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
loais Genryme
2013
Defibrotide(Definelio)
HVOD
Jazz
2016
Eteplirsen(Exondys 51)
Duchenne muscular dystrophy (Exon 51 of SMN2 gene)
Sarepa
Nusinersen(Spinraza)
spinal muscular atrophy (Exon 7 of DMD gene)
lonis Biogen
Patiseran(Oupattro)
Transmissible parathyroid amyloidosis
siRNA
Alnylam
2018
Inotersen(Tegsedi)
lonis/Akce
Waylivra(volmesorsen)
Familial chylomicron syndrome
lonis/Akcea
2019
Givosaran(Givlaari)
Acute hepatic porphyria
Alnyfam
Golodirsen(Vyondys 53)
Duchenne muscular dystrophy (Exon 53 of DMD gene)
Sarepta
Viltolarsen(Vilepso)
Nippon Shinyaku
2020
Lumasiran(Oxlumo)
Primary hyperoxaluria type
Alnyiam
Inclisiran(Leqvio)
Hypercholesterolemia and mixed dyslipidemia in adults
Novartis
Casamersen(Amnndys 45)
Duchenne muscular dystrophy (Exon 45 of DMD gene)
2021
Advantages of oligonucleotide drugs
Compared with traditional drugs, oligonucleotide drugs have multiple technical advantages, including:
Classification of oligonucleotide drugs
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.
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).
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).
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.