Analytical characterization of DNA and RNA oligonucleotides by hydrophilic interaction liquid chromatography-tandem mass spectrometry

Liquid chromatography-mass spectrometry has been widely implemented as a powerful tool for providing in-depth characterization of nucleic acid therapeutic modalities, such as anti-sense oligonucleotides and small interfering RNAs (siRNAs).

Journal of Chromatography A 1648 (2021) 462184 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Analytical characterization of DNA and RNA oligonucleotides by hydrophilic interaction liquid chromatography-tandem mass spectrometry Ming Huang, Xiaobin Xu∗, Haibo Qiu∗, Ning Li Regeneron Pharmaceuticals Inc., Tarrytown, NY 10591, USA a r t i c l e i n f o Article history: Received January 2021 Revised 28 March 2021 Accepted 18 April 2021 Available online 27 April 2021 Keywords: Oligonucleotides siRNAs Hydrophilic interaction liquid chromatography Tandem mass spectrometry Phosphorothioate Synthetic metabolites a b s t r a c t Liquid chromatography-mass spectrometry has been widely implemented as a powerful tool for providing in-depth characterization of nucleic acid therapeutic modalities, such as anti-sense oligonucleotides and small interfering RNAs (siRNAs) In this study, we developed a generic hydrophilic interaction liquid chromatography (HILIC) hyphenated with tandem mass spectrometry method in the absence of ionpairing reagents and demonstrated its capability as an attractive and robust alternative for oligonucleotide and siRNA analysis HILIC separation of mixtures of unmodified and fully phosphorothioatemodified DNA oligonucleotides and their synthetic 3’ exonuclease-digested metabolites were also assessed High-resolution mass spectrometric (HRMS) analysis was used to determine the deconvoluted masses of oligonucleotide and siRNA standards and their impurities To enable unbiased sequence characterization with tandem mass spectrometry (MS/MS), we also optimized higher-energy C-trap dissociation (HCD) on improving the sequence coverage of DNA and RNA oligonucleotides Lastly, we evaluated on-column sensitivity for a phosphorothioate oligonucleotide by performing targeted analysis with either targeted selected ion monitoring (tSIM) or parallel reaction monitoring (PRM) Higher on-column sensitivity of 13 ng, equivalent to 2.0 pmol, of a phosphorothioate oligonucleotide was achieved by tSIM analysis as compared to PRM analysis © 2021 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction The continued optimism for synthetic nucleic acid drugs has fueled an increasing interest in antisense oligonucleotide therapy and RNA interference (RNAi) therapy Due to their mechanisms of gene silencing and modulation of gene expression, oligonucleotides and small interfering RNAs (siRNAs) represent a distinct class of therapeutic molecules Recently, an increasing number of these drug candidates have been approved or have progressed to later-stage clinical trials [1,2] However, there are different regulatory strategies for the approval pathway of nucleic acid therapeutics These drug candidates are categorized by the Food and Drug Administration (FDA) as small molecule drug candidates while they are categorized as biologics by European Medicines Agency (EMA) [3] Both oligonucleotides and siRNAs are large, highly negatively charged ∗ Corresponding authors E-mail addresses: xiaobin.xu@regeneron.com (X Xu), haibo.qiu@regeneron.com (H Qiu) molecules and show fundamentally different chemical and physiological properties relative to small molecule and antibody drugs Driven by drug development needs and regulatory requirements, numerous analytical strategies have been explored to provide indepth characterization of various drug facets, such as synthetic impurities, chemical modifications, and metabolites In 1990 Andrew Alpert coined the term “hydrophilic interaction chromatography” (HILIC) to describe a liquid chromatographic technique for separating polar or ionized analytes The HILIC retention mechanisms involved the partition of solutes between the bulk mobile phase and an enriched water layer partially immobilized on the stationary phase surface, as well as secondary electrostatic and hydrogen bonding interactions [4] Since then, HILIC coupled to mass spectrometry (HILIC−MS) has been broadly adopted in the characterization of different classes of biomolecules, including proteins, glycopeptides, and small molecules, such as glycans and saccharides [5,6] Because polar ribosyl residues and ionizable phosphate groups in nucleic acid structures are hydrophilic, early applications of HILIC include bioanalysis of nucleosides and https://doi.org/10.1016/j.chroma.2021.462184 0021-9673/© 2021 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) M Huang, X Xu, H Qiu et al Journal of Chromatography A 1648 (2021) 462184 LC−MS grade) were purchased from Sigma-Aldrich (St Louis, MO, USA) HPLC-grade acetonitrile was purchased from Honeywell (Charlotte, NC, USA) Deionized water was provided by a Milli-Q integral water purification system installed with a MilliPak Express 20 filter (Milli-Q system, Millipore, El Passo, TX, USA) All DNA and RNA oligonucleotides used in this study were purchased from Integrated DNA Technologies Inc (IDT; Coralville, IA, USA), and their sequences are listed in Supplementary Table All synthetic oligonucleotides contained - and -terminal hydroxyl groups The luciferase-targeted siRNA (siLuc) and its corresponding sense and anti-sense strands were purchased from IDT (Supplementary Table 1) MISSION® siRNA Universal Negative Control #1 (SIC001, 13317 g/mol) and MISSION® siRNA Universal