Application of LC-MS in the Bioassay of Oligonucleotide Drugs

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)

Disease

Drug Category

Company

Approval Year

Fomvirsen(Vitravene)

Tile cell virus retinitis

Antisense oligonucleotide

lonas Novartis

1998

Pegaptanib(Macugen)

age-related macular degeneration

Aptamer

NeXstarEyelech

2004

Mipomersen(Kynamro)

hoFH

Antisense oligonucleotide

loais Genryme

2013

Defibrotide(Definelio)

HVOD

Antisense oligonucleotide

Jazz

2016

Eteplirsen(Exondys 51)

Duchenne muscular dystrophy (Exon 51 of SMN2 gene)

Antisense oligonucleotide

Sarepa

2016

Nusinersen(Spinraza)

spinal muscular atrophy (Exon 7 of DMD gene)

Antisense oligonucleotide

lonis Biogen

2016

Patiseran(Oupattro)

Transmissible parathyroid amyloidosis

siRNA

Alnylam

2018

Inotersen(Tegsedi)

Transmissible parathyroid amyloidosis

Antisense oligonucleotide

lonis/Akce

2018

Waylivra(volmesorsen)

Familial chylomicron syndrome

Antisense oligonucleotide

lonis/Akcea

2019

Givosaran(Givlaari)

Acute hepatic porphyria

siRNA

Alnyfam

2019

Golodirsen(Vyondys 53)

Duchenne muscular dystrophy

Antisense oligonucleotide

Sarepta

2019

Viltolarsen(Vilepso)

Duchenne muscular dystrophy (Exon 53 of DMD gene)

Antisense oligonucleotide

Nippon Shinyaku

2020

Lumasiran(Oxlumo)

Primary hyperoxaluria type

siRNA

Alnyiam

2020

Inclisiran(Leqvio)

Hypercholesterolemia and mixed dyslipidemia in adults

siRNA

Novartis

2020

Casamersen(Amnndys 45)

Duchenne muscular dystrophy (Exon 45 of DMD gene)

Antisense oligonucleotide

Sarepta

2021

Advantages of oligonucleotide drugs

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

l High specificity and efficiency

l Long-lasting efficacy, reducing dosing frequency

l The research period is short, and the success rate of clinical transformation and development is relatively high

l Rich targets and broad indications.

Classification of oligonucleotide drugs

lASO: single-stranded DNA or modified RNA oligonucleotides; modulates target RNA function

lsiRNA: siRNA can induce gene silencing

lmiRNA: Can regulates the translation process of mRNA

lAptamer: Specific binding to ligand proteins using three-dimensional structures

lOthers: saRNA can activate gene expression; TRAN, sgRNA, and U1 snRNA.

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.

BOC Sciences is a brand of BOCSCI Inc. We leverage our wide spectrum of business in the fields of development, manufacturing, marketing, and distribution to help you make best-informed decisions tailored to your evolving needs for premium chemicals.