Identification of GalNAc-Conjugated Antisense Oligonucleotide

May 18, 2017 - Antisense oligonucleotides linked by phosphorothioates are an important class of therapeutics under investigation in various pharmaceut...
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Identification of GalNAc-Conjugated Antisense Oligonucleotide Metabolites Using an Untargeted and Generic Approach Based on High Resolution Mass Spectrometry Christophe Husser, Andreas Brink, Manfred Zell, Martina B. Müller, Erich Koller, and Simone Schadt* Roche Pharma Research and Early Development, Roche Innovation Center Basel, Grenzacherstr. 124, CH-4070 Basel, Switzerland ABSTRACT: Antisense oligonucleotides linked by phosphorothioates are an important class of therapeutics under investigation in various pharmaceutical companies. Antisense oligonucleotides may be coupled to high-affinity ligands (triantennary N-acetyl galactosamine = GalNAc) for hepatocyte-specific asialoglycoprotein receptors (ASGPR) to enhance uptake to hepatocytes and to increase potency. Since disposition and biotransformation of GalNAc-conjugated oligonucleotides is different from unconjugated oligonucleotides, appropriate analytical methods are required to identify main cleavage sites and degradation products of GalNAc conjugated and unconjugated oligonucleotides in target cells. A highly sensitive method was developed to identify metabolites of oligonucleotides using capillary flow liquid chromatography with column switching coupled to a high resolution Orbitrap Fusion mass spectrometer. Detection of GalNAc-conjugated oligonucleotides and their metabolites was achieved by combining full scan MS with two parallel MS2 experiments, one data-dependent scan and an untargeted MS2 experiment (all ion fragmentation) applying high collision energy. In the all ion fragmentation scan, a diagnostic fragment originating from the phosphorothioate backbone (O2PS-: m/z 94.936) was formed efficiently upon collisional activation. Based on this fragment an accurate determination of metabolites of oligonucleotides was achieved, independent of their sequence or conjugation in an untargeted but highly selective manner. The method was effectively applied to investigate uptake and metabolism of GalNAcconjugated oligonucleotides in incubations of primary rat hepatocytes; the elucidation of expected and unexpected degradation products was achieved in subnanomolar range.

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to be more complex than that of phosphodiester containing oligonucleotides.4 The biotransformation of phosphorothioate oligonucleotides depends on the sequence, stereochemistry and length, and is mediated mainly by 3′ exonucleases and, to a lesser extent, by 5′ exonucleases and endonucleases.4 The asialoglycoprotein receptor, which is located on hepatocytes, is used for liver targeting. To target this receptor, oligonucleotide drugs can be coupled via a linker to triantennary N-acetyl galactosamine (GalNAc).5−7 With this, new sites for potential biotransformations are introduced into the oligonucleotide drug. To date, there is only limited information on the biotransformation of GalNAc-conjugated oligonucleotides available in the literature. To our knowledge, only one comprehensive report has been published on the in vivo fate of the GalNAc-trishexylaminolinker part of the GalNAcconjugated oligonucleotide ION-681257 after subcutaneous dosing to rat and cynomolgus monkey using radio-labeled compound.8 Understanding the biotransformation and in vivo fate of new therapeutics is essential for drug development, in

n recent years, pharmaceutical companies’ interest in research on oligonucleotide therapeutics has increased significantly. Currently, various oligonucleotide therapeutics are evaluated in preclinical and clinical trials for the therapy of many diseases.1,2 Spinraza, used for the treatment of children and adults with spinal muscular atrophy, is the most recent antisense oligonucleotide therapeutic to be approved by the FDA.3 Like small molecule drugs, oligonucleotide therapeutics also undergo biotransformation, but in completely different pathways. While for small molecule drugs, biotransformation is mainly mediated by cytochrome P450 (CYP) and various phase II enzymes like glucuronosyl or sulfo transferases, the biotransformation of oligonucleotide drugs mainly consists of cleavage of the phosphodiester bonds between nucleic acids by nucleases. To improve the stability of oligonucleotide drugs toward nucleases, typically the phosphodiester bond in the backbone is modified to a phosphorothioate. Another common modification, the incorporation of locked nucleic acids (LNA) that contain bridged sugar rings, further enhances nuclease stability and increases RNA affinity.1 The biotransformation of phosphorothioate linked oligonucleotides has been studied in liver homogenate, and it was found © XXXX American Chemical Society

