34S-SIL of PCSK9-Active Oligonucleotide as Tools for Accurate Quantification by Mass Spectrometry.

Rouven Stulz,Anders Dahlén,Fiona Milligan,Roger Strömberg,Iain Love,Shalini Andersson,Craig Stovold

doi:10.1089/nat.2020.0915

Abstract

Stable isotope labeling (SIL) of active pharmaceutical ingredients (API) is a well-established technique for the accurate quantification of small-molecule drugs. As the scope of active ingredients is expanding into areas of larger molecules, such as oligonucleotides (ONs), the development of new quantification techniques is critical. Herein, we describe the analysis of a 34S-SIL anti-PCSK9 gapmer-type antisense ON. A new method for the quantification of this API in complex biological matrices was developed and applied to mouse, dog, and monkey tissue homogenates, which gave improved accuracy and reproducibility compared with the use of auxiliary ONs as internal standard.

Highlights

Therapeutic oligonucleotides (ONs) have a history spanning over many decades
All ONs were purified using ion pair high performance liquid chromatography (HPLC) (Supplementary Data) and analyzed by ultra performance liquid chromatography (UPLC) using a gradient from 30% to 80% acetonitrile in 10 mM tributylammonium acetate over 20 min on a XBridge C18 column at 60°
The isotope-labeled antisense ONs (ASOs) 2 was demonstrated to be appropriate as internal standard for quantitative measurement of 1 in tissue samples

Summary

Introduction

Therapeutic oligonucleotides (ONs) have a history spanning over many decades. After the discovery that short DNA sequences could inhibit viral RNA translation in 1978, the field started growing rapidly [1]. The specific mechanism of action of a biologically active ON depends on the type of ON and cellular target. In the early days of ON drug development, many problems were encountered, such as unfavorable pharmacokinetics, lack of oral bioavailability, poor cell penetration, and off-target effects that led to preclinical and clinical failures [7]. A modification that is very prevalent in therapeutic antisense ONs (ASOs) is the phosphorothioate (PS) linkage, which replaces a nonbridging oxygen of a phosphodiester linkage by a sulfur atom. This increases resistance toward nuclease degradation and increases protein binding, which leads to improved tissue distribution and longer half-life [8,9]

Methods

Results

Conclusion