An Accurate and Fast ³¹P qNMR Assay Method for Oligonucleotide Therapeutics

Area of Science:

• Analytical Chemistry
• Biochemistry
• Pharmaceutical Sciences

Background:

• Oligonucleotide therapeutics require accurate quality assurance assays.
• Conventional UV-260 nm quantification is inaccurate due to structural modifications and higher-order structures (HOS).
• Gravimetric analysis is affected by water and counterions, impacting accuracy.

Purpose of the Study:

• To develop a fast, accurate, and flexible quantitative assay for oligonucleotide therapeutics.
• To compare the performance of a novel 31P quantitative-NMR (qNMR) method with UV-260 nm and gravimetric analysis.
• To evaluate the suitability of 31P qNMR as a primary assay method for oligonucleotide drugs.

Main Methods:

• Development of an externally referenced 31P quantitative-NMR (qNMR) assay with a run time under 1 hour.
• Validation of the qNMR method using single-stranded DNA standards.
• Comparative analysis of 31P qNMR and UV-260 nm methods on intact oligonucleotide drug products (eteplirsen, inotersen, inclisiran).

Main Results:

• The 31P qNMR assay showed results within 1-5% of UV-260 nm for DNA standards.
• Compared to drug labels, 31P qNMR underestimated concentrations by 7 ± 2% (eteplirsen), 8 ± 1% (inotersen), and 12 ± 1% (inclisiran).
• UV-260 nm underestimated concentrations significantly more: 28 ± 3% (eteplirsen), 10 ± 3% (inotersen), and 10 ± 1% (inclisiran), suggesting issues with extinction coefficients, especially for PMO structures.

Conclusions:

• The 31P qNMR method provides accurate and reliable quantification of oligonucleotide therapeutics.
• The method overcomes the limitations of UV-260 nm assays, particularly for modified oligonucleotides.
• 31P qNMR is proposed as a primary assay method for oligonucleotide drugs and reference standards, offering improved accuracy, efficiency, and flexibility.