Received: April 4, 2017 Accepted: May 18, 2017 Published: May 18, 2017 A

DOI: 10.1021/acs.analchem.7b01244 Anal. Chem. XXXX, XXX, XXX−XXX

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indicate locked nucleic acids (LNA) containing a 2′-O-,4′-Cbridged ribose moiety. Chemicals, Reagents, and Materials. Guanidinium thiocyanate (GTC), hexafluoroisopropanol (HFIP), N,Ndiisopropylethylamine (DIPEA), and triethylamine (TEA) were purchased from Sigma-Aldrich (Buchs, Switzerland). Fetal bovine serum, penicillin, streptomycin, and Williams’ E medium (WME) were purchased from Gibco (Grand Island, NY, U.S.A.), and RLT buffer was purchased from Qiagen (Hilden, Germany) Water used for preparation of buffers and mobile phases for chromatography was Lichrosolv grade from Merck (Darmstadt, Germany). Acetonitrile (HPLC grade S) was obtained from Rathburn (Walkerburn, U.K.) and methanol (HPLC grade) was purchased from (Merck, Darmstadt, Germany). Hepatocyte Incubations. Hepatocytes were isolated from 10−14 weeks old male Wistar rats by a two-step collagenase liver perfusion method as previously described.18 Freshly isolated primary rat hepatocytes were suspended in WME supplemented with 10% fetal bovine serum, penicillin (100 U/ mL), and streptomycin (0.1 mg/mL) and seeded into collagencoated 24-well plates (Becton Dickinson AG, Allschwil, Switzerland) at a density of 2 × 105 cells/well. Cells were precultured for 2−3 h allowing for attachment to cell culture plates before start of treatment with phosphorothioate antisense oligonucleotide compounds. A total of 10 μM compound was added to cells in WME supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 mg/mL) (complete medium) and left on the cells for 3 h. Cells were washed with complete medium and further incubated for 3, 21, and 69 h, respectively. Cells were then lysed in RLT buffer. Sample Preparation. The cells lysed in RLT buffer were sonicated for 5 min, then aliquots of 150−300 μL were mixed with 100 μL of 4 M GTC in 0.1 M Tris (pH 7.5) and incubated at ambient temperature for 10 min with 1050 rpm in the Thermomixer (Eppendorf, Hamburg, Germany). Then, 600 μL of H2O/HFIP/DIPEA (100:2:0.2, v/v/v) were added and incubated at ambient temperature for 2 h with 1050 rpm in the Thermomixer. Then, the samples were subjected to solid phase extraction (SPE) on OASIS HLB 1 cm3 cartridges (Waters, Wexford, Ireland). Prior to use, the cartridges were primed with 1 mL of acetonitrile, followed by 1 mL of H2 O/HFIP/DIPEA (100:2:0.2, v/v/v). Then the samples were loaded, washed with 1 mL of H2O/HFIP/DIPEA (100:2:0.2, v/v/v), and eluted with 400 μL of H2O/acetonitrile/TEA (40:60:1, v/v/v). The eluate was evaporated in a vacuum centrifuge (RVC 2−33 IR Christ, Osterode am Harz, Germany) at 45 °C at 10 mbar for 1−2 h to near but incomplete dryness in order to avoid solubility issues and then redissolved in 150−200 μL of H2O/ MeOH/HFIP/DIPEA (90:10:1:0.1, v/v/v) by shaking for 20 min at 150 rpm in the Thermomixer. LC-MS Instrumentation. A Thermo Scientific Dionex UltiMate NCP-3200RS Binary Rapid Separation HPLC system was used in combination with a Pal autosampler (CTC Analytics AG, Zwingen, Switzerland) and a Thermo Scientific Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Scientific, Bremen, Germany) equipped with an electrospray ionization (ESI) source. The components of the UltiMate system were specifically designed for operating at pressures up to 800 bar. The UltiMate UPLC system was used in a column-switching mode (single pump trapping) as displayed in Figure 1. The trapping column (Xbridge BEH C4, 300 μm ID × 50 mm, 5 μm,

