The impact of low adsorption surfaces for the analysis of DNA and RNA oligonucleotides

The goal of the present paper was to comprehensively evaluate various types of bioinert materials used in ion-pairing reversed-phase (IPRPLC) and hydrophilic interaction chromatography (HILIC) to mitigate this issue for 15- to 100-mer DNA and RNA oligonucleotides.

Journal of Chromatography A 1677 (2022) 463324 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma The impact of low adsorption surfaces for the analysis of DNA and RNA oligonucleotides Honorine Lardeux a,b, Alexandre Goyon c, Kelly Zhang c, Jennifer M Nguyen d, Matthew A Lauber d, Davy Guillarme a,b, Valentina D’Atri a,b,∗ a Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, Geneva 1211, Switzerland School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, Geneva 1211, Switzerland Small Molecule Pharmaceutical Sciences, Genentech Inc., DNA Way, South San Francisco, CA 94080, USA d Waters Corporation, 34 Maple Street, Milford, MA 01757, USA b c a r t i c l e i n f o Article history: Received 18 March 2022 Revised July 2022 Accepted July 2022 Available online July 2022 Keywords: Oligonucleotides Ion-pairing reversed-phase chromatography (IP-RPLC) Hydrophilic interaction chromatography (HILIC) Bioinert surfaces Low adsorption surfaces a b s t r a c t As interest in oligonucleotide (ON) therapeutics is increasing, there is a need to develop suitable analytical methods able to properly analyze those molecules However, an issue exists in the adsorption of ONs on different parts of the instrumentation during their analysis The goal of the present paper was to comprehensively evaluate various types of bioinert materials used in ion-pairing reversed-phase (IPRPLC) and hydrophilic interaction chromatography (HILIC) to mitigate this issue for 15- to 100-mer DNA and RNA oligonucleotides The whole sample flow path was considered under both conditions, including chromatographic columns, ultra-high-performance liquid chromatography (UHPLC) system, and ultraviolet (UV) flow cell It was found that a negligible amount of non-specific adsorption might be attributable to the chromatographic instrumentation However, the flow cell of a detector should be carefully subjected to sample-based conditioning, as the material used in the UV flow cell was found to significantly impact the peak shapes of the largest ONs (60- to 100-mer) Most importantly, we found that the choice of column hardware had the most significant impact on the extent of non-specific adsorption Depending on the material used for the column walls and frits, adsorption can be more or less pronounced It was proved that any type of bioinert RPLC/HILIC column hardware offered some clear benefits in terms of adsorption in comparison to their stainless-steel counterparts Finally, the evaluation of a large set of ONs was performed, including a DNA duplex and DNA or RNA ONs having different base composition, furanose sugar, and modifications occurring at the phosphate linkage or at the sugar moiety This work represents an important advance in understanding the overall ON adsorption, and it helps to define the best combination of materials when analyzing a wide range of unmodified and modified 20-mer DNA and RNA ONs © 2022 The Authors Published by Elsevier B.V This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Introduction Therapeutic oligonucleotides have gained increasing attention thanks to their high potential to treat a large variety of diseases [1,2] By the end of 2021, 16 oligonucleotide-drug therapies have been approved by Food and Drug Administration (FDA) or European Medicines Agency (EMA), with twelve of them having received approval since 2016 [3] Milasen, a personalized oligonucleotide specifically developed for a single patient suffering from ∗ Corresponding author at: Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, Geneva 1211, Switzerland E-mail address: valentina.datri@unige.ch (V D’Atri) Batten disease, is a promising example of oligonucleotide-based customized medicine [4,5] To support the development of these complex drugs, robust and sensitive analytical methods are required Ion-pairing reversedphase liquid chromatography (IP-RPLC), also known as ion-pair chromatography, is recognized as the gold standard method for the characterization of oligonucleotide products and related impurities [6,7] Being complex amphiphilic molecules, ONs present hydrophilic and negatively-charged backbone Therefore, they are not sufficiently retained on hydrophobic RPLC stationary phases For this reason, ion-pairing (IP) agents such as N-alkyl amines are added to the mobile phase, forming oligonucleotide ion-pairs that may be separated based on their related hydrophobicity At elevated temperatures applied to these separations, oligonucleotides https://doi.org/10.1016/j.chroma.2022.463324 0021-9673/© 2022 The Authors Published by Elsevier B.V This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 adopt a linear form such that a length-based separation is primarily observed [8–10] Even if the ion-pair formation in solution is a commonly depicted mechanism, the chromatographic separation may also be explained by the initial adsorption of the IP agent on the hydrophobic stationary phase via its alkyl chains, followed by an ion-exchange process between the charged surface and the analyte [11] It is generally accepted that both mechanisms coexist to explain the retention model of the so-called “ion-pair chromatography” [12,13] IP-RPLC of oligonucleotides has been widely explored over the years First achieved by Fritz et al in 1978, IP-RPLC separation of oligonucleotides historically used triethylamine (TEA) as the IP agent [8,14,15] To overcome some limitations in terms of ESI-MS sensitivity, Apffel et al suggested in 1997 the addition of hexafluoroisopropanol (HFIP) to facilitate the ESI process as well as the IP efficiency [11,16–21] While alternative IP agents have been widely evaluated [22–31], TEA-HFIP mobile phase remains widely used for oligonucleotide characterization [6,7,16,32] The highly polar nature of ONs makes it possible to also consider hydrophilic interaction chromatography (HILIC) In a HILIC separation, charged oligonucleotides can be