 9.470 9.421 9.428 45.553 45.127 45.485
Accuracy %
100.5 100.6 100.6 101.6 100.4 101.1 101.5 101.0 101.1 97.7 96.8 97.5
Average Accuracy %
RSD %
100.6
0.06
101.1
0.60
101.3
0.26
97.3
0.49
J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179
5
Fig. 3. Evaluation of method linearity for phosphodiester to phosphorothioate ratio determination.
Table 5 Evaluation of the accuracy of phosphodiester to phosphorothioate ratio determination of DTP and oligonucleotide samples. Sample
Number of P/mol
Number of S/mol
Theoretical P/S ratio
Experimental P/S ratio
Accuracy %
RSD (n = 3)
O,O-diethyl thiophosphate Oligonucleotide 1 (20 mer, full 2 -OMe full PS) Oligonucleotide 2 (20 mer, full PS) Oligonucleotide 3 (20 mer, full 2 -OMe full PS) Oligonucleotide 4 (22 mer, full PS) Oligonucleotide 5 (22 mer, full PS) Oligonucleotide 6 (23 mer, full 2 -OMe, 6 PS, 5 -Cholesteryl)
1 19 19 19 21 21 23
1 19 19 19 21 21 6
1.000 1.0000 1.0000 1.0000 1.0000 1.0000 3.833
0.9913 1.010 0.9999 1.0032 0.9884 0.9935 3.876
99.1 101.0 100.0 99.7 101.2 100.7 101.1
0.1 0.4 0.4 0.3 0.3 0.8 0.3
4. Conclusion
CRediT authorship contribution statement
We developed an ICP-MS/MS method based on the quantification of phosphorus and sulfur after microwave digestion for the absolute quantification of oligonucleotides (based on phosphorus content), and for the determination of phosphodiester to phosphorothioate ratio in purified therapeutic oligonucleotide samples, in the same analytical run. Although the quantification of phosphorus and sulfur by ICP-MS/MS was previously described, this is to the best of our knowledge the first application of this technology to therapeutic phosphorothioate oligonucleotides for both absolute quantification and phosphodiester-to-phosphorothioate ratio determination. Around 10 mg of sample are required to perform the test, and results can be obtained within one day of analytical work, including sample preparation. Even if additional investigations may be needed, preliminary results are very encouraging, with excellent precision and accuracy obtained on reference standards and therapeutic oligonucleotide samples. As there is no chromatographic separation prior to ICP-MS/MS analysis, this method is intended to be applied to highly-purified therapeutic-grade oligonucleotides. The absolute quantification method will actually quantify the oligonucleotide and all the potential impurities, while another purity method (usually a chromatographic method with optical or MS detection) may be used to determine the purity profile. The method could be used in routine testing of oligonucleotides in a GMP environment, as an orthogonal method to 31 P-NMR.
Juliusz Bianga: Methodology, Investigation, Formal analysis, Writing - original draft. Magali Perez: Supervision. Damien Mouvet: Supervision. Caroline Cajot: Resources. Philippe De Raeve: Conceptualization, Methodology, Supervision. Arnaud Delobel: Project administration, Writing - review & editing.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments The authors would like to thank Kaneka Eurogentec for providing oligonucleotide samples.
References [1] C.A. Stein, D. Castanotto, FDA-approved oligonucleotide therapies in 2017, Mol. Ther. 25 (2017) 1069–1075, http://dx.doi.org/10.1016/j.ymthe.2017.03. 023, PMID - 28366767. [2] C.F. Bennett, Therapeutic antisense oligonucleotides are coming of age, Annu. Rev. Med. 70 (2019) 307–321, http://dx.doi.org/10.1146/annurev-med041217-010829, PMID - 30691367.