light of compound optimization risk assessment, understanding drug disposition and comparison between animal species used in toxicity testing and human. Another important aspect warranting a thorough understanding of the biotransformation of oligonucleotide therapeutics is bioanalytical quantification. The gold standard for bioanalytical quantification of oligonucleotides is hybridization ELISA and quantitative PCR (qPCR) in support of PK/PD studies due to excellent sensitivity.9,10 However, hybridization ELISA quantifies all molecular entities containing the oligonucleotide sequence, whether or not still conjugated to the intact triantennary GalNAc or degradation products thereof. So far, methods described in the literature for the investigation of the biotransformation of oligonucleotides are mainly based on high-performance liquid chromatography (HPLC) in combination with mass spectrometry (MS).11−17 These methods mostly consist of ion-pair reversed-phase (RP) HPLC coupled to quadrupole or ion trap MS. There is one study describing the investigation of the biotransformation of an siRNA duplex using Orbitrap MS.17 More recently, a method has been described for the study of the biotransformation of phosphorothioate containing oligonucleotides that is based on higher resolution mass spectrometry using a quadrupole timeof-flight (qTOF) MS.12 In this article, we describe a highly sensitive and selective LCMS method for investigating the biotransformation of phosphorothioate-containing oligonucleotide drugs that was inspired by small molecule analytical workflows for metabolite identification. The method is based on capillary flow liquid chromatography with column switching coupled to highresolution Orbitrap Fusion mass spectrometry.

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EXPERIMENTAL SECTION Reference Compounds. The phosphorothioate antisense oligonucleotides RO-A and RO-B, their corresponding GalNAcconjugates and potential metabolites (see Table 1) were Table 1. Phosphorothioate Antisense Oligonucleotides, Their Corresponding GalNAc-Conjugates, and Potential Metabolites name

sequence (5′−3′)

length

GalNAc-RO-A RO-A 3′ n-1 3′ n-2 3′ n-3 5′ n-1 5′ n-2 5′ n-3 GalNAc-RO-B RO-B 3′ n-5 3′ n-6 3′ n-7

GalNAc-GCattggtatTCA GCattggtatTCA GCattggtatTC GCattggtatT GCattggtat CattggtatTCA attggtatTCA ttggtatTCA GalNAc-(PO)-c(PO)a(PO)-GAGttacttgccaACT GAGttacttgccaACT GAGttacttgc GAGttacttg GAGttactt

13 13 12 11 10 12 11 10 16 16 11 10 9

provided by Roche Innovation Center Copenhagen, Denmark. The oligonucleotides were dissolved in phosphate buffered saline (PBS) or water to obtain a concentration of 3, 1, and 0.5 mM used as stock solution. Unless indicated otherwise by PO (phosphorodiester), the sequences are linked by phosphorothioates. Capital letters B

