Studying in vitro metabolism of the first and second generation of antisense oligonucleotides with the use of ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

Abstract

The aim of the present investigation was the analysis and identification of antisense oligonucleotide metabolism products after incubation with human liver microsomes regarding four different oligonucleotide modifications. Separation and detection methods based on the use of liquid chromatography coupled with quadrupole time-of-flight mass spectrometry were developed for this purpose. Firstly, the optimization of mass spectrometer parameters was done to select those which ensure the highest possible sensitivity of oligonucleotide analysis. This step was conducted for two chromatographic modes—ion pair chromatography and hydrophilic interaction liquid chromatography—due to their common application in oligonucleotide analysis. Based on sensitivity results, ion pair chromatography coupled with mass spectrometry was selected for the separation of model oligonucleotide mixtures in order to verify its selectivity for N-deleted metabolite separation. Next, the developed method was applied in the examination of oligonucleotides in vitro metabolism. First, wide optimization of incubation parameters was conducted including the concentration of the reaction buffer components. Obtained results indicated that both 3′-exonucleases and 5′-exonucleases contributed to the biotransformation of oligonucleotides. Moreover, it may be concluded that the number of metabolites depends on oligonucleotide modification and consequently its resistance to enzymatic attack. Thus, the number of the oligonucleotide metabolites decreased with the decrease of the resultant polarity of oligonucleotide caused by chemical modification.

Development of the Method for Nusinersen and Its Metabolites Identification in the Serum Samples of Children Treated with Spinraza for Spinal Muscular Atrophy

“…Despite the well-known fact that phosphorothioate and 2′- O -methoxyethyl modifications decrease nuclease-mediated degradation, nusinersen was metabolized [ 15 , 18 , 19 , 29 , 30 ]. The 3′end shortmers and 5′end shortmers were generated and identified in serum.…”

“…All of these parameters were changed for similar chromatographic conditions: 5–80 % v / v MeOH in 15 min for 5 mM DMBA/150 mM HFIP; flow rate 0.3 mL/min; 1 μL injection; autosampler temperature 30 °C, column temperature 50 °C, column Kinetex 1.7 µm EVO C18 (100 × 2.1 mm) (Phenomenex, Torrance, CA, US). The other Q-TOF-MS parameters, such as drying gas flow (10 L/min), shielding gas flow (10 L/min), octopole voltage (800 V), drying gas temperature (350 °C), and shielding gas temperature (400 °C) were selected based on our previous experience with antisense OGNs studies using Q-TOF-MS [ 29 , 30 ].…”

Targeting SAM-I Riboswitch Using Antisense Oligonucleotide Technology for Inhibiting the Growth of Staphylococcus aureus and Listeria monocytogenes

“…These PS-modified ASOs can induce an RNase H-related cleavage of mRNA. The second generation represents oligonucleotides in which the structural modification is not limited to the backbone linkage but includes structural modifications of the ribose: ASO with 2′-O-alkyl modifications of the ribose were developed [ 11 ]. These modifications improve binding affinity, increase efficacy, decrease the protein binding of oligonucleotides, and enhance nuclease resistance.…”

“…Our research in this paper is focused on developing new efficient strategies for antibacterial drug discovery that may lead to a shorter development time and a higher success rate of drug candidate discovery. Our approach combines synthetic biology methods, bioinformatics [ 4 , 5 ], and rational drug design of antisense oligonucleotides (ASOs) [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. Herein, we employ ASO that binds explicitly with the complementary sequence of the S-adenosyl methionine (SAM-I) riboswitch located in the 5′-untranslated region (UTR) of mRNAs and inhibits the growth of pathogenic human bacteria, such as S. aureus and Listeria (L.) monocytogenes [ 15 ].…”

Assessing the Impact of Nonspecific Binding on Oligonucleotide Bioanalysis

“…Husser and co-workers have shown that LC-MS can be sensitive enough to determine the cleavage points and metabolites of oligonucleotides conjugated with GalNAc, a ligand that can grant greater potency through high affinity liver targeting but also causes changes in the biotransformation of oligonucleotides [9,10]. More recent publications by Kim and co-authors have described the development of an LC-MS method for the 2 -O-methyl modified phosphorothioate ASO eluforsen and its metabolites [8], and Kilanowska and co-authors have described the in vitro metabolism of various modified and different length ASOs with human liver microsomes using LC-MS [11]. Nevertheless, there remain challenges in using LC-MS for quantitation.…”