separated on a polar stationary phase, usually bonded with polar groups such as amide or diol moieties, using a highly-organic mobile phase that contains salts to enhance retention capabilities and selectivity The separation mechanism involves the partitioning of analytes between the bulk mobile phase and a water-rich layer immobilized on the stationary phase surface Retention is further achieved through ionic and hydrogen bonding interactions [33] HILIC was first introduced by Alpert in 1990, but HILIC for ON analysis has grown exponentially in the last few years [34] The popularization of MS-friendly, ion-pairing free buffers such as ammonium acetate or formate that substitute the previous use of triethylammonium acetate in HILIC mode further encourages the development of HILIC [35–44] It has been widely reported that oligonucleotides, because of their electron-rich backbone, suffer from undesired, and often adsorptive, interactions with materials traditionally used in chromatographic analyses The main construction material of chromatographic systems and columns is stainless-steel to ensure pressure resistance Despite its mechanical strength, easy manufacturability and compatibility with most eluents, stainless-steel was found to be susceptible to corrosion with many diverse chromatographic eluents [45,46] The resulting positively-charged metal oxide layer at the surface of the metallic components may cause problems of metal leaching, impacting the chromatographic and MS performance, as well as leading to irreversible adsorption of analytes [47] This non-specific adsorption is even more critical when working at low to neutral pH, being that these are conditions under which metals are more electropositive and most likely to cause ionic interactions with negatively-charged species such as oligonucleotides [42,48–51] These unwanted ionic interactions with the oligonucleotides are further increased as the stainlesssteel surface becomes more corroded and as the number of phosphate groups increases [51,52] Non-specific adsorption may also be a result of polarity-based interactions between hydrophobic ion-pairs and hydrophobic materials from the flow path This phenomenon negatively impacts chromatographic performance by reducing recovery and altering peak shapes (tailing, asymmetry) Consequently, sensitive detection as well as accurate quantitation are hindered, and reliability and reproducibility become compromised [53,54] Several approaches have traditionally been used in an attempt to minimize oligonucleotide adsorption In one case, a strong acid or a sacrificial sample can be used to mask active sites of metallic surfaces and thereby passivate a chromatographic system or column [55,56] Chelators such as ethylenediaminetetraacetic acid (EDTA) may also be used to trap metal ions and prevent adsorption However, their use can come with certain drawbacks, such as ion suppression and persistence in the system In addition, these techniques are time-consuming and not longstanding [57–60] In the last few years, chromatographic instrument manufacturers have focused their developments on strategies to permanently mitigate adsorption of problematic analytes Low adsorption systems and columns have been offered and are based on the use of novel surface technologies Made of bioinert and/or biocompatible materials, they provide a solution to suppress interactions of oligonucleotides with surfaces [53–55,60–63] In general, the term “bioinert” refers to a surface that hampers adsorption, while the term “biocompatible” is used to define a corrosion-resistant material [61,64] Therefore, oligonucleotide analyses require the use of bioinert materials, which are also biocompatible In this work, we present a comprehensive evaluation of bioinert strategies to prevent non-specific adsorption of oligonucleotides in IP-RPLC and HILIC In IP-RPLC mode, three columns made of different bioinert hardware (i.e titanium-lined, PEEK-lined and hybrid organic/inorganic surface columns) were compared to their stainless-steel counterparts using model oligonucleotide samples (DNA and RNA oligonucleotides ranging from 15- to 100-mer) Similarly, bioinert HILIC columns were compared to their stainlesssteel counterparts As bioinert HILIC columns are just recently emerging, only PEEK-lined and hybrid organic/inorganic surface columns are available To our knowledge, HILIC columns in a titanium-lined hardware are not yet offered Finally, the impact of instrumentation hardware was also investigated Three chromatographic systems with different fluidic path material (i.e stainlesssteel, MP35N and titanium, and hybrid organic/inorganic surface) were considered and the impact of the UV flow cell was also highlighted The last part of the study deals with the extension of our observations with the analysis of a wide range of unmodified and modified 20-mer oligonucleotides To our knowledge, a systematic comparison of the impact of bioinert columns consisting of different column hardware has never been reported before More importantly, the evaluation of non-specific adsorption of DNA and RNA oligonucleotides in HILIC mode has been here comprehensively investigated for the first time by using bioinert column hardware This work was essential to understand the contribution of each hardware parameter on the overall oligonucleotide adsorption and conclude on a combination of materials to preferentially use in future studies Experimental 2.1 Chemicals and reagents Oligonucleotides were purchased from Eurogentec (Seraing, Belgium) and Integrated DNA Technologies (IDT, Leuven, Belgium) Type water was obtained from a Milli-Q purification system from Millipore (Bedford, MA, USA) LC-MS grade methanol (art M/4062/17) and acetonitrile (art A/0638/17) were purchased from Thermo Fisher Scientific (Reinach, Switzerland) Ammonium acetate (≥98%, art 32301), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, ≥99%, art 105228), triethylamine (TEA, ≥99.5%, art 90340), and RNase-free water (art 95289) were purchased from Sigma-Aldrich (Buchs, Switzerland) 2.2 Sample preparation Eppendorf DNA LoBind® tubes, Eppendorf Dualfilter T.I.P.S® and polypropylene vials were systematically used during this work to eliminate any risk of