5 Table 2: QTOF-LC-MS analytical condition of Mipomersen analogues. Method
The oligonucleotide to be analysed was Mipomersen, a drug for treatment of familial hypercholesterolemia. Mipomersen is a 20-base antisense oligonucleotide targeted to apolipoprotein B-100 mRNA that inhibits transcription and has a molecular weight of approximately 7000. Three types of analogues of Mipomersen in total were used: an oligonucleotide obtained by substituting all the 2’-O-(2-Methoxyethyl)- modified bases contained in Mipomersen with DNA base; an oligonucleotide obtained by substituting it with a 2’-O-methyl nucleic acid base; and an oligonucleotide obtained by substituting it with a locked nucleic acid (LNA) base. The respective oligonucleotide sequences and modifications are shown in Table 1.
LC/MS conditions
Analysis using a high-performance liquid chromatography-quadrupole time-of-flight mass spectrometer (Q-TOF-LC-MS)
medicines combining the properties of both low-molecular drugs and biopharmaceutics [2].
Therapeutic oligonucleotides are classified into antisense, siRNA, miRNA, aptamer, decoy and CpG oligo, depending on their structure and mechanism of action. Currently available therapeutic oligonucleotides include antisense and siRNAs targeting mRNAs and miRNAs in cells, aptamers that interact directly with proteins outside cells, and CpG oligos that activate innate immunity.
Natural oligonucleotides are unstable to enzymes in vivo such as nucleases, and it is known that their membrane permeability is low due to their molecular weight and negative charge [3]. These oligonucleotides are chemically modified, and phosphorothioated (Sizing) in which an oxygen atom of a phosphate moiety is replaced with a sulphur atom, modification of a sugar moiety to the 2’position such as 2’-O-methoxyethyl (2’ -MOE) and 2’-O-methyl (2’-OMe), and 2’,4’-BNA/ LNA in which crosslinking is performed to the 2’-4 ‘position are known.
The molecular weight of these oligonucleotides’ ranges from approximately 1000 to several tens of thousands, larger than that of small molecule drugs and smaller than that of antibody drugs. The molecular weight of antisense drugs is approximately 1000. Ligand binding assays (LBA) have been used to quantify therapeutic oligonucleotides with chromatography and mass spectrometry providing other modes of analysis. In general, LBA has the advantage of high sensitivity and high throughput yet takes time to establish these methods and they experience difficulty in identifying metabolites and impurities [4].
Liquid chromatography-mass spectrometry based methods on the other hand can be developed in a short period of time and can identify molecules with different molecular weights or partial structures. These favourable traits support the use of LC-MS in both the screening environment in the drug discovery stage and metabolite analysis in the development stage.
To establish an LC-MS based analytical method for quality characterisation and kinetic evaluation of therapeutic oligonucleotides using a liquid chromatograph mass spectrometer, molecular weight confirmation is reported by multivalent ion mass spectrometry deconvolution of Mipomersen and its related compounds and quantitative analysis by MRM.
QTOF-LC-MS is a high-performance liquid chromatography (LC) tandem mass spectrometer (LC-MS/MS) combined with a quadrupole time-of- flight mass spectrometer (Q-TOF). Utilising high mass resolution, it is used mainly for quality characterisation and quality control in the analysis of nucleic acid drugs. The body of this work utilised a high-performance liquid chromatograph (Nexera) coupled with a Q-TOF system (LCMS- 9030) both by Shimadzu. In a typical reversed-phase separation of oligonucleotides, a mobile phase containing an ion-pair reagent is generally applied. Here, the ion pair reagents hexafluoro-2-propanol (HFIP) and N,N-diisopropylethylamine (DIPEA) were selected, which can measure phosphorothioate oligonucleotides with higher sensitivity, was used. The analytical conditions for both the HPLC and MS components are shown in Table 2.
Table 3: TQ-LC-MS analytical condition of Mipomersen analogues.
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