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tion and ionization efficiency are mainly complicated by the polyanionic backbone. A compromise between MS sensitivity and chromatographic retention needs to be found. The method of choice is ion pair RP-HPLC using ESI-MS in negative ion mode with compatible ion pairing reagents.9,19 Our optimized mobile phase that consisted of 98 mM HFIP and 5.8 mM DIPEA in H2O/methanol is comparable to methods that have been published previously.12,20,21 A major advance with respect to sensitivity could be achieved by employing a capillary flow rate of 10 μL/min and by introducing column switching HPLC to increase the amount of sample which could be injected. With this setup, a 50 μL aliquot of the sample following solid phase extraction could be loaded onto a trapping column and then eluted onto the analytical column, leading to a significant concentration step. Ultimately, this setup led to a significantly improved sensitivity of the method of 0.8 ng/mL, as compared to 1−1.3 μg/mL described previously for a full scan MS based method.12 Analytical Strategy for Identification of Oligonucleotide Biotransformation Products. Traditionally, the biotransformation of oligonucleotides has been investigated using mass spectrometers that generate nominal mass resolution data, while for small molecules, nowadays high resolution mass spectrometry is state-of-the-art. Only recently has a method been described that is based on higher resolution mass spectrometry using a quadrupole time-of-flight (qTOF) MS.12 The method we developed is inspired by the typical small molecule analytical workflows for metabolite identification and is based on high-resolution Orbitrap Fusion Mass Spectrometry. For small molecule biotransformation studies, a typical LC-MS approach includes full-scan high resolution MS data and product ion spectra, either from data-dependent MS/MS acquisition methods or pseudo MS/MS experiments where all ions are fragmented in an untargeted way simultaneously or a combination of both.22 In analogy, the MS method developed for oligonucleotide biotransformation investigation includes full-scan high resolution MS data, a high resolution all ion fragmentation scan, and data-dependent product ion spectra with detection in the ion trap at nominal resolution. The application of this novel MS method is described here for two GalNAc-conjugated oligonucleotides, RO-A and RO-B, which have been incubated with rat hepatocytes to generate metabolites. Detection of Biotransformation Products in All Ion Fragmentation (AIF) Mode. In negative ionization mode, phosphorothioate containing oligonucleotides efficiently undergo collision-induced dissociation on the phosphorothioate backbone, resulting in characteristic fragment ions at m/z 94.9362 and 192.97299 Da (Figure 2B). In particular, the fragment ion at m/z 94.9362 was formed efficiently and could be used as a sensitive diagnostic fragment for postacquisition data analysis (Figure 2B). At the retention times where a signal at 94.9362 is observed in the AIF scan, the corresponding full scan data provides the precursor m/z envelope of the analyte of interest at this retention time (Figure 2A). Such an untargeted postacquisition approach based on a characteristic and unique collision-induced fragment ion generic for all phosphorothioate-containing oligonucleotides facilitates the identification of biotransformation products in the absence of radiolabeled compound. It further reduces the risk of missing metabolites and, due to the presence of multiple negatively

Figure 1. Schematic configuration of the LC-MS UltiMate columnswitching system.

300 A) was connected via a stainless-steel tee device with the analytical column (Xbridge BEH C18, 300 μm ID × 50 mm, 5 μm, 130 A) from Waters (Baden, Switzerland). The system included an UltiMate NCP-3200RS Binary Rapid Separation Nano Flow Pump preconfigured for capillary flow (5−50 μL/ min). The column-switching between the trapping column (TC) and analytical column (AC) was performed using an external six-port Rheodyne MX Series II divert/inject valve at ambient temperature. The column-switching valve was controlled by the XCalibur software as a timed event. Chromatographic Separation of Oligonucleotide Metabolites. A 50 μL aliquot of the sample following solid phase extraction was loaded onto the trapping column. The loading eluent was a mixture of 2% mobile phase B and 98% mobile phase A, delivered at a flow rate of 20 μL/min for 8.0 min and at a flow rate of 10 μL/min for further 2 min, with mobile phase A consisting of H2O/MeOH/HFIP/DIPEA, 90/ 10/1/0.1 and mobile phase B of H2O/MeOH/HFIP/DIPEA, 10/90/1/0.1, corresponding to 98 mM HFIP and 5.8 mM DIPEA. After loading, the trapping column was switched in line with the analytical column and the retained analytes were transferred to the analytical column by gradient elution at a flow rate of 10 μL/min. At the start of the linear gradient, eluent B was kept at 2% for 4 min and then raised to 35% within 20.5 min. Thereafter, eluent B was increased to 70% and maintained for 2.4 min and then decreased again to 2% for re-equilibration of the column. The trapping column and the analytical column were kept at 60 °C during the whole analysis sequence. Prior to the next injection, the trapping was decoupled from the analytical column. The overall run time of the analysis cycle was 42.5 min. Mass Spectrometric Detection of Oligonucleotide Metabolites. The oligonucleotide metabolites were analyzed in negative ionization mode. The following ion source parameters were used: sheath gas, 10; auxiliary gas, 0; sweep gas, 0; ion transfer tube temperature, 300 °C; spray voltage negative ion, 2200 V. The MS method consisted of three experiments. For the fullscan, the resolution for the Orbitrap was set on 120000 and the scan range was 550−1700. Based on this fullscan, a data-dependent HCD fragmentation experiment at a collision energy of 35% with detection in the ion trap at nominal mass resolution and a scan range of 150−2000 was triggered on the most intense multiply charged ions in top speed mode. The third experiment was an all ion fragmentation in HCD mode (with a collision energy of 35%), the Orbitrap resolution was set to 30000, the isolation range to 600−1800, and the scan range to 80−1200.

