4 Buyers’ Guide 2021


LC-MS Analysis of Therapeutic Oligonucleotide and Related Products


A comparison of TQ and Q-TOF Systems


Tairo Ogura1 , Toshiya Matsubara2 , Noriko Kato3


1,2MS Business Unit, Life Science Business Department, Analytical & Measuring Instruments Division, Shimadzu Corporation, Kyoto, Japan 3


Global Application Development Center, Analytical & Measuring Instruments Division, Shimadzu Corporation, Kyoto, Japan Background


The purpose of this study is the qualitative analysis and quantitation of a phosphorothioate oligonucleotide. Therapeutic oligonucleotides are synthetic oligonucleotides composed of 10-30 nucleic acid bases. They are aimed directly at targets such as mRNAs, miRNAs or target proteins that are involved in disease pathogenesis. Both sample preparation and method development are simplified using MS detection to analyse oligonucleotides when compared to immune-based detection methodologies such as ELISA. MS detection also provides the added benefit of being the only method capable of detecting slight differences in modifications and resembling impurities based on accurate molecular weight information.


In this study, a molecular weight determination and quantitation of a therapeutic oligonucleotide and related products was performed utilising liquid chromatography-mass spectrometry. The therapeutic oligonucleotides used in the analysis were Mipomersen (Kynamro) and its analogues. From the results of the charge state deconvolution, the molecular weight of the oligonucleotides was confirmed with a mass error of less than 1 ppm.


In the development of a quantitative method using Q-TOF LC-MS or Triple Quadrupole LC-MS, a product ion derived


from phosphorothioate was selected as a multi-reaction monitoring (MRM) transition, and a highly-sensitive MRM-quantification was successfully achieved. Since the phosphorothioate modification is one of the most common methods used to increase the stability of oligonucleotides in vivo, it is expected that quantitative methods constructed in the same way as this can be applied to a variety of therapeutic oligonucletotides.


Introduction Therapeutic oligonucleotides are synthetic Table 1: Oligonucleotide sequences of Mipomersen analogues.


oligonucleotides with a chemical backbone structure composed of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) [1]. They are ‘molecular-targeted drugs’ aiming specific mRNAs, miRNAs and proteins related to the onset of diseases and regulating their expression and function. Although they work as a molecular target drug, these therapeutic oligonucleotides share the favourable attribute of antibody therapeutics and low- molecular drug molecules in that they can be chemically synthesised in the laboratory, making it possible to reduce manufacturing costs compared to an antibody. They are expected to be the next-generation


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