additional adsorption that would bias our results 100-μM oligonucleotide aliquots were initially prepared by H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 Table Sequences, molecular masses and modification types of investigated oligonucleotides Phosphorothioate (PS) linkages are indicated by a ∗ , 2’-O-methoxyethyl modifications (MOE) by a X, 2’-O-methyl (OMe) modifications by a Y, and locked nucleic acids (LNA) by a Z Compound Name Length (mer) DNA/ RNA Sequence (5’-3’) Molecular weight (g.mol−1 ) Modification dT15-35: Equimolar mixture of dT15, dT20, dT25, dT30, dT35 15 20 25 DNA TTT (TTT)3 TTT TTT (TTT)5 TT TTT (TTT)7 T 4500.9 6021.9 Unmodified dT40-100: Equimolar mixture of dT40, dT60, dT80, dT100 rU15-30: Equimolar mixture of rU15, rU20, rU30 dT20 dA20 dG20 dC20 rU20 dT20-PS rU20-PS rU20-MOE rU20-OMe dT20-LNA 30 35 40 60 80 100 15 20 30 20 20 20 20 20 20 20 20 20 20 DNA DNA DNA DNA DNA DNA RNA DNA RNA RNA RNA DNA TTT (TTT)8 TTT TTT (TTT)10 TT TTT (TTT)12 T TTT (TTT)18 TTT TTT (TTT)25 TT TTT (TTT)32 TT UUU (UUU)3 UUU UUU (UUU)5 UU UUU (UUU)8 UUU TTT TTT TTT TTT TTT TTT TT AAA AAA AAA AAA AAA AAA AA GGG GGG GGG GGG GGG GGG GG CCC CCC CCC CCC CCC CCC CC UUU UUU UUU UUU UUU UUU UU T∗ T∗ T∗ TTT TTT TTT TTT TT∗ T ∗ T∗ T U∗ U∗ U∗ UUU UUU UUU UUU UU∗ U ∗ U∗ U XXX UUU UUU UUU UUU UUX XX YYY UUU UUU UUU UUU UUY YY ZZZ TTT TTT TTT TTT TTZ ZZ reconstituting lyophilized material in the appropriate volume of RNase-free water and stored at – 20°C (DNA oligonucleotides) or – 80°C (RNA oligonucleotides) Oligonucleotides samples were prepared by diluting the oligonucleotide material to μM in RNasefree water or 10:90 H2 O/ACN prior to RPLC or HILIC analysis, respectively Equimolar oligonucleotide mixtures were prepared by mixing aliquots and diluting the oligonucleotide product to μM Table lists investigated oligonucleotides and their characteristics A 20-mer double-stranded DNA oligonucleotide was also studied in HILIC mode A single-stranded ON, having 5’-TTC GCC TCG CAG TGC GCC TT-3’ sequence and molecular weight of 12 237 g.mol−1 , and its complementary sequence were annealed to form the duplex by using the following protocol 100-μM singlestranded oligonucleotides were mixed in an annealing buffer consisting of 100 mM ammonium acetate in H2 O, heated at 85°C for min, then allowed to slowly cool down to room temperature and aliquoted The final concentration of the duplex was 50 μM Duplex sample was then prepared by diluting the material to μM in 10:90 H2 O/ACN 7542.9 9063.8 10584.8 12105.6 18189.8 24273.7 30357.6 4530.6 6061.4 309123.1 6021.9 6202.2 6522.2 5721.6 6061.4 6118.4 6157.8 6494.2 6145.6 6190.0 Unmodified Unmodified Unmodified Unmodified Unmodified Unmodified Unmodified Phosphorothioate (6) Phosphorothioate (6) 2’-O-methoxyethyl (6) 2’-O-methyl (6) Locked Nucleic Acid (6) flow-through-needle injector The TUV detector was equipped with a 500-nL analytical flow cell (10 mm path length) The flow path of this instrument contains hybrid surface technology (HST), which is described as MaxPeakTM High Performance Surfaces by Waters It is an ethylene bridge hybrid siloxane surface that is applied to materials by vapor deposition For sake of consistency and proper data comparison, the same TUV detector was used on the three instruments while being equipped with different flow cells In all cases, absorbance data were acquired at 260 nm Data acquisition and instrument control were performed by Empower software (Waters) 2.3.4 Columns RPLC and HILIC columns used for this study, including bioinert columns and their stainless-steel analogs, have been listed in Table The complete history of RPLC and HILIC column injections is reported in Table S1 and Table S2, respectively 2.4 Chromatographic conditions 2.3 Instrumentation and columns Mobile phases for IP-RPLC analyses were composed of 15 mM TEA, 400 mM HFIP in water, pH 7.9 (mobile phase A) and a mixture of 50:50 mobile phase A and methanol (mobile phase B) The flow rate was set at 0.4 mL/min and the column temperature at 70°C A gradient of 40–50%B in 15 was used for dT40– 100, while a gradient change of 20%B in 20 was used for the other oligonucleotides Gradients of 30–50%B were used for dT1535, dT20, dA20, dG20, dC20, dT20-PS, rU20-MOE, dT20-LNA; and 20-40%B for rU15-30, rU20, rU20-PS, rU20-OMe Mobile phases for HILIC analyses were composed of 50 mM ammonium acetate in water, pH 6.9 (mobile phase A, no adjustment of pH) and acetonitrile (mobile phase B) The flow rate was set at 0.3 mL/min and the column temperature at 40°C Gradient conditions were the same for all oligonucleotides A gradient of 55–25%B in 30 was used for Waters columns, while a gradient of 75– 45%B in 30 was used for YMC columns It should be noted that the goal of this work was to evaluate the contribution of each hardware parameter on the overall oligonucleotide adsorption, therefore possible effects of mobile phase compositions and pH were not investigated 2.3.1 H-Class The Acquity UPLCTM H-Class system (Waters, Milford, MA, USA) was equipped with a quaternary solvent delivery pump, an autosampler including a 10-μL flow-through-needle injector and a tunable ultraviolet (TUV) detector with a 500-nL analytical flow cell (10 mm path length) The flow path of the instrument was made of stainless-steel 2.3.2 H-Class Bio The Waters Acquity UPLCTM H-Class Bio system was equipped with a biocompatible quaternary solvent delivery pump, an autosampler including a 10-μL flow-through-needle injector The TUV detector was equipped with a 1500-nL titanium flow cell (5 mm path length) The flow path of this instrument primarily consists of an MP35N alloy 2.3.3 Premier system The Waters Acquity PremierTM system was equipped with a binary solvent delivery pump and an autosampler including a 10-μL Diol Diol Ethylene-bridged hybrid organic-inorganic particles Ethylene-bridged hybrid organic-inorganic particles 1.7 1.7 1.9 1.9 150 × 2.1 150 × 2.1 150 × 2.1 150 × 2.1 130 130 2.6 150 × 2.1 100 Results and discussions 3.1 Impact of column material Column hardware represents more than 70% of the sample accessible surfaces during an analysis [60] Generally composed of a stainless-steel tube and stainless-steel frits, it is often assumed that the LC column will introduce the