6
J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179
[3] L. Echevarría, P. Aupy, A. Goyenvalle, Exon-skipping advances for Duchenne muscular dystrophy, Hum. Mol. Genet. 27 (2018) R163–R172, http://dx.doi. org/10.1093/hmg/ddy171, PMID - 29771317. [4] C.I.E. Smith, R. Zain, Therapeutic oligonucleotides: state of the art, Annu. Rev. Pharmacol. Toxicol. 59 (2018) 1–26, http://dx.doi.org/10.1146/annurevpharmtox-010818-021050, PMID - 30285540. [5] O. Khorkova, C. Wahlestedt, Oligonucleotide therapies for disorders of the nervous system, Nat. Biotechnol. 35 (2017) 249–263, http://dx.doi.org/10. 1038/nbt.3784, PMID - 28244991. [6] K.E. Lundin, O. Gissberg, C.I.E. Smith, Oligonucleotide therapies: the past and the present, Hum. Gene Ther. 26 (2015) 475–485, http://dx.doi.org/10.1089/ hum.2015.070, PMID - 26160334. [7] K. Takakura, A. Kawamura, Y. Torisu, S. Koido, N. Yahagi, M. Saruta, The clinical potential of oligonucleotide therapeutics against pancreatic cancer, Int. J. Mol. Sci. 20 (2019) 3331, http://dx.doi.org/10.3390/ijms20133331, PMID - 31284594. [8] F. Eckstein, Phosphorothioates, essential components of therapeutic oligonucleotides, Nucleic Acid Ther. 24 (2014) 374–387, http://dx.doi.org/10. 1089/nat.2014.0506, PMID - 25353652. [9] D. Michaud, Oligonucleotide assay and potency, in: Handb. Anal. Oligonucleotides Relat. Prod., CRC Press, 2011, pp. 285–305, http://dx.doi.org/ 10.1201/b10714-10. [10] V. Murugaiah, Determination of extinction coefficient, in: Handb. Anal. Oligonucleotides Relat. Prod., CRC Press, 2011, pp. 351–359, http://dx.doi.org/ 10.1201/b10714-13. [11] H. Aygün, Determination of Base composition, in: Handb. Anal. Oligonucleotides Relat. Prod, CRC Press, 2011, pp. 439–452, http://dx.doi.org/ 10.1201/b10714-18. [12] B.L. Hirschbein, K.L. Fearon, 31P NMR spectroscopy in oligonucleotide research and development, Antisense Nucleic Acid Drug Dev. 7 (1997) 55–61, http://dx.doi.org/10.1089/oli.1.1997.7.55, PMID - 9055040. [13] M. DeRider, D. Brooks, G. Burt, Structural Determination by NMR, Handb. Anal. Oligonucleotides Relat. Prod., 2011, pp. 361–384, http://dx.doi.org/10. 1201/b10714-14. [14] B.J. Bergot, W. Egan, Separation of synthetic phosphorothioate oligodeoxynucleotides from their oxygenated (phosphodiester) defect species by strong-anion-exchange high-performance liquid chromatography, J.
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
Chromatogr. A 599 (1992) 35–42, http://dx.doi.org/10.1016/00219673(92)85456-4. V. Metelev, S. Agrawal, Ion-exchange high-performance liquid chromatography analysis of oligodeoxyribonucleotide phosphorothioates, Anal. Biochem. 200 (1992) 342–346, http://dx.doi.org/10.1016/00032697(92)90476-n, PMID - 1632499. O. Chahrour, J. Malone, Inductively coupled plasma mass spectrometry (ICP-MS) applications in quantitative proteomics, Protein Pept. Lett. 23 (2016) 1, http://dx.doi.org/10.2174/0929866523666161213094936, PMID 27964701. H.-S. Lee, S.H. Kim, J.-S. Jeong, Y.-M. Lee, Y.-H. Yim, Sulfur-based absolute quantification of proteins using isotope dilution inductively coupled plasma mass spectrometry, Metrologia 52 (2015) 619–627, http://dx.doi.org/10. 1088/0026-1394/52/5/619. L. Cid-Barrio, F. Calderón-Celis, P. Abásolo-Linares, M.L. Fernández-Sánchez, J.M. Costa-Fernández, J.R. Encinar, A. Sanz-Medel, Advances in absolute protein quantification and quantitative protein mapping using ICP-MS, TrAC Trends Anal. Chem. 104 (2018) 148–159, http://dx.doi.org/10.1016/j.trac. 2017.09.024. L. Balcaen, E. Bolea-Fernandez, M. Resano, F. Vanhaecke, Inductively coupled plasma – tandem mass spectrometry (ICP-MS/MS): a powerful and universal tool for the interference-free determination of (ultra)trace elements – a tutorial review, Anal. Chim. Acta 894 (2015) 7–19, http://dx.doi.org/10.1016/j. aca.2015.08.053. S.D. Fernández, N. Sugishama, J.R. Encinar, A. Sanz-Medel, Triple quad ICPMS (ICPQQQ) as a new tool for absolute quantitative proteomics and phosphoproteomics, Anal. Chem. 84 (2012) 5851–5857, http://dx.doi.org/10. 1021/ac3009516. Q. Tu, E.N. Guidry, F. Meng, T. Wang, X. Gong, A high-throughput flow injection inductively coupled plasma mass spectrometry method for quantification of oligonucleotides, Microchem. J. 124 (2016) 668–674, http:// dx.doi.org/10.1016/j.microc.2015.10.011. ´ ´ S. Studzinska, S. Mounicou, J. Szpunar, R. Łobinski, B. Buszewski, New approach to the determination phosphorothioate oligonucleotides by ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry, Anal. Chim. Acta 855 (2015) 13–20, http://dx.doi. org/10.1016/j.aca.2014.12.010, PMID - 25542085.