Negative Control #2 (SIC002, 13302 g/mol) were purchased from MilliporeSigma (Burlington, MA, USA), and their sequences are proprietary All samples were dissolved in sterile nuclease-free OmniPur® Water (MilliporeSigma, Burlington, MA, USA) and stored as 200 μM stock solutions at –20°C Aliquots were prepared to avoid repeated freeze-thaw cycles HILIC mobile phases were prepared by pre-mixing 500 mM aqueous ammonium acetate (AA) or ammonium formate (AF), water and acetonitrile (ACN) to yield appropriate salt concentrations Mobile phase A (MPA) was composed of 70% ACN buffered with AA or AF, and mobile phase B (MPB) was composed of 30% ACN buffered with AA or AF For HILIC analysis, sample diluent with identical buffer composition with MPA (weak solvent) was used for all analyzed oligonucleotides, and the on-column injection amount was 20 pmol (2 μL) if not otherwise specified The (n – x) oligonucleotide mixture was prepared by reconstitution of lyophilized equimolar mixtures of each oligonucleotide with sample diluent at a nominal concentration of 10 μM nucleotides in different types of sample matrices [7–9] Recently, HILIC has gained more attention as a promising alternative to the widely used ion-pairing reversed-phase (IP-RP) LC methods for oligonucleotide analysis in an effort to mitigate ion-pairing (IP) reagent disadvantages, including reduced MS signal intensity caused by ionization suppression and potential contamination introduced to multipurpose instruments [10] In addition, HILIC exhibits high MS compatibility because it uses volatile mobile phase additives (e.g ammonium acetate), whereas another commonly used chromatographic method, anion-exchange chromatography (AEX), requires high concentrations of non-volatile salts which impair the coupling to MS detection [10] Easter et al first demonstrated the applicability of HILIC to oligonucleotide analysis [11,12] HILIC in the presence of ionpairing reagents (IP-HILIC) was also pursued to render selective and orthogonal analysis of oligonucleotides [11,13] Although these studies provided proof-of-concept evidence that HILIC can be utilized for the separation of oligonucleotides, these methods still suffer from poor column stability, long equilibration time between runs, and limited resolution for oligonucleotides of similar chain lengths Till recently, oligonucleotide LC−MS analyses facilitated by HILIC in the absence of ion-pairing reagents has presented promise for characterizing oligonucleotide therapeutics [14–17] From the development of different HILIC stationary phases, increased applications of HILIC in oligonucleotide characterization emerged To date, different types of column chemistries have been successfully applied to oligonucleotide analysis to promote selectivity, including weak ion-exchange and neutral (silica-, diol-, amide- and cyanofunctionalized) stationary phases Easter et al first presented the use of a diol-bonded HILIC column that couples crossed-linked diol functional groups with the neutral, silica stationary phase [11] Loube et al also explored HILIC separation of oligonucleotides using a diol-derivatized polymer-based column and showed moderate separation efficiency for separating (n – x) shortmers of unmodified or phosphorothioate (PS) oligonucleotides from the fulllength products (FLPs) [14] Demelenne et al’s more recent study further demonstrated the excellent performance of a BEH amide column functionalized with nonionic carbamoyl groups in rendering high peak capacities for unmodified and PS oligonucleotides [16] Besides, with good orthogonality, HILIC has been coupled to IP-RPLC or AEX in a two-dimensional (2D)-LC fashion to comprehensively characterize synthetic impurities of therapeutic oligonucleotides [10] In this study, we developed a generic HILIC hyphenated with high-resolution mass spectrometric (HILIC−HRMS) method to provide rapid, robust, and in-depth analytical characterization of DNA/RNA oligonucleotides This approach could be further extended to the characterization of duplexed siRNAs Furthermore, HILIC separations of structurally similar synthetic metabolites of unmodified and PS-modified DNA oligonucleotides were assessed, and the coupling of HILIC to HRMS in profiling impurities and degradants was explored To facilitate unbiased sequence characterization, we also optimized higher-energy C-trap dissociation (HCD) fragmentation conditions in sequencing DNA and RNA oligonucleotides by MS/MS The study presented herein demonstrated the analytical robustness of HILIC as an alternative chromatographic approach for in-depth characterization of oligonucleotides and siRNAs, when completely liberated from any IP reagents 2.2 LC–MS/MS analysis The LC–MS/MS platform was composed of a Waters Acquity I-class ultra-performance liquid chromatography (UPLC) system (Waters, Milford, MA, USA) interfaced to a Q Exactive Hybrid quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) by a heated electrospray ionization (H-ESI) source Samples were analyzed on a BEH Amide UHPLC column (Waters, Milford, MA, USA; 2.1 mm × 150 mm, 1.7 μm particle size, 130 A˚ pore size) For regular analyses, DNA and RNA oligonucleotides were eluted with a linear gradient that was increased from 20% to 70% MPB over 10 at a flow rate of 0.25 mL/min with column temperature set as 30°C siRNAs were eluted with a 10-min linear gradient that was increased from 60% to 70% MPB For oligonucleotide impurity characterization, analytes were eluted with a 15-min linear gradient that was increased from 20% to 65% MPB The gradient was then ramped to 80% MPB over and held for min, before dropping to 