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RESULTS AND DISCUSSION Optimization of Chromatography. When developing LC-MS methods for oligonucleotides, chromatographic retenC

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length and the corresponding GalNAc-conjugates was in a similar range (Table 2), with a slightly lower MS response for Table 2. Relative Peak Areas by Different Integration Modes of 5 nM Reference Compounds in PBS for the RO-B Series and the RO-A Series, Referenced to RO-A or RO-B, Respectively

GalNAc-RO-A RO-A 3′ n-1 3′ n-2 3′ n-3 5′ n-1 5′ n-2 5′ n-3 GalNAc-RO-B RO-B 3′ n-5 3′ n-6 3′ n-7

Figure 2. Extracted ion chromatogram (XIC m/z 94.9362) of the all ion fragmentation (AIF) experiment of a rat hepatocyte incubation of GalNAc-RO-B, together with the corresponding full scan XIC data of GalNAc-RO-B and its metabolites eluting between 31 and 32 min (A). RO-B spectrum of the AIF experiment at 29 min with assignment of key fragments in the lower mass range (B).

monoisotopic

most intense ion

sum of charge states

deconvoluted

0.3 1.0 1.3 1.0 0.9 1.3 1.7 3.2 0.2 1.0 2.8 3.3 2.3

0.8 1.0 1.2 0.8 0.6 1.2 1.2 1.4 0.5 1.0 1.3 1.3 0.7

0.8 1.0 1.2 0.8 0.5 1.1 1.0 0.7 0.4 1.0 1.2 1.1 0.9

0.8 1.0 1.1 0.8 0.5 1.1 1.0 1.3 0.6 1.0 1.3 1.4 1.3

the GalNAc conjugates. The data shown here indicate that a good estimation of the relative amounts of metabolites formed is possible based on peak integration. Quantitative estimations in the absence of reference compounds are especially important for earlier phases of drug discovery, when reference compounds are typically not yet available. The mass spectra (Figure 3B) contained a series of peaks corresponding to the different charge states of the oligonucleotides. The mass spectra were then deconvoluted using the Xtract Software in Xcalibur in the protein mode which is based on an average theoretical amino acid called averagine23 to generate the isotope table (Figure 3C). The mass accuracy was typically within 1 ppm, allowing the unambiguous assignment of structural changes in the molecules due to biotransformation. Interestingly, a good quantitative estimation was possible by either integrating the most intense ions, or summation of the most intense ions for different charge states, or the deconvoluted MS signal, but not by integrating the monoisotopic peak of the most abundant charge state (Table 2).The sensitivity was approximately 0.8 ng/mL for phosphorothioate oligonucleotides. Opportunities and Limitations of Product Ion Data for the Structure Elucidation of Oligonucleotide Metabolites. The use of product ion data of oligonucleotides for structure elucidation has been discussed controversly in the literature.12,24 In contrast to peptides, which typically fragment at the amide bond between amino acids, thus, yielding product ion data with series of peptide fragments that differ by one amino acid, from which the sequence can be deduced,25 oligonucleotides do not easily fragment into series that allow the reconstruction of the original sequence.12 However, the generation of fragment series and their use for the structural characterization of oligonucleotides has been demonstrated.24 Also, we found that product ion data can be helpful for qualitative analysis and support the structure elucidation of metabolites, as illustrated in Figure 4. Even though we did not observe fragment ion series resulting from backbone cleavage that allowed the reconstruction of the sequence, we found that phosphorothioate nucleic acids were

charged phosphorothioate groups in the oligonucleotide molecules, yields excellent sensitivity. For ubiquitous endogenous oligonucleotides, we observed mainly PO3− (78.95 Da) and, to a much lesser extent, PO4H2− (96.97 Da) as fragments; however, we cannot exclude the formation of PO4− (m/z 94.95) under other experimental conditions. To enable the distinction between PO2S− (m/z 94.9362) and PO4− (m/z 94.9540), a minimum resolving power of 5500 would be required, which can be easily achieved using FTICR, Orbitrap, or TOF mass analyzers. Interpretation of Full Scan Data. Examples of full scan chromatograms, together with the corresponding mass spectra are displayed in Figure 3. The ionization efficiency within a series of phosphorothioate oligonucleotides of different chain