most significant source of adsorption problems Several strategies exist to minimize sample losses and distorted peaks While a “sample conditioning” protocol is often used prior to analysis, bioinert columns have recently become available, and they are meant to be a permanent solution to non-specific adsorption on column hardware Columns featuring titanium, PEEK and hybrid organic/inorganic surface technologies are now commercially available as alternatives to conventional stainless-steel columns To examine currently available techniques, we compared three different bioinert RPLC columns (one of each technology) to their stainless-steel analog The first technology was a hybrid surface technology (HST) column described as MaxPeakTM High Performance Surfaces by Waters (Wpr), which is the bioinert version of the BEH C18 column (Wss) The second one is a PEEK-lined column from YMC (Ypk), which is the bioinert version of the YMC-Triart C18 column (Yss) Last one is a titanium-lined column described as bioZenTM by Phenomenex (Pti), which is the bioinert version of the Kinetex® EVO C18 (Pss) Besides, bioinert HILIC alternatives are still emerging To the best of our knowledge, commercially available bioinert HILIC columns are currently limited to hybrid organic/inorganic surface and PEEK technologies Therefore, we compared the two existing bioinert HILIC column hardware types with their stainless-steel analogs The first one is a HST column from Waters (Wpr_H) that is the bioinert version of a BEH Amide column (Wss_H) The second one is a PEEK-lined column from YMC (Ypk_H), which is the bioinert version of the YMC-Triart Diol (Yss_H) A Premier system comprised of hybrid surface flow path components was systematically used for this column comparison study YMC-Triart DIOL-HILIC YMC-Triart DIOL-HILIC Metal-free Yss_H Ypk_H 3.1.1 Sample conditioning of columns A sample conditioning step was incorporated into the comparison of these columns The dT15-35 sample (mixture of polydeoxythymidylic acids of 15-, 20-, 25-, 30- and 35-mer), which has many times been used for column and system performance testing, was chosen as the “conditioning” sample [53,60] Consecutive injections of a solution containing pmol of each oligodeoxyribonucleotide (ODN) were made on each brand-new column until consistent peak areas were achieved For the IP-RPLC mode, results have been reported in Fig for Waters (Wss vs Wpr, Fig 1A, B), YMC (Yss vs Ypk, Fig 1C, D) and Phenomenex (Pss vs Pti, Fig 1E, F) columns For each column, the first injection was taken as the reference value (100%) and relative peak areas of each ODN were expressed as % to plot the evolution of peak areas over injections Fig also presents the overlaid chromatograms of the first and last injections of the sample conditioning protocol With stainless-steel columns, very low peak areas were obtained for the first injection on the columns, with early eluting peaks particularly affected, as shown in Fig 1A and C for the Wss and Yss columns This was even worse for the Pss column (Fig 1E) which resulted in nearly complete sample loss regardless of the ODN Because of non-specific adsorption, the use of a brand-new YMC YMC Acquity UPLC BEH Amide Acquity Premier BEH Amide Wss_H Wpr_H Waters Waters Phenomenex bioZenTM Oligo Pti Biocompatible titanium (BioTi) Stainless-steel MaxPeakTM High Performance Surfaces Stainless-steel PEEK-lined stainless-steel 1.9 1.9 2.6 150 × 2.1 150 × 2.1 150 × 2.1 YMC YMC Phenomenex Yss Ypk Pss Wpr Waters Acquity UPLC Oligonucleotide BEH C18 Acquity Premier Oligonucleotide BEH C18 YMC-Triart C18 YMC-Triart C18 Metal-free Kinetex EVO C18 Wss Waters All oligonucleotides were concentrated at μM and injection volume was μL Gradient conditions were optimized during preliminary studies All gradients were systematically followed by an 8-min re-equilibration to the initial conditions 120 120 Amide Amide C18 C18 C18 C18 Ethylene-bridged hybrid organic-inorganic particles Ethylene-bridged hybrid organic-inorganic particles Organo-silica core-shell particles with ethane cross-linking Organo-silica core-shell particles with ethane cross-linking Ethylene-bridged hybrid organic-inorganic particles Ethylene-bridged hybrid organic-inorganic particles 1.7 150 × 2.1 MaxPeakTM High Performance Surfaces Stainless-steel PEEK-lined stainless-steel Stainless-steel 120 120 100 C18 Ethylene-bridged hybrid organic-inorganic particles 1.7 150 × 2.1 Stainless-steel 130 C18 Ethylene-bridged hybrid organic-inorganic particles Particle size (μm) Column dimensions (mm) Name Manufacturer Column Hardware (tube and frits material) 130 Journal of Chromatography A 1677 (2022) 463324 Acronym Table List of investigated chromatographic columns and their properties Pore size ˚ (A) Particle type Ligand type H Lardeux, A Goyon, K Zhang et al H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 Fig Monitoring of peak area increases during sample conditioning in IP-RPLC mode using the Premier system Overlaid chromatograms from the first injection (before conditioning) and the last injection (after conditioning) of the mixture dT15-35 when using (A) stainless-steel Waters (Wss), (B) bioinert Waters (Wpr), (C) stainless-steel YMC (Yss), (D) PEEK-lined YMC (Ypk), (E) stainless-steel Phenomenex (Pss), or (F) titanium-lined Phenomenex (Pti) columns 15 injections (300 pmol) and injections (80 pmol) were required for sample-based conditioning of the stainless-steel and bioinert columns, respectively 100% peak area corresponds to first injection (inj 1) column without conditioning cannot give reliable results In the subsequent injections, the peak areas gradually increased with a plateau reached after 15 injections This corresponds to an actual mass load of 300 pmol, required to mask the active sites of stainless-steel material from the columns We can also notice differences in terms of adsorption behavior between ODNs The shortest oligodeoxythymidines (dT15 and dT20) systematically showed the greatest increase in peak areas over conditioning time, with relative peak areas up to 4200% in the 15th injection This may be explained by their elution order rather than length Early eluting compounds will sacrificially saturate adsorption sites as they go through the