20% MPA and then getting re-equilibrated at 20% MPA for 10 before the next run The eluents were monitored at a wavelength of 260 nm using either a photodiode array (PDA) or tunable ultraviolet (TUV) detector and then electro-sprayed into MS The following parameters were used for MS analysis: negative polarity (static spray, 3.0 kV), ion funnel radiofrequency (RF) level at 60%, sheath gas (40 a.u.), auxiliary gas (15 a.u.), sweep gas (0 a.u.), ion transfer tube temperature of 325 °C, vaporizer temperature of 350 °C, mass range 40 0–20 0 m/z, mass resolution 70,0 0 full-width half maximum (FWHM), automatic gain control (AGC) 5e5, injection time (IT) 100 ms, intensity threshold 1e3, charge state selection 1–4, and each full scan spectrum consisted of accumulation of microscan Data dependent MS/MS (top 7) were acquired by employing higher-energy C-trap dissociation (HCD) with a 2.0 m/z quadrupole isolation window, AGC of 1e5, IT 200 ms, and each MS/MS spectrum consisted of accumulation of microscan For Experimental 2.1 Materials and reagents For mobile phase preparation, glacial acetic acid, ammonium hydroxide (25% for LC−MS LiChropurTM ), ammonium formate (LiChropurTM LC−MS grade) and ammonium acetate (LiChropurTM M Huang, X Xu, H Qiu et al Journal of Chromatography A 1648 (2021) 462184 HCD fragmentation, normalized collision energy (NCE) was set at 15% and first mass m/z was set as 70 at a mass resolution of 30,0 0 FWHM For on-column sensitivity assessment, targeted selected ion monitoring (tSIM) and parallel reaction monitoring (PRM) acquisition were performed at a mass resolution of 70,0 0 FWHM and a mass isolation window of 2.0 m/z, AGC of 1e6, and IT of 100 ms In PRM mode, the data were acquired according to a predetermined inclusion list containing the accurate mass and normalized collisional energies (NCE) of analytes Additionally, other MS parameters were set as the AGC of 2e5, IT of 100 ms and isolation window of 2.0 m/z 2.3 Data processing High-resolution mass spectra were deconvoluted by the PMIIntact MassTM software (Protein Metrics, San Carlos, CA, USA) to obtain both deconvoluted monoisotopic mass and average mass Automated MS/MS fragment annotation was achieved by using BioPharma FinderTM 4.0 (Thermo Fisher Scientific, Waltham, MA, USA) as described in the text Other DDA or tSIM raw files were analyzed by Xcalibur Qual Browser software version 2.2 (Thermo Fisher Scientific, Waltham, MA, USA) To generate response curves for sensitivity evaluation, extracted ion chromatograms (EICs) were generated for each standard injection from the most abundant charge state with an m/z extraction window of 10 ppm The peak areas of the EICs were plotted versus concentration to generate linear regression results (slope and R2 ) The limit of detection (LOD) of each method was determined based on a target signal-to-noise ratio (S/N) above 3, as calculated in the Qual Browser software For impurity profiling, the 5’ and 3’ shortmer sequences were calculated by Mongo Oligo Mass Calculator v2.08 (University at Albany, SUNY) PRM data was analyzed by Skyline version 4.2.0 (University of Washington) Results and discussion 3.1 BEH amide column evaluation in oligonucleotide analysis Fig HILIC retention of siRNAs, RNA oligonucleotides, DNA oligonucleotides and phosphorothioate DNA oligonucleotides (grouped by color shaded areas) on a BEH amide column in mobile phases with (A) 25 mM ammonium formate or ammonium acetate; (B) ammonium acetate (pH 6.8) of various concentrations (2.5 mM, 7.5 mM, 15 mM, and 25 mM); (C) 15 mM of ammonium acetate of different pH (pH 5.5, pH 6.8, pH 9.0) Many efforts have been made to understand the HILIC retention mechanisms of oligonucleotides by exploring the use of different HILIC stationary phases Among them, the superior separation efficiency obtained using BEH amide column independently reported by several research groups encouraged us to further evaluate the performance of this column for oligonucleotide analysis [10,15,16] Mechanistically, the polar amide functional groups may be more effective at interacting with the aqueous portion of the mobile phase and forming the stagnant water layer required for HILIC The carbamoyl groups within the amide phase can participate in hydrogen-bonding as hydrogen-bond donors and further interact with hydroxyl groups in the analytes [18] Additionally, with smaller diameters of packing materials, improved chromatographic performance was achieved due to more effective mass transfer (Van Deemter equation) As a result, we selected the BEH amide column to develop a generic HILIC method suitable for routine oligonucleotide analysis To validate the method robustness, we analyzed both single-stranded DNA and RNA oligonucleotides as well as double-stranded siRNAs in five replicates with a 10-min gradient and a column re-equilibration time of 10 between each injection High repeatability of retention times and peak areas were obtained with RNA oligonucleotide > DNA oligonucleotide > PS-modified DNA oligonucleotide Besides, the elution order of DNA oligonucleotides was dependent on their chain length, which could be attributed to the net negative charges carried This is consistent with previous studies that used poly-deoxy(thymidylic) acids (dT) ladders for benchmarking LC separations [14–16] Meanwhile, DNA oligonucleotides carrying backbone PS modifications showed diminished HILIC retention due to the higher hydrophobicity comparing to phosphate backbone, with the displacement of oxygen with the M Huang, X Xu, H Qiu et al Journal of Chromatography A 1648 (2021) 462184 further increasing the charge, in proportion