Figure 3. Base Peak Chromatogram (BPC), deconvoluted BPC, and AIF XIC (m/z 94.9362) of 5 nM GalNAc-RO-B, RO-B, and 3 RO-B shortmers (A), mass spectra (B), and deconvoluted mass spectra of GalNAc-RO-B (C). D

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were not taken up by the hepatocytes, and subsequent further incubation for 21 h led to the formation of mainly four metabolites, as can be seen in Figures 2 and 6. The main

Figure 4. Product ion spectra in the lower mass range (310−380) of RO-A (A) and its metabolite where the 2′-O,4′-C-adenosinethiophosphate at the 3′ end has been cleaved off (B).

formed as fragment ions. The fragment ions that are labeled in green in Figure 4 are common fragments between RO-A and its metabolite. The fragment in red, the 3′ end A, is not present in the product ion spectrum of the metabolite. Since, by design, in modern gapmer oligonucleotide therapeutics, the terminal nucleotides differ structurally from the nucleotides within the sequence, they consequently have a different mass and can therefore be distinguished. Biotransformation of GalNAc-RO-A in Rat Hepatocytes. GalNAc-RO-A was incubated with rat hepatocytes for 3 h to allow for uptake into the liver cells, then the cells were washed to remove oligonucleotides that were not taken up, and subsequently further incubated for 3, 21, and 69 h. Several metabolites were formed, mainly by cleavage of 1−3 GalNAc moieties and part of the linker. RO-A was not detected, probably due to the increased stability of the phosphorothioate bond between the aminohexanol and the LNA. Interestingly, cleavage of the locked nucleic acid A at the 3′ end of the sequence mediated most likely by 3′ exonucleases was detected in significant amounts. Formation of these metabolites occurs within 3 h; further incubation up to 21 or 69 h leads only to a subtle change in the metabolic pattern insofar as the degradation of the GalNAc moiety progresses (Figure 5). Biotransformation of GalNAc-RO-B in Rat Hepatocytes. Incubation of GalNAc-RO-B with rat hepatocytes for 3 h, followed by washing of the cells to remove oligonucleotides that

Figure 6. Biotransformation of GalNAc-RO-B after incubation with rat hepatocytes for 3 h, followed by a washing step and subsequent incubation for 21 h.

biotransformation product is RO-B indicating that the triantennary GalNAc is cleaved off at the cleavable phosphodiester bond. Further biotransformation products result from the cleavage of 1, 2, or 3 GalNAc sugars (Figure 6). The oligonucleotide sequence in itself is relatively stable, and only traces of degradation of the phosphorothioate chain were detected. This finding supports the fact that both the phosphorothioate and the LNAs are introduced into the molecule to render it more stable toward nuclease cleavage.1 The triantennary GalNAc structure, in contrast, is introduced for targeting of oligonucleotides to the liver, and after uptake into the liver has served its purpose and can be cleaved off. The data shown here also support a strategy where the chemical synthesis of shortmer metabolites is only triggered after initial assessment of the biotransformation. In both examples presented here, shortmer metabolites are only formed in trace amounts, therefore, an estimation based on peak area integration is sufficient to reach this conclusion, and bioanalytical quantification using reference compounds is not necessary. Complementary Methods for the Study of GalNAc Conjugated Oligonucleotides. The method described in this article is complementary to the work described by Shemesh et al.8 Their study on the in vivo fate of the GalNAc-conjugated oligonucleotide ION-681257 after subcutaneous dosing to rat and cynomolgus monkey focused on understanding the biotransformation of the GalNAc-linker part that carried the radio-label. A total of 14 linker associated metabolites were identified, including mono-, di-, tri-, and tetraoxidation on the different branching arms, and three very interesting cyclic metabolites formed by internal esterification. For the study of these metabolites, mobile phases on the basis of ammonium bicarbonate and acetonitrile were used, and the mass spectrometer was operated in positive ion mode. In contrast, the method presented here was optimized for the analysis of the oligonucleotide part of the molecule, with mobile phases containing ion pairing reagents, the mass spectrometer operated in negative ion mode, and a diagnostic fragment ion derived from the phosphorothioate backbone used instead of a radiolabel.