column, leading to later eluting oligonucleotides being less adsorbed to the column hardware [54] As a result, dT35 relative peak area showed a 175 to 580% value in the last injection, which is moderately high compared to dT15 or dT20 Despite differences in adsorption behavior, these results demonstrate the need to carefully condition stainless-steel columns Contrary to their stainless-steel analogs, nearly full recovery for all oligonucleotides was achieved upon the first injection on bioinert RPLC columns To verify this observation, four successive injections of the ODN mixture were performed, corresponding to 80 pmol loaded on column Fig 1B, D and 1F showed that relative peak areas over the four injections varied from 98 to 104% regardless of the ON length and type of bioinert column Adsorption was reduced upon the first injection, and reliable results were obtainable without sacrificing time or samples In addition, resolution of minor peaks from failure sequences can be seen to be afforded with the bioinert columns upon the first injection However, this was not achieved with brand-new stainless-steel columns, where sample conditioning was needed to detect such minor impurities The same sample conditioning protocol was applied to both stainless-steel HILIC (Fig 2A and C) and bioinert HILIC (Fig 2B and D) columns Different than with IP-RPLC, each HILIC column showed different behavior and did not require the same amount of sample to be effectively passivated As shown in Fig 2C, Yss_H columns showed the greatest number of injections required to reach a plateau in terms of peak area, with 20 injections corresponding to 400 pmol of oligonucleotide in total In any case the utility of sample conditioning the stainless-steel columns to limit undesired interactions with the metallic column hardware was again demonstrated However, despite the conditioning protocol, the increase in peak areas over conditioning is not as pronounced as in IP-RPLC mode (up to 440% vs up to 4200%, respectively) Surprisingly, bioinert HILIC columns also required some conditioning injections to achieve consistent peak areas for the dT15-35 sample (Fig 2B and D) while it was barely necessary in IP-RPLC Indeed, 10 and 14 injections (200/280 pmol) were performed to reach the plateau of peak area on Wpr_H and Ypk_H columns, respectively, demonstrating that this sample conditioning protocol was quite slow with a very gradual increase in peak areas Finally, these findings highlight the benefits of using a bioinert column for the analysis of metal-sensitive analytes from a “sample conditioning” point of view Sample conditioning was found to be required with standard RPLC columns, while bioinert RPLC columns seem to show ready to use performance upon the first H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 Fig Monitoring of peak area increases during sample conditioning in HILIC mode using the Premier system Overlaid chromatograms from the first injection (before conditioning) and the last injection (after conditioning) of the mixture dT15-35 when using (A) stainless-steel Waters (Wss_H), (B) bioinert Waters (Wpr_H) column, (C) stainless-steel YMC (Yss_H), (D) PEEK-lined YMC (Ypk_H) columns 15 injections (300 pmol) and 10 injections (200 pmol) were required for sample-based conditioning of the Wss_H and Wpr_H columns, respectively, while 20 injections (400 pmol) and 14 injections (280 pmol) were required for sample-based conditioning of the Wss_H and Ypk_H, respectively 100% peak area corresponds to the first injection (inj 1) injection Besides, the use of bioinert HILIC columns did not completely suppress non-specific adsorption, but required a reduced number of conditioning injections in comparison with stainlesssteel HILIC columns the two remaining stainless-steel columns (Yss and Pss) produced a peak area equal to only 60–82%, with increasing values for the larger ODNs such as dT35 (similar behavior to what was already explained in Section 3.2.1.) This confirms that adsorption of ONs (equal to 20–40%) is still taking place on these two columns, despite the column conditioning procedure This could be due to differences in metal surface areas, microsite corrosion across the various materials or column manufacturing procedures, but ultimately it seems to be an indication of the number of active sites where ONs can adsorb Batch-to-batch testing of different hardware lots was not possible here, and it could be equally possible that the behavior of stainless-steel columns in this regard is highly variable As reported in Fig 3B, more pronounced differences were observed between the RPLC columns when analyzing the dT40–100 sample, most likely due to the increasing sizes of the ONs Some differences were observed between the reference bioinert hybrid surface (Wpr) column and its stainless-steel counterpart (Wss) Indeed, relative areas varied from 90% for dT40 to only 40% for dT100, and the first eluted peak was not the one to show the lowest recovery In addition, the PEEK column (Ypk) behaves very well for dT40 (relative peak area of 101%), but adsorption was significantly more pronounced when increasing the ON size (relative peak area of 46% for dT100) On the contrary, the stainless-steel column from the same provider (Yss) has a relatively constant behavior independent of the ON size, with relative peak area comprised between 70 and 82% Finally, the titanium column (Pti) has the exact opposite behavior to the PEEK column (Ypk), with a significant reduction of adsorption from dT40 (relative peak area of 44%) to dT100 (relative peak area of 97%) The stainless-steel column from the same provider (Pss) showed similar behavior, but adsorption was slightly less pronounced, in particular for the smaller ONs of this sample (dT40 and dT60) Interpretation of these results is quite difficult since there is likely to be an interplay between several different factors (mostly related to the larger size of these ONs and their molecular masses ranging between 10 and 30 kDa) However, it is also important to keep in mind that the columns were initially 3.1.2 Analysis of unmodified oligonucleotides In addition to the initial column conditioning of the different columns employed in this work, the behavior of