to the T(U) + G content [21] Interestingly, we also observed slightly more noticeable peak tailing at pH 5.5 while a more symmetrical peak profile at pH 9.0, which was also reported by Goyon et al [10] Alterations of mobile phase pH, however, did not have obvious effects on modulating LC peak resolution less electronegative sulfur [13] Moreover, AF-containing mobile phases displayed slightly greater HILIC elution strength and diminished retention on the BEH amide column, as reflected by a decrease of the retention factor or k values (Fig 1A) Such a phenomenon was, however, opposed to the previous findings made on a TSK-gel Amide-80 column that AA poses greater elution strength compared to AF [11,17] Weaker elution strength of AF in comparison to AA was also indicated on a modified diol column, as the analyte retention is more attributed to the pH rather than ionic strength of the mobile phase [14] The differential elution strength between AF- and AA-containing mobile phases on the BEH amide column may be attributed to stronger ion exchange interactions of the formate ions Adjustments of ionic strengths in HILIC mobile phases can alter method selectivity, column retention, and separation efficiency [19] Lobue et al previously investigated buffer salt concentrations ranging from 2.5 mM to 15 mM and determined 15 mM AA (pH 5.5) as the optimal concentration for good chromatographic peak shapes and MS response on a polymer-based diol-bonded column [14] The authors found that lower AA concentrations generally gave rise to broadened LC peaks and worse peak shape Similar mobile phase conditions, with 15 mM AA (pH 5.5) being chosen as an optimal mobile phase additive, were reported in an independent study from Demelenne et al on a BEH amide column [16] Moreover, in another two studies that used BEH amide column, MacNeill et al employed 10 mM AF (pH 9.0) for quantification of a PS oligonucleotide, while Goyon et al chose 25 mM AA with no pH adjustment [10,15] Taken together, we reckoned that a systematic screening of salt concentrations and pH could benefit HILIC method development for oligonucleotide using the BEH amide column In the present investigation, four different salt concentrations of AA were tested: 2.5 mM, 7.5 mM, 15 mM and 25 mM As shown in Fig 1B, the observed k values or HILIC retention increased with elevated salt concentrations, consistent with previous findings made on different HILIC stationary phases [14,17,20] Such a phenomenon may suggest that solvation instead of ion exchange remains the dominating retention mechanisms [19] For BEH amide column, the underlying mechanisms accountable for this observation are likely to be the expansion of the aqueous layer adsorbed on the stationary phase surface and as such, more solvated salt ions accumulate in this layer and consequently contribute to a thickening of the water layer and increased retention via hydrogen bonding [17,19,20] Packed with hybrid silica particles, the BEH amide column exhibits greater tolerance to high pH (>8) than pure silica particles, allowing more flexible method development By testing mobile phases composed of 15 mM AA at different pH (pH 5.5, pH 6.8, and pH 9.0), we observed that elevated mobile phase pH with addition of ammonium hydroxide (pH 9.0) led to lower HILIC retention of most oligonucleotides, whereas addition of acetic acid (pH 5.5) generally gave rise to higher HILIC retention (Fig 1C) Our findings were in line with a recent study by Kilanowska et al, in which a minor increase of k was observed with decrease of pH values independent of salt types [17] However, opposing results indicating increasing of HILIC retention of a PS oligonucleotide with increase of pH were also seen, which was justified by higher charge carried by oligonucleotides at higher pH [10] In theory, nucleotide subunits contain both basic nitrogen atoms within the nucleobase and acidic phosphate groups and, as such, mobile phase pH determines the ionization and charge state This can in turn modulate the polarity or hydrophilicity of the analyte and likewise the HILIC retention [19] At pH < 8, each nucleotide contributes to one negative charge, so each additional nucleotide increases the overall charge on the molecule and consequently increases HILIC retention As the eluent pH further increases to > 8, the tautomeric oxygen on each G and T (U for RNA) becomes an oxyanion, thereby 3.3 Assessing effects of column temperature on HILIC analysis of oligonucleotides and siRNAs In HILIC mode, column temperatures can be optimized to enhance selectivity, lower solvent viscosity, and increase mass transfer rates Most HILIC applications in oligonucleotides use relatively low temperature (40°C) for separation [14,16] In a study by Goyon et al, it was shown that higher column temperature of a BEH amide column led to increased peak capacity but lower signal response when coupled to UV detection [10] In the present investigation, a panel of different column temperatures ranging from 30°C to 80°C (with 10°C intervals) were tested As suggested by the HILIC–UV traces in Figure S1, increased column retention and peak capacity yet lowered UV response were observed with elevated temperature, which is in keeping with the trend observed by Goyon et al The mechanisms accounting for higher peak resolution at elevated column temperature could be attributed to minimized non-specific interactions that may arise from internal hydrogen bonding [22] However, analyte adsorption may occur at the surface of HILIC stationary phase through electrostatic interactions or hydrogen bonding, and increasing temperature may result