Application of LC-MS in the Bioassay of Oligonucleotide Drugs

Introduction of oligonucleotide drugs

Oligonucleotides are a class of nucleic acid molecules consisting of dozens of nucleotides with short sequences. They mainly inhibit the expression of target proteins through gene silencing so as to achieve the purpose of treating diseases.

The world's first antisense nucleic acid drug was approved in 1998, which started the journey of oligonucleotide drug marketing. Patisiran, the world's first siRNA drug, was certificated in 2018, which was more of a milestone. Recent years witnessed the approval of 4 siRNA drugs and 15 oligonucleotide drugs, and more than 400 compounds are under development so far.

Name (Trade Name)DiseaseDrug CategoryCompanyApproval Year
Fomvirsen(Vitravene)Tile cell virus retinitisAntisense oligonucleotidelonas Novartis1998
Pegaptanib(Macugen)age-related macular degenerationAptamerNeXstarEyelech2004
Mipomersen(Kynamro)hoFHAntisense oligonucleotideloais Genryme2013
Defibrotide(Definelio)HVODAntisense oligonucleotideJazz2016
Eteplirsen(Exondys 51)Duchenne muscular dystrophy (Exon 51 of SMN2 gene)Antisense oligonucleotideSarepa2016
Nusinersen(Spinraza)spinal muscular atrophy (Exon 7 of DMD gene)Antisense oligonucleotidelonis2016
Patiseran(Oupattro)Transmissible parathyroid amyloidosissiRNAAlnylam2018
Inotersen(Tegsedi)Transmissible parathyroid amyloidosisAntisense oligonucleotidelonis/Akce2018
Waylivra(volmesorsen)Familial chylomicron syndromeAntisense oligonucleotidelonis/Akcea2019
Givosaran(Givlaari)Acute hepatic porphyriasiRNAAlnyfam2019
Golodirsen(Vyondys 53)Duchenne muscular dystrophy (Exon 53 of DMD gene)Antisense oligonucleotideSarepta2019
Viltolarsen(Vilepso)Duchenne muscular dystrophy (Exon 53 of DMD gene)Antisense oligonucleotideNippon Shinyaku2020
Lumasiran(Oxlumo)Primary hyperoxaluria typesiRNAAlnyiam2020
Inclisiran(Leqvio)Hypercholesterolemia and mixed dyslipidemia in adultssiRNANovartis2020
Casamersen(Amnndys 45)Duchenne muscular dystrophy (Exon 45 of DMD gene)Antisense oligonucleotideSarepta2021

Advantages of oligonucleotide drugs

Compared with traditional drugs, oligonucleotide drugs have multiple technical advantages, including:

  • High specificity and efficiency
  • Long-lasting efficacy, reducing dosing frequency
  • The research period is short, and the success rate of clinical transformation and development is relatively high
  • Rich targets and broad indications, etc.

Classification of oligonucleotide drugs

oligonucleotide drugsDescription
ASOSingle-stranded DNA or modified RNA oligonucleotides; modulates target RNA function
siRNAsiRNA can induce gene silencing
miRNACan regulates the translation process of mRNA
AptamerSpecific binding to ligand proteins using three-dimensional structures
OtherssaRNA can activate gene expression; TRAN, sgRNA, U1 snRNA, etc.

Bioanalysis of oligonucleotide

At present, the biological analysis methods of oligonucleotide mainly include HPLC, LC-MS, ELISA, and RT-qPCR.This paper mainly introduces the application of LC-MS technology in the biological analysis of oligonucleotide drugs.

(1) Sample pretreatment

In the biological matrix, there are various complex components including proteins, phospholipids, a large number of salts, and other organic and inorganic substances, which will produce an obvious matrix effect when LC-MS is used for detection. In addition, oligonucleotides have a high plasma protein binding rate after modification. Therefore, to make LC-MS analysis proceed smoothly, salt and protein in biological samples need to be removed in the sample pretreatment stage.

Conventional sample pretreatment mainly includes protein precipitation (PPT), liquid-liquid extraction (LLE), and solid-phase extraction (SPE).

(2) Chromatographic separation

Oligonucleotides are highly acidic and polar combinations, which are difficult to retain on common chromatographic columns. Oligonucleotide chromatography mainly includes ion-pair reverse phase chromatography (IP-RPLC), ion-exchange chromatography (IEC), and hydrophilic interaction chromatography (HILIC). Ion-pair reverse phase chromatography is the most commonly used method.

There are many kinds of ion-pair reagents. The most commonly used ion-pair reagents are amine acetate-based reagents and amine ion-pair reagents based on Hexafluoroisopropanol (HFIP). However, HFIP-based ion-pair reagents are more commonly used than acetic acid-based ions, because compared with acetic acid, HFIP has a weaker boiling point and acidity, which means that HFIP is more volatile in the gas phase of electrospray ionization, has less interference with oligonucleotide ionization and is more compatible with electrospray mass spectrometry. Common ion-pair reagents are mainly triethylamine (TEA), triethylamine acetate (TEAA), triethylamine carbonate (TEAB), and hexafluoroisopropanol (HFIP).

(3) Mass spectrometric detection

Due to its special structure, oligonucleotides have one acidic proton (PKa~1) on each phosphodiester bond, and these compounds respond better in the negative ion mode of ESI source. Because oligonucleotides contain multiple nucleotide groups, deprotonation occurs in the ESI source, forming multi-charged ions in negative ion mode, and at the same time easily binding metal ions, thus dispersing the mass spectral signal. In addition, with the increase in molecular weight of oligonucleotides, the detection response will be seriously lost. Therefore, it is necessary to optimize the mass spectrum conditions for these compounds, improve the multi-charge distribution, and reduce the formation of adduct ions in the mass spectrum analysis. Mainly by adjusting the pH value of the solution, the concentration of cations, and organic solvent composition to eliminate or reduce the influence of the above factors. It has been mentioned in chromatographic separation that ion-pair reagents need to be used. At the initial stage of method development, ion-pair reagents must also be used to search for parent ions, otherwise, it is difficult to find the corresponding parent ion fragments. Then, based on this condition, mass spectrometry parameters can be optimized, which may obtain a better signal response.

Due to the wide range of oligonucleotide drug indications, including tumors, rare diseases (amyotrophic lateral sclerosis, Duchenne muscular dystrophy, spinal muscular atrophy), viral diseases, kidney diseases, cardiovascular diseases, inflammatory diseases, and metabolic diseases (diabetes) are expected to bring about the small molecular drugs, antibody drugs after a wave of new drug research and development. While there are many challenges in bioanalysis, BOC Sciences has a professional mass spectrometry team that can provide customers with solutions to solve various analytical challenges and contribute to the development of new drugs.

* Only for research. Not suitable for any diagnostic or therapeutic use.
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