these columns was evaluated for the analysis of three different types of model oligonucleotide (ON) samples These included the previously analyzed dT15-35 sample, but also a mixture of larger ODNs (dT40100, a mixture of poly-deoxythymidylic acids of 40-, 60-, 80and 100-mer), and finally a mixture of small oligoribonucleotides (ORNs, rU15-30, a mixture of poly-uridylic acids of 15-, 20- and 30-mer) To obtain a consistent comparison and draw reliable conclusions, the six RPLC columns and the four HILIC columns shared the same history in terms of usage before the injections of the three model ON samples were carried out (as reported in Table S1 and Table S2) In Fig 3, the relative peak areas of the different ON products are provided, while the chromatograms related to these data for bioinert columns are reported in Fig S1 For each individual ON and in both IP-RPLC and HILIC modes, the Waters Premier column (Wpr and Wpr_H, respectively) was taken as the reference value (100%) for the calculations The columns were already conditioned, and a plateau was reached as described in Section 3.1.1 and reported in Figs and Concerning the results obtained in IP-RPLC mode (Fig 3A–C), no significant differences were observed between the stainlesssteel and bioinert (Wss and Wpr) Waters columns when analyzing the dT15-35 sample (Fig 3A) The behavior of the two other bioinert columns (Ypk and Pti) was also in line with our expectations, and values of 105–110% (not significantly different from 100%) were experimentally observed, which means that all three bioinert type columns produced the same peak areas for the dT1535 mixture On the other hand, despite the column conditioning, H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 Fig RPLC bioinert columns composed of hybrid surfaces, PEEK- or titanium-lined hardware (Wpr, Ypk and Pti) in comparison with their stainless-steel analogs (Wss, Yss, Pss), and HILIC bioinert columns composed of hybrid surfaces and PEEK-lined hardware (Wpr_H, Ypk_H) in comparison with their stainless-steel analogs (Wss_H, Yss_H).One injection of each mixture, namely dT15-35 (A, D), dT40-100 (B, E), and rU15-30 (C, F), was performed on each previously conditioned column using the Premier system Relative peak areas for each oligonucleotide are reported 100% corresponds to peak area using the Wpr and Wpr_H column for the IP-RPLC and HILIC mode, respectively conditioned with a dT15–35 sample (see Section 3.1.1.) Since the chemical nature of the dT40-100 sample is different from the material used for conditioning, the column adsorption sites may not have been perfectly masked and lead to partial adsorption of larger ONs (Fig 3B) This itself highlights an inherent drawback to having to rely on sample-based column conditioning Fig 3C shows the adsorption data experimentally obtained for small ORNs, namely the rU15–30 sample In this case, the differences observed between the RPLC columns were more pronounced than for the dT15-35 sample (Fig 3A), but less than for the dT40-100 sample (Fig 3B) It is important to mention that two bioinert columns (Wpr and Ypk) showed comparable behavior, with no issue related to adsorption for the small RNA products The third bioinert column (Pti) offered very good performance in terms of adsorption, with relative peak area values ranging from 75 to 90%, and there was no problem with peak shape for this class of molecules (see Fig S1) For the three stainless-steel columns (Wss, Yss and Pss), undesired adsorption became systematically more pronounced versus their bioinert counterparts, with an average increase of 20 to 40% As expected, losses appeared to be increasingly less pronounced for the larger ON species Concerning the results obtained in HILIC mode (Fig 3D–F), sample conditioning was not sufficient to permanently mitigate adsorption For the dT15-35 sample (Fig 3D), the relative peak areas from the stainless-steel columns (Wss_H and Yss_H) varied from 20 to 90% and from 30 to 40% respectively That means that nonspecific adsorption was still significant on the HILIC stainless-steel columns, even after a conditioning procedure (see previous section) However, these two columns behave quite differently Indeed, the Yss_H column offers consistent analyte recovery whatever the ON size, while sample losses become more and more significant with increasing ON size when using the Wss_H Some significant differences were also observed between the bioinert columns (HST or PEEK) and those made from stainlesssteel On average, the HST material (Wpr_H) offered 20–30% better recovery than the PEEK-coated column (Ypk_H) that was found to result in almost stable relative peak areas whatever the ON length Despite some differences in terms of peak areas, peak shapes were excellent on the two bioinert columns for the dT15-35 sample, as illustrated in Fig S1 Similar results were obtained for the mixture of small ORNs (rU15-30, relative peak areas presented in Fig 3F) vs small ODNs (Fig 3D), but the recoveries experimentally obtained were improved compared to what was observed for all the columns for the small ODNs ORN relative peak areas were measured to be 50–80% for the Wss_H, 50–60% for the Yss_H and 80–95% for the Ypk_H column Values for the small ODNs were 20–90% for the Wss_H, 30–40% for the Yss_H and 70–80% for the Ypk_H column These results demonstrate that ORNs are less prone to adsorption than ODNs under HILIC conditions Besides some changes in peak areas, it is important to notice that peak shapes were again excellent on the two bioinert columns (Fig S1) Finally, some larger ODNs (dT40–100) were also analyzed on the four HILIC columns, and these results are reported in Fig 3E Here, the performance of the columns ranked the same as with small ONs The Wpr_H always showed better results compared to the other ones in terms of adsorption (reference column, relative peak area values of 100%) followed by the other bioinert HILIC col7 H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 umn (Ypk_H, values of 70–85%), the Waters stainless-steel column (Wss_H, values of 60–70%) and the YMC one (Yss_H, values of 40–50%) Non-specific adsorption did not vary according to the length of oligonucleotides across any of the tested HILIC columns