in greater exposure of polar groups present in analytes [10,18] Besides, higher column temperature could result in reduction of the adsorbed water-rich layer on the stationary phase, thereby modulating the partition of the analytes between the bulk mobile phase and adsorbed water-rich layer and consequently retention behavior [23] Consequently, 30°C was chosen as the column temperature for the HILIC separation of single-stranded oligonucleotides In its native conformation, the sense (S) and anti-sense (AS) strands of duplex siRNA are primarily associated through noncovalent interactions such as hydrogen bond interaction and base stacking Increased column temperature is therefore expected to impact the peak shapes of duplexed siRNA compounds by disrupting the double-stranded structures For further investigation, column temperatures ranging from 30°C to 80°C were applied and the duplex, S and AS strands of an siRNA that targets luciferase (siLuc) were subject to HILIC–UV analysis As shown in Figure S2, we observed complete or partial melting of the duplex strands with elevated column heating at 70°C The results demonstrated that elevating column temperature to as low as 70°C led to peak broadening due to duplex melting, yet the conformation of S/AS strands remained largely unchanged (Figure S2) The denaturation or melting of the duplexed structures was further confirmed by MS analysis As such, it is generally advisable to employ a column temperature below the melting point (Tm ) of the double-stranded nucleic acids, under which the duplex structures remain largely intact That said, on-column Tm depression can occur in the presence of an organic solvent (i.e., ACN) in the mobile phase, and optimal column temperature should, therefore, be carefully evaluated [24] Furthermore, it is generally recognized that siRNAs appear to be more hydrophilic compared to their corresponding S/AS strands, as their hydrophobic bases are shielded from solvents in duplex conformation [25] Indeed, we observed substantially elevated HILIC retention of the duplexed structures in contrast to the corresponding S and AS strands (Figure S2), inferring that our current HILIC conditions could preserve non-covalent duplexes of nucleic acids under a lower column temperature (30°C) M Huang, X Xu, H Qiu et al Journal of Chromatography A 1648 (2021) 462184 Fig Full-scan and deconvoluted mass spectra for six DNA oligonucleotides (CMV-F, M13-R, SP6, TRC-F, T3, and T7) analyzed by HILIC−MS 3.4 Impacts of mobile phase additives on full-scan MS analysis of oligonucleotides and siRNAs matographic performance and reasonable MS signal intensities, 15 mM AA was chosen as the final mobile phase salt concentration Moreover, oligonucleotide precursor ions with longer chain lengths were populated in higher charge states (Figure S4A), and distinct differences in charge envelope profiles were noticed for three mobile phase pH tested: AA (pH 5.5), AA (pH 9.0) and AF (pH 5) (Figure S4B) The impacts of mobile phase composition on charge states were not solely dependent on buffer pH, but also can be related to salt types Collectively, to ensure that less water content was needed for analyte elution and thereby higher ESI desolvation efficiency, we tend to use 15 mM AA (pH 9.0) as mobile phase additives optimized for the BEH amide column, which provided an equivalent chromatographic performance to the one obtained at pH 5.5 Furthermore, we also showed that the acquired full-scan mass spectra of DNA/RNA oligonucleotides and siRNA duplexes acquired could be successfully deconvoluted using the PMI-Intact MassTM software (Protein Metrics, San Carlos, CA, USA) The accurate mass for each of the DNA oligonucleotides (Fig 2) could be readily determined, with additional peaks of sodium (Na), potassium (K) and acetate (Ac) adducts observed Moreover, full-scan mass spectra The advantages of coupling HILIC to mass spectrometric (MS) analysis include more efficient sample desolvation and ionization facilitated by the high organic content in the eluents, as such contributing to superior electrospray ionization (ESI)-MS sensitivity Because nucleic acid drugs are a class of molecules that exhibit dominant negative charges, their charge state distribution could be impacted by the generic pKa of the molecules, conformational changes, and formation of cationic adducts We first compared the MS response obtained with the use of mobile phases containing AF (25 mM) and different concentrations of AA: 2.5 mM, 7.5 mM, 15 mM and 25 mM The highest MS response for most DNA oligonucleotides was achieved with a salt concentration of 15 mM AA, except for a longer 25-mer TRC-F (Figure S3) The lowest MS response was observed for 25 mM AF (Figure S3) In addition, with decrease of salt concentration, we observed a systematic shift of charge state distributions of oligonucleotide polyanions to lower m/z values, which align with the results shown by Guo et al via direct infusion experiments [26] Together, to balance good chro- M Huang, X Xu, H Qiu et al Journal of Chromatography A 1648 (2021) 462184 Fig Full-scan and deconvoluted mass spectra for intact siRNA duplexes (NT siRNA#1, NT siRNA#2 and siLuc) analyzed by HILIC−MS Fig (A) HILIC–UV chromatograms showing the separation of the mixture of synthetic 3’ (n – x) truncated sequences of a DNA oligonucleotide TRC-F (B) HILIC–UV chromatograms showing the separation of the 4-oligo (upper panel) and 9-oligo (lower panel) mixture of synthetic 3’ (n – x) truncated sequences of a PS oligonucleotide TRC-FPS of siRNA S/AS strands that were individually analyzed or partially denatured during ESI