Figure 5. Biotransformation of GalNAc-RO-A after incubation with rat hepatocytes (A) and MS peak area percentages (based on integration of the most intense ion) of the identified metabolites after 3 h incubation, followed by a washing step and subsequent incubation for 3, 21, and 69 h (B). E

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ACKNOWLEDGMENTS The authors thank Nathalie Schaub and Eveline Durr for providing rat hepatocytes, Franz Schuler and Christoph Funk for helpful discussions and critical reading of the manuscript, Roche Innovation Center Copenhagen for synthesizing antisense oligonucleotide compounds, and Jon Kyle Bodnar for thorough review and language editing of the manuscript.

Therefore, complementary information can be obtained by both methods. Consistently, both for RO-A and RO-B as well as for ION-681257, cleavage of 1, 2, or 3 GalNAc sugars was observed. In the case of RO-B and ION-681257, the GalNAclinker was cleaved from the oligonucleotide; in the case of ROA, no such cleavage was observed, most likely due to the increased stability of the phosphorothioate bond between the aminohexanol and the LNA.

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CONCLUSIONS This article describes a novel LC-MS/MS method for the investigation of the biotransformation of phosphorothioatecontaining oligonucleotide drugs. While the biotransformation of oligonucleotides has been investigated traditionally using nominal resolution mass spectrometry data, for small molecules biotransformation high resolution mass spectrometry is state-ofthe-art. The methodology presented here is inspired by small molecule analytical workflows for metabolite identification and includes full scan high resolution MS data and product ion spectra, both from data-dependent MS/MS acquisition and from pseudo MS/MS experiments. In addition, a major advance with respect to sensitivity could be achieved by low flow rates and by introducing column switching HPLC. With this setup, a larger aliquot of the sample following solid phase extraction could be loaded onto a trapping column and then eluted onto the analytical column, leading to a significant concentration step that enabled the sensitive detection of biotransformation products. Understanding the biotransformation of new therapeutics is an essential part of drug development, also in light of characterization of the bioanalytical quantification method. Hybridization ELISA and qPCR quantify the sum of all molecular entities containing the oligonucleotide sequence and, in some cases, also if one or two nucleotides are cleaved off. In many cases, these moieties quantified by the hybridization ELISA will correspond to all pharmacologically active compounds. The two examples presented here, however, demonstrate that the most abundant drug-related compounds might not be the GalNAc-conjugated oligonucleotide or the oligonucleotide itself but metabolites thereof. Correlating the pharmacokinetic profile of a drug with the pharmacological response requires understanding and characterization of what exactly is quantified by the methods applied.