At most, there was 10–15% variation of relative peak area with a given column This behavior is in line with the previously obtained results, except on the Wss_H column where the decrease of relative peak areas with oligonucleotide size was not any longer observed with large oligonucleotides (dT40-100) These results suggest that sample losses on the Wss_H column are very dependent on the size of the oligonucleotide between a 15- and 40-mer length, but that differences in size beyond 40 residues might have a diminishing effect As illustrated in Fig S1, all peaks remained symmetrical on the two bioinert columns for the mixture of large ODNs Broader peaks were observed on the Wpr_H vs the Ypk_H column Nevertheless, selectivity and resolution were always greater on the Wpr_H column, which might in part be tied to its stationary phase and its corresponding retentivity In summary, it appears that the smaller DNA and RNA samples (15- to 35-mer) behaved quite similarly in terms of non-specific adsorption in both modes, with adsorption being generally more pronounced under HILIC conditions The larger oligodeoxyribonucleotides (40- to 100-mer) showed different behavior on the different columns investigated in this work Most of the observed chromatographic differences are related to the size of these ON species, which are clearly more difficult to characterize nL PEEK is used as the tubing material on the inside of the flow cell apparatus that is itself mostly made of Teflon The second one is commonly used with the Waters Acquity H-Class Bio system to limit adsorption of biopharmaceutical products during their analysis It is a titanium flow cell having a path length of only mm and a volume of 1500 nL Fig shows the results obtained for the dT15-35, dT40-100 and dT15-35 samples with each combination of UHPLC instrument and UV flow cell material when analyzing the mixtures in HILIC mode Results concerning IP-RPLC mode have been instead reported in the Supplementary Information (Fig S2) 3.2.1 Impact of UV flow cell conditioning Some preliminary HILIC experiments were performed with the UV analytical flow cell mounted on a Waters Premier instrument before and after oligonucleotide sample conditioning Fig shows HILIC chromatograms of the small (dT15-35) and large (dT40–100) ODNs samples as obtained with the original and then the conditioned UV flow cell The experiments with the original UV flow cell were performed on a brand-new Premier instrument, as received from manufacturing This means that the UV flow cell had not seen any ON sample before the experiment reported in Fig (blue trace) On the other hand, the conditioned UV flow cell corresponds to a part that had been used for about one month and exposed to numerous injections of ONs This corresponds to the black trace in Fig As shown, some clear differences were observed between the black and the blue traces and were seen upon zooming in on the baseline (bottom chromatograms in Fig 5) Differences were not drastic for small ODNs of around 15–20 nucleotides (nt), even though some minor species (probably corresponding to shortmers and longmers) were less resolved and/or hardly visible on the UV flow cell that was not conditioned (blue trace) Differences between the UV flow cells were amplified for the larger ODNs of the dT15-35 sample corresponding to dT25 to dT35, where there was a significant loss of resolution between minor species and a significant drift in baseline The situation was at its worst with the mixture of large ODNs (dT40-100) Here, weakly retained components in the sample were hardly detected when using the unconditioned, original UV cell In addition, the four main peaks (dT40, dT60, dT80 and dT100) were poorly resolved and a strong baseline drift was observed On the contrary, the sample-conditioned UV flow cell provided better signal intensity, improved peak symmetry, higher resolution and less baseline drift These observations clearly demonstrate the need to properly condition the UV flow cell before its first use This also proves that non-specific adsorption within the UV flow cell was most pronounced with large ONs 3.2 Impact of instrumentation The instrument could also be responsible for non-specific adsorption and its impact might very well be different under HILIC vs IP-RPLC conditions since mobile phase compositions are quite different For this part, a previously conditioned bioinert Waters column (Wpr or Wpr_H) was systematically employed to minimize as much as possible non-specific adsorption within the column and to thereby more sensitively investigate the impact of the UV flow cell and UHPLC instrumentation Indeed, in the last few years, all LC instrument manufacturers have released several different chromatographic systems, which have been referred to as bioinert, biocompatible and iron-free [55,61,64] These systems have been designed to minimize nonspecific adsorption losses due to metal interactions and/or to offer a better compatibility with mobile phases containing high amount of salts Historically, bioinert HPLC systems were made of PEEK, but with the emergence of UHPLC conditions, advanced materials such as titanium and MP35N alloys have also been applied to build instrumentation that can withstand elevated pressures In addition, a new bioinert UHPLC system was recently released by Waters where the flow path is covered with a hybrid surface technology that is created through the vapor deposition of ethylene bridged hybrid inorganic/organic surfaces To have a clear view of what can be done with the currently available UHPLC instruments for the analysis of ON products, three different systems from the same manufacturer were compared The first one was a regular stainless-steel UHPLC instrument (Waters Acquity H-Class) The second one was a biocompatible UHPLC system (Waters Acquity HClass Bio), where the flow path is composed of corrosion resistant MP35N alloy Finally, the last one was a new UHPLC system (Waters Acquity Premier) constructed with hybrid surface flow path components Besides the evaluation of three different UHPLC instruments, two different UV flow cells were also tested The first one is included on the commercial Waters Acquity H-Class and Waters Acquity Premier instruments It