process could also be deconvoluted (Figure S5) While the analysis of duplexed nucleic acids by HILIC–MS appears to be absent from the current body of literature, it was demonstrated by others that effective IP-RPLC–MS analysis of intact duplex RNA could be achieved by using suitable mobile phases under non-denaturing conditions [27–29] Nevertheless, some IP reagents used in IP-RPLC applications can result in duplex dissociation in both chromatographic and MS detection, presumably due to the disruption of hydrogen bonds with higher ionic strength [29,30] We showed in the previous section that the siRNA duplexes could be retained under HILIC conditions More importantly, full-scan and deconvoluted mass spectra for native siRNA duplexes corroborated the preservation of gas-phase duplex conformation by MS analysis (Fig 3) Taken together, the results have evidenced that HILIC–MS could provide an attractive alternative to analyzing oligonucleotides and siRNAs, and this is the first demonstra- tion of HILIC–MS application in providing native analyses of siRNA duplexes Besides, for both siRNA duplexes and single-stranded oligonucleotides, the metal adduction peaks observed in HILIC−MS were of relatively low abundance (Figs 2, and S5) In line with our results, it was previously reported that the addition of ammonium acetate during ESI process can aid in the removal of alkali metal adducts with volatile ammonium ions (NH4 + ) bound to the negatively charged backbone of oligonucleotides [31] Together, these observations support another advantage of HILIC in mitigating the adduct formation issues that are commonly encountered in IPRPLC applications [32] 3.5 HILIC separation of synthetic 3’ (n − x) truncated sequences of oligonucleotides In-depth characterization of nucleic acid drug products necessitates unambiguous profiling of structural variants of oligonu6 M Huang, X Xu, H Qiu et al Journal of Chromatography A 1648 (2021) 462184 Fig (A) Full view and 60× zoom-in view illustrating the HILIC–UV profiles of the DNA oligonucleotide CMV-F The dashed square highlights the peak region that was further resolved by MS analysis Extracted ion chromatograms depicting a panel of (B) 3’ and (C) 5’ (n – x) shortmer impurities present in CMV-F using the most abundant [M – 4H]4– precursor (D) Summary of the identified impurity sequences of CMV-F and their relative abundance present in the sample cleotides or siRNA arising from synthetic impurities, degradants, and metabolites generated in vitro or in vivo [33] Oligonucleotide and siRNA therapeutics are degraded mainly by cleavage of the phosphodiester linkages by nucleases In vitro experiments showed that cleavage at the -terminus resulting from 3’exonucleolytic activities is the primary form of metabolites, followed by -exonuclease and endonuclease cleavage products [34] Chemical modifications made to the ribose or phosphate backbone are, hence, routinely incorporated into oligonucleotide and siRNA molecule design in an effort to mitigate exo- and endo-nuclease activity in order to ensure sustained drug efficacy [35] In addition to metabolites, process-related impurities can arise from chemical synthesis and often include families of shortmer (n – x) and longmer (n + x) sequences For example, an n – family of impurities would consist of multiple n – base (B), where B can be any base in the sequence [36] Structurally similar synthetic (n – x) sequences of oligonucleotides have been largely employed to examine HILIC separations of shortmer impurity or metabolite mimics [14,16] Therefore, we first evaluated separations of an equimolar mixture of up to (n – 8) 3’ truncated synthetic sequences of a 25-mer DNA oligonucleotide TRC-F By extending to a 20-minute linear HILIC gradient, we successfully achieved good separation of all (n – x) shortmer sequences and the FLP (Fig 4A) However, when it comes to a fully PS-modified version of TRC-F, i.e TRC-FPS, it was particularly difficult to resolve the 3’ (n – 1) sequence and the FLP sequences Specifically, the 3’ (n – 3) and 3’ (n – 2) sequences could be well resolved from the FLP, with the other 3’ (n – x) sequences eluted closer together (Fig 4B) The challenge of separating PS oligonucleotides arises from the compromised LC peak resolution caused by the inherent stereochemical configuration of the PS linkages (“Sp” or “Rp”) One single PS linkage can introduce a chiral center at phosphorus in addition to the D-ribose chiral centers, giving rise to 2N diastereoisomers (number of PS bonds = N) and chromatographic peak splitting or broadening In IP-RPLC, negative charges on the oligonucleotide phosphate backbone are neutralized by positively charged alkylammonium ions in the mobile phase Hydrophobic interactions between the oligonucleotide bases and the reversed-phase column play a major role in the separation, and the different hydrophobicity of the individual bases contributes to differences in retention In comparison, HILIC chromatography depends on hydrophilicity, which is not highly variable between oligonucleotide units largely due to the presence of phosphate groups Therefore, HILIC separation of oligonucleotides is not as robust as it is with IP-RPLC approaches Further modifications with mobile phase modifiers or derivatization are needed to improve selectivity 3.6 Oligonucleotide impurity analysis by HILIC–MS Although extensive purification process removes most of impurities from oligonucleotide therapeutics, low levels of remaining impurities or degraded products could significantly impact both drug safety and efficacy The commonly identified impurities of oligonucleotide