Bianga 2020
Bianga 2020
Bianga 2020
Short communication
a r t i c l e i n f o a b s t r a c t
Article history: A new analytical method based on ICP-MS/MS is proposed for the characterization of synthetic phos-
Received 15 December 2019 phorothioate oligonucleotides. Absolute quantification of oligonucleotides is challenging, as well as the
Received in revised form 13 February 2020 determination of phosphodiester to phosphorothioate ratio for phosphorothioate oligonucleotides. Both
Accepted 14 February 2020
are considered as critical quality attributes and should be determined using robust validated methods.
Available online 15 February 2020
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
Keywords:
of the phosphodiester to phosphorothioate ratio (based on the quantification of phosphorus and sulfur)
Absolute quantification
ICP-MS/MS
and optionally determination of sodium (or any other metal) as a counter ion. The performance of the
Mass spectrometry method was demonstrated on O,O-diethyl thiophosphate potassium salt, a well characterized model sub-
Oligonucleotide stance that possesses similar composition to phosphorothioate oligonucleotides. Method was also tested
Phosphorothioate on different synthetic phophorothioate oligonucleotides, showing excellent accuracy and precision.
© 2020 Elsevier B.V. All rights reserved.
https://doi.org/10.1016/j.jpba.2020.113179
0731-7085/© 2020 Elsevier B.V. All rights reserved.
2 J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179
of impurities. A missing substitution may play a role in the potency As sodium is the most common counter-ion for oligonu-
and the in vivo stability of the oligonucleotide versus nucleases cleotides, we also measured its concentration during the same
activity. 31 P-NMR (Nuclear Magnetic Resonance) is the common analytical run, in order to be able to perform mass-balance cal-
way to determine this ratio [12]. This technique requires expen- culations (vide infra).
sive equipment and skilled operators, is relatively slow, sequential We present the preliminary results obtained for the develop-
and lacks sensitivity [13]. Moreover, it cannot be easily imple- ment of an ICP-MS/MS method for the absolute quantification of
mented in a GMP (Good Manufacturing Practices) environment if therapeutic oligonucleotides in drug substances and drug products
this method has to be used for batch release. Another possibil- and for the determination of phosphodiester to phosphorothioate
ity is to use strong-anion exchange chromatography (SAX) with ratio in a single method.
UV detection [14,15]. This technique is more sensitive than NMR
but shows strong limitations in the presence of other process- or 2. Material
product-related impurities.
ICP-MS is mainly used to analyze metals in a wide range of 2.1. Reagents and material
applications, from environmental analysis to quality control of
pharmaceuticals and biopharmaceuticals. However, it can also be ICP/MS-grade standard solutions of sodium, phosphorus and
used in biotechnology and proteomics [16] thanks to its ability to sulfur were obtained from Merck Chemicals. ICP/MS-grade stan-
quantify elements such as phosphorus (for phosphoproteins) or dard solution of yttrium was obtained from VWR International.
sulfur [17,18] (for virtually all proteins). Sulfur determination by Nitric and hydrochloric acid (TraceSELECTTM grade) were obtained
ICP-MS has long been a challenge because of the high ionization from Honeywell Fluka. O,O-Diethyl thiophosphate potassium salt
potential of this element (10.4 eV) and the interferences from poly- (DTP) and ammonium hydroxide solution (28.0–30.0%) were
atomic ions for all sulfur isotopes. The same issue is observed for obtained from Merck Chemicals. Ultrapure deionized water
the determination of phosphorus. (resistivity > 18 M.cm) from a Milli-Q system (Millipore) was
Triple-quadrupole ICP-MS [19] can easily circumvent these used throughout the experiment. Oligonucleotide samples were
issues, with the formation of sulfur and phosphorus oxide cations obtained from Kaneka Eurogentec (Seraing, Belgium).
in the reaction cell, as shown previously by Diez-Fernandez et al.