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Article

REFERENCES

(1) Juliano, R. L. Nucleic Acids Res. 2016, 44 (14), 6518−48. (2) Lundin, K. E.; Gissberg, O.; Smith, C. I. Hum. Gene Ther. 2015, 26 (8), 475−85. (3) https://www.fda.gov/NewsEvents/Newsroom/ PressAnnouncements/ucm534611.htm, accessed on 31st March, 2017. (4) Crooke, R. M.; Graham, M. J.; Martin, M. J.; Lemonidis, K. M.; Wyrzykiewiecz, T.; Cummins, L. L. J. Pharmacol. Exp. Ther. 2000, 292 (1), 140−9. (5) Kanasty, R.; Dorkin, J. R.; Vegas, A.; Anderson, D. Nat. Mater. 2013, 12 (11), 967−77. (6) Ming, X.; Laing, B. Adv. Drug Delivery Rev. 2015, 87, 81−9. (7) Prakash, T. P.; Graham, M. J.; Yu, J.; Carty, R.; Low, A.; Chappell, A.; Schmidt, K.; Zhao, C.; Aghajan, M.; Murray, H. F.; Riney, S.; Booten, S. L.; Murray, S. F.; Gaus, H.; Crosby, J.; Lima, W. F.; Guo, S.; Monia, B. P.; Swayze, E. E.; Seth, P. P. Nucleic Acids Res. 2014, 42 (13), 8796−807. (8) Shemesh, C. S.; Yu, R. Z.; Gaus, H. J.; Greenlee, S.; Post, N.; Schmidt, K.; Migawa, M. T.; Seth, P. P.; Zanardi, T. A.; Prakash, T. P.; Swayze, E. E.; Henry, S. P.; Wang, Y. Mol. Ther.–Nucleic Acids 2016, 5, e319. (9) van Dongen, W. D.; Niessen, W. M. Bioanalysis 2011, 3 (5), 541− 64. (10) Yu, R. Z.; Baker, B.; Chappell, A.; Geary, R. S.; Cheung, E.; Levin, A. A. Anal. Biochem. 2002, 304 (1), 19−25. (11) Lin, Z. J.; Li, W.; Dai, G. J. Pharm. Biomed. Anal. 2007, 44 (2), 330−41. (12) Studzinska, S.; Rola, R.; Buszewski, B. Anal. Bioanal. Chem. 2016, 408 (6), 1585−95. (13) Ewles, M.; Goodwin, L.; Schneider, A.; Rothhammer-Hampl, T. Bioanalysis 2014, 6 (4), 447−64. (14) Dai, G.; Chan, K. K.; Liu, S.; Hoyt, D.; Whitman, S.; Klisovic, M.; Shen, T.; Caligiuri, M. A.; Byrd, J.; Grever, M.; Marcucci, G. Clin. Cancer Res. 2005, 11 (8), 2998−3008. (15) Gaus, H. J.; Owens, S. R.; Winniman, M.; Cooper, S.; Cummins, L. L. Anal. Chem. 1997, 69 (3), 313−9. (16) Wei, X.; Dai, G.; Liu, Z.; Cheng, H.; Xie, Z.; Marcucci, G.; Chan, K. K. AAPS J. 2006, 8 (4), E743−55. (17) Zou, Y.; Tiller, P.; Chen, I. W.; Beverly, M.; Hochman, J. Rapid Commun. Mass Spectrom. 2008, 22 (12), 1871−81. (18) Roth, A.; Boess, F.; Landes, C.; Steiner, G.; Freichel, C.; Plancher, J. M.; Raab, S.; de Vera Mudry, C.; Weiser, T.; Suter, L. J. Biochem. Mol. Toxicol. 2011, 25 (3), 183−94. (19) Apffel, A.; Chakel, J. A.; Fischer, S.; Lichtenwalter, K.; Hancock, W. S. Anal. Chem. 1997, 69 (7), 1320−5. (20) Chen, B.; Bartlett, M. G. J. Chromatogr A 2013, 1288, 73−81. (21) Gong, L.; McCullagh, J. S. Rapid Commun. Mass Spectrom. 2014, 28 (4), 339−50. (22) Zhu, M.; Zhang, H.; Humphreys, W. G. J. Biol. Chem. 2011, 286 (29), 25419−25. (23) Senko, M. W.; Beu, S. C.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1995, 6 (4), 229−33. (24) Huang, T. Y.; Kharlamova, A.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2010, 21 (1), 144−53. (25) Steen, H.; Mann, M. Nat. Rev. Mol. Cell Biol. 2004, 5 (9), 699− 711.

AUTHOR INFORMATION

Corresponding Author

*Phone: +41 61 687 24 07. E-mail: [email protected]. ORCID

Simone Schadt: 0000-0001-5932-9423 Author Contributions

C.H. and A.B. contributed equally to the work. Study was planned by C.H., A.B., M.Z., E.K., and S.S. Experiments were conducted by C.H. and M.B.M. Data was analyzed and interpreted by C.H., A.B., M.Z., and S.S. The manuscript was written by S.S. All authors have given approval to the final version of the manuscript. Notes

The authors declare the following competing financial interest(s): All co-authors are, or have been, employees of F. Hoffmann-La Roche Ltd. F

DOI: 10.1021/acs.analchem.7b01244 Anal. Chem. XXXX, XXX, XXX−XXX