therapeutics usually include chain shortened (n – x) products [37] Many hybridization-based oligonucleotide assays not accurately distinguish large impurities or degradants from the full-length oligonucleotide of interest, despite their ultra-high sensitivity in quantifying impurities or metabolites [38] In recent years, MS has gradually become the method of choice in that it can provide unambiguous and comprehensive impurity identification/quantitation and metabolite profiling for both oligonucleotide and siRNA modalities [39] As illustrated by the HILIC–UV traces in Figure S6, various small impurity peaks could be readily separated with an extended linear gradient (20% to 65% MPB in 15 min) These impurities could result from degradation through freeze-thaw cycles and the presence of trace nuclease in the samples Interestingly, for the PS oligonu7 M Huang, X Xu, H Qiu et al Journal of Chromatography A 1648 (2021) 462184 Fig (A) Annotated HCD-MS/MS fragmentation for the [M – 5H]5– precursor (m/z 1309.2167) of a DNA oligonucleotide CMV-F (NCE 15%) (B) MS/MS fragment coverage map for the results shown in (A), generated from Thermo Biopharma Finder 4.0 cleotide T7-FPS, there seemed to be a lesser amount of impurities compared to the amount of impurities for unmodified DNA or RNA oligonucleotides, possibly due to the increased resistance of PS bonds against nucleolytic activities Taking advantage of the downstream HRMS analysis, we attempted to further characterize the identities of some of these impurity peaks In the elution window highlighted by the dashed box in the HILIC–UV profile of the 20-mer DNA oligonucleotide CMV-F (Fig 5A), we performed targeted MS extraction using the m/z value of the most abundant precursor ions ([M – 4H]4– ) of each (n – x) sequence from either 3’ or 5’ terminus to calculate signal intensities Based on the peak intensities extracted for the corresponding 3’ and 5’ (n – x) shortmer sequences (Fig 5B, C), our results evidenced that the generation of 3’ truncated degradants are more prevalent than those generated from the 5’ end for CMV-F (Fig 5D) For other impurity peaks with earlier elution times, we speculated that there could be both exo- and endo-nuclease activities present that led to oligonucleotide impurities with shorter chain length Overall, we demonstrated the feasibility of coupling the HILIC method to UV/MS detection for impurity profiling of oligonucleotides 3.7 Oligonucleotide sequence characterization by HILIC–MS/MS Sequencing elucidation of DNA or RNA oligonucleotides composed of up to 70 residues by MS/MS fragmentation of multiply charged oligonucleotide precursor ions in negative ESI mode has been intensively studied in the past few decades McLuckey et al indicated that the major fragmentation pathways by collisionalinduced dissociation (CID) of oligonucleotides can proceed with multiple pathways [40] Among the backbone cleavage products, a– B and w-type ions are the most dominant species for DNA oligonucleotides, while y- and c-type ions remain more favored for RNA oligonucleotides [41] The Q-Exactive mass spectrometer features beam-type CID, sometimes referred to as higher-energy C-trap dissociation (HCD), which relies on an octupole collision cell that operates at higher collisional energies than CID [42] Furthermore, it is known that precursor charge states can affect the efficiency of fragmentation, as described in previous studies on peptides and other biomolecules [43] As such, we attempted to evaluate how the commonly observed precursor charge states in the HILIC–MS mode and the normalized collision energy (NCE) applied for HCD8 M Huang, X Xu, H Qiu et al Journal of Chromatography A 1648 (2021) 462184 Fig Assessment of on-column sensitivity and linearity range of the HILIC–MS method by (A) tSIM scan and (B) PRM scan MS/MS fragmentation could impact signature fragment ions and sequence coverage for both DNA and RNA oligonucleotides The complexity of a typical oligonucleotide mapping experiment resulting from a great number of structurally diverse fragmentation ions presents a significant throughput hurdle Fortunately, this challenge has been mitigated via recent developments and advancements in software tools that facilitate automated and unbiased data analysis In this study, a newly developed data analysis module incorporated into Biopharma FinderTM 4.0 (Thermo Fisher Scientific) was utilized to achieve automated data processing and sequence annotation for HCD-MS/MS data acquired from oligonucleotides Benefited from this tool, the overall % sequence coverage was systematically examined for oligonucleotides with various base composition (deoxyribonucleic acid, ribonucleic acid), chain length (17-mer to 25-mer), backbone modification (unmodified, partially or fully PS-modified), and sequence composition The most abundant precursors ([M – 4H]4– , [M – 5H]5– and [M – 6H]6– ) observed in the HILIC–MS mode were chosen for analysis, and a panel of % normalized collisional energy (%NCE) values ranging from 13% to 23% were examined In general, precursor ions of higher charge states require higher NCE to achieve good sequence coverage, and an opposite trend was observed for lowercharge-state precursors (Figure S7) For most DNA or RNA oligonucleotides selected in this study, the optimal NCE was determined to be 15% for their most abundant charge states For higher charge states such as [M – 6H]6– , only partial fragmentation of parent ions was observed with low %NCE applied, resulting in low sequence coverage As depicted in Fig 6A, Biopharma FinderTM 4.0 enabled rapid and accurate fragment annotations (predominantly