[20]. 2.2. Sample and standard preparation
Different groups had already mentioned the use of ICP/MS for
the quantification of oligonucleotides, but in other contexts [21,22]. Sample solutions were obtained by gravimetric reconstitution
The ability of triple quadrupole ICP-MS to quantify both phospho- of the oligonucleotide lyophilizates or DPT in water. Then, the
rus and sulfur appeared to us as an opportunity for the absolute solutions were aliquoted into digestion vessels mixed with 0.1 mL
quantification and the efficient determination of phosphodiester of internal standard (50 ppm Yttrium solution), 2.5 mL of concen-
to phosphorothioate ratio in phosphorothioate oligonucleotides, if trated nitric acid and 0.5 mL of concentrated hydrochloric acid and
possible in the same analytical run. covered with caps. Digestion was carried out using a single reaction
Provided the structure of the oligonucleotide is known (which chamber Ultrawave MW digestion system (Milestone Srl, Sorisole
is always the case for oligonucleotide drug substances and drug (BG), Italy), equipped with 15 position sample rack.
products), the phosphorus content can be easily calculated. There- The sample rack with the digestion vessels was installed in
fore, measuring the phosphorus content by ICP-MS/MS can give the reaction chamber containing outer bath (150 mL of water and
access the oligonucleotide concentration, without the need for a 5 mL concentrated nitric acid). The reaction chamber was closed
standard. Only a certified solution of phosphoric acid is needed. and pressurized to 40 bars with compressed nitrogen, heated to
Moreover, by measuring both the phosphorus and the sulfur con- reach 220 ◦ C in 35 min and then the temperature was maintained
tent, the phosphodiester to phosphorothioate ratio can also be at 220 ◦ C for 30 min. After digestion all the solutions for analysis
readily determined. were transferred to polypropylene metal free tubes and diluted to
In an ICP-MS/MS instrument, samples are introduced through 50 mL with water.
a nebulization device, but nebulization and transport efficiency For each digestion cycle at least one procedure blank was
are not equivalent for small and large molecules. As the standard- included.
ization is done using sulfuric acid and phosphoric acid, a very Digested samples were analyzed versus an external five-point
accurate method cannot be obtained by introducing directly the calibration with an internal standardization. Calibration was pre-
oligonucleotide solution in the nebulizer. Therefore, all samples pared by dilution of the reference standards of phosphorus, sulfur
were digested with nitric acid and hydrochloric acid in a microwave and optionally of sodium, in the matrix matched solvent (water
oven, after addition of yttrium as an internal standard. 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 3
Table 2
Results obtained for the evaluation of absolute quantification method performance.
Theoretical DTP Replicate Phosphorus concentration Accuracy (expressed as % Average accuracy (%) RSD% (n = 3)
concentration (mmol/L) measured (mmol/L) recovery)
1 0.198 102.9
0.192 2 0.196 102.0 102.4 0.4
3 0.197 102.3
1 0.297 100.9
0.295 2 0.299 101.3 101.2 0.2
3 0.298 101.2
1 0.387 100.7
0.384 2 0.386 100.6 100.7 0.2
3 0.388 100.9
1 0.475 99.8
0.475 2 0.477 100.2 99.8 0.4
3 0.473 99.4
1 0.579 100.3
0.578 2 0.581 100.6 100.3 0.3
3 0.577 99.9
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.
Table 4
Results obtained for the evaluation method performance for the determination of phosphodiester to phosphorothioate ratio.
P S Theoretical Experimental
Table 5
Evaluation of the accuracy of phosphodiester to phosphorothioate ratio determination of DTP and oligonucleotide samples.
We developed an ICP-MS/MS method based on the quantifica- Juliusz Bianga: Methodology, Investigation, Formal analysis,
tion of phosphorus and sulfur after microwave digestion for the Writing - original draft. Magali Perez: Supervision. Damien Mou-
absolute quantification of oligonucleotides (based on phosphorus vet: Supervision. Caroline Cajot: Resources. Philippe De Raeve:
content), and for the determination of phosphodiester to phospho- Conceptualization, Methodology, Supervision. Arnaud Delobel:
rothioate ratio in purified therapeutic oligonucleotide samples, in Project administration, Writing - review & editing.
the same analytical run. Although the quantification of phospho-
rus and sulfur by ICP-MS/MS was previously described, this is to
the best of our knowledge the first application of this technology Declaration of Competing Interest
to therapeutic phosphorothioate oligonucleotides for both abso-
lute quantification and phosphodiester-to-phosphorothioate ratio The authors declare that they have no known competing finan-
determination. Around 10 mg of sample are required to perform the cial interests or personal relationships that could have appeared to
test, and results can be obtained within one day of analytical work, influence the work reported in this paper.
including sample preparation. Even if additional investigations may
be needed, preliminary results are very encouraging, with excel-
Acknowledgments
lent precision and accuracy obtained on reference standards and
therapeutic oligonucleotide samples.
The authors would like to thank Kaneka Eurogentec for provid-
As there is no chromatographic separation prior to ICP-MS/MS
ing oligonucleotide samples.
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 References
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