a–B and w-type ions) for the DNA oligonucleotide CMV-F By employing 15% NCE for the [M – 5H]5– precursor (m/z 1309.2167), 90% sequence coverage was achieved, with the intensities of each fragment highlighted in the sequence coverage map (Fig 6B) and HILIC–MS in the field Loube et al showed enhanced MS signal response by HILIC–MS for oligonucleotides of shorter chain-lengths and lower hydrophobicity, as compared to IP-RPLC–MS [14] Nevertheless, Kilanowska et al also pointed out that ACN in the mobile phase may actually cause ionization suppression of oligonucleotides due to its aprotic properties [17] In the work by Easter et al, the LOD values were observed at the picomolar level with the coupling of inductively coupled plasma (ICP)–MS to monitor the signature 31 P16 O+ ion [11] In another study, analyses of oligonucleotides were performed by HILIC–MS analysis with an LOD value of 2.5 pmol loaded on-column for a 20-mer oligonucleotide [12] A similar LOD value of 2.0 pmol on-column was also reported for a 22-mer oligonucleotide using 2D-LC–MS analysis [45] We first determined the optimal HCD energy for chemically modified oligonucleotides including the most prevalent therapeutic oligonucleotide design that involve full PS backbone modifications on T7 (T7-FPS) and TRC-F (TRC-FPS) Notably, CID- or HCD-MS/MS fragmentation of oligonucleotides with PS modifications gave rise to a diagnostic fragment ion PSO2 – (m/z 94.93), which was broadly used for targeted MS analysis such as multiple-reaction monitoring (MRM) [17,20] Our PRM results shown in Figure S8 indicated that higher %NCE may favor generation of the signature PSO2 – fragment ions for quantification purpose (sensitivity), whereas lower %NCE could benefit more comprehensive sequence annotation (specificity) Next, we sought to examine the on-column sensitivity for T7-FPS by performing tSIM or PRM analysis As displayed in Fig 7, tSIM analysis displayed higher S/N ratios or sensitivity in comparison to ratios or sensitivity of PRM analysis in all injections analyzed Even with the lowest injection of 13 ng (equivalent to 2.0 pmol), a clear S/N profile was achieved for the top two most abundant charge states Furthermore, we believe that PRM analysis could warrant higher selectivity for the analysis of structurally similar variants in complex mixtures by incorporating signature fragment ions 3.8 Quantitative analysis of oligonucleotides Conclusions Quantitative analysis of therapeutic oligonucleotides often represents a fundamental part in determining their pharmacokinetic and pharmacodynamic (PK/PD) properties during drug development LC–MS based bioanalytical method can achieve the sensitivity of ng/mL when coupled to upstream sample preparation techniques such as solid-phase extraction (SPE) [44] Thus, we reckon that it is essential to evaluate the sensitivity of our HILIC–MS method to better suit bioanalysis requirements There have been controversies on the sensitivity comparison between IP-RPLC–MS Until now, IP-RPLC remains the most frequently used technique in providing analytical characterization of oligonucleotides There is, however, a drastically increasing trend of using HILIC applications to render complementary and more in-depth analysis of oligonucleotides and other nucleic acid therapeutics In this study, we established a universal HILIC–MS/MS method that could provide a comprehensive solution for the analysis of oligonucleotides and siRNAs, including separation, mass determination, sequence M Huang, X Xu, H Qiu et al Journal of Chromatography A 1648 (2021) 462184 characterization, impurity profiling, as well as the potential in quantitative analysis of oligonucleotides and siRNAs to support drug development We have evaluated the impacts of mobile phases additives on both chromatographic separation and MS response of the HILIC–MS method Accurate intact mass measurement for oligonucleotides was successfully achieved by HRMS analysis More importantly, for the first time, we presented the utility of this HILIC– MS method that preserves the gas-phase duplexed conformation in rendering native analysis of siRNA duplexes Furthermore, the HILIC–MS described herein could facilitate straightforward impurity analysis of oligonucleotides Another highlight of the established method was the hyphenation of HILIC with MS/MS for unbiased sequence annotation, which could be further applied to oligonucleotides with backbone or ribose chemical modifications for structural characterization We showed that employing low NCE (15%) on an orbitrap instrument could facilitate comprehensive HCD-MS/MS sequence coverages for 17- to 25-mer oligonucleotides, and that automated MS/MS annotation could be achieved with improved ease Additionally, we have demonstrated that the use of HILIC–MS based on tSIM mode, as compared to PRM mode, improved the overall on-column sensitivity However, for complex mixtures of structurally similar oligonucleotide impurities and metabolites, selectivity could be further enhanced with PRM analysis by selecting structure-related signature fragment ions In-depth characterization and identification of synthetic impurities present in drug products and metabolites generated in vitro or in vivo remain critical tasks in risk mitigation and clinical studies Extensive work that relies on LC–MS approach has been done previously to identify trace impurities [46,47], in 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their characterization by mass spectrometry, 2019 n/a [40] S.A McLuckey, G.J Van Berkel, G.L Glish, Tandem mass spectrometry of small, multiply charged oligonucleotides, J Am Soc Mass