is a regular light-guided analytical flow cell with a path length of 10 mm and a volume of only 500 3.2.2 Impact of instrumentation material The impact of UHPLC instrumentation on the non-specific adsorption of ONs was assessed using three different Waters chromatographic systems The H-Class and Premier instrument are originally equipped with an analytical UV flow cell (mostly made of Teflon wetted parts) that was already conditioned with the dT15-35 sample (see Section 3.2.1.), while the H-Class Bio is originally equipped with a titanium UV flow cell Therefore, the original instrument configurations were compared with the use of the analytical flow cell for the H-Class Bio instrument Importantly, to have adsorption data that can be reliably compared between the three instruments, the same UV detector was used on the three instruments Differences in sensitivity due to the detector itself and in particular the UV lamp were therefore avoided HILIC experimental results have been summarized in Fig for the three model mixtures of ONs ON relative peak areas were plotted for the four different systems, and the instrument configura8 H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 Fig Comparison of instruments namely H-Class, H-Class Bio and Premier equipped with the same UV detector with an analytical flow cell, except for the H-Class Bio where the use of a titanium flow cell was also discussed Letter M indicates that the configuration is the one that is commercially available Histograms corresponding to HILIC-UV chromatograms of the three mixtures using the Waters bioinert column (Wpr_H) Fig HILIC-UV chromatograms of DNA mixtures of oligonucleotides showing the impact of sample conditioning of the UV analytical flow cell on oligonucleotide adsorption a The UV analytical flow cell was used for the first time b The same analytical flow cell was used after being sample conditioned A previously conditioned Wpr_H column was used on the Premier system H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 tion combining the Premier system with the conditioned analytical flow cell was taken as the reference (100%) Regardless of the UHPLC instrument, the peak areas obtained with the titanium UV flow cell were about 2-fold lower than with the analytical UV flow cell (as a result of its vs 10 mm path length) Since the analytical UV flow cell was already conditioned, no significant differences were observed between ONs varying in size and type As illustrated in Fig 4, there were almost no differences for large ODNs and small ORNs between the three UHPLC instruments equipped with the analytical flow cell (relative peak areas values ranged from 95 to 105%) However, some slight differences were observed between the Premier system and the two remaining UHPLC instruments when analyzing small ODNs (dT15-35) In this particular case, relative peak areas on the two other instruments were equal to 85-90% vs 100% on the Premier system The Premier system having being used for the column conditioning studies, it therefore saw significantly higher quantities of samples and in particular the dT1535 sample This might explain the slightly improved recoveries of short ODNs with as much likelihood as the hybrid surfaces of the instrument flow path inherently contributing such a sizable effect In the end, it appears that the non-specific adsorption of oligonucleotides on any type of UHPLC instrument can be negligible, at least with the mass loads applied here for the sake of sample characterization In this type of work, care should at least be taken to condition the UV flow cell The impact of the instrumentation on non-specific adsorption of ONs was also evaluated in IP-RPLC mode Fig S2 shows the results obtained for the dT15-35, dT40-100, and rU15-30 samples with each combination of UHPLC instruments and UV flow cell material (3 systems and UV flow cells) As in HILIC mode, the two-fold decrease of UV signal observed for each ON when modifying the analytical UV flow cell for a titanium flow cell can be attributed to the shorter path length (5 mm vs 10 mm) When looking at chromatograms of 15- to 35-mer ONs (Fig S2A and S2C), peak shapes remain strictly identical whatever the type of instrument and UV flow cell material employed meaning that no significant adsorption issues were observed, even when using the Waters Acquity H-Class system which is composed of stainless-steel Fig S2B shows the results obtained for the mixture of larger ODNs (dT40-100 sample) Here, the instrument had a clear impact on adsorption and above all peak shapes of the largest ONs Interestingly, the peak shapes of the largest ONs (60- to 100-mer) were strongly degraded with severe tailing and broadening observed when using the regular vs titanium UV flow cell To understand this behavior, it is relevant to mention that there are some significant differences between the two UV flow cells in terms of their surface exposed materials Indeed, the analytical flow cell is mostly composed of Teflon which is a hydrophobic material where the complexes of ON and TEA (which are also hydrophobic) can adsorb, despite their short residence time, and that can lead to peak shape distortion Besides the UV flow cell, some additional (more limited) differences were also observed for dT80 and dT100 between the different UHPLC systems, especially when using the analytical flow cell Indeed, the H-Class system offered worse performance (asymmetry at 10% for dT100 was 4.39) vs the H-Class Bio (asymmetry at 10% was 1.85) or the Premier instrument (asymmetry at 10% was 2.18) This confirms that large ONs (60- to 100-mer) are more prone to adsorption and that the latter two UHPLC systems should be preferentially used Based on these findings, it is clear that the analytical UV flow cell of 10 mm is suitable for use with small ONs (15- to 40-mer) in order to achieve maximum sensitivity (namely a 2-fold improvement) On the contrary, despite the more limited sensitivity, the titanium flow cell, even with its mm path length, should be preferably used in IP-RPLC mode to achieve suitable peak shapes for large ONs (60- to 100-mer) 3.3 Application to the analysis of unmodified and modified 20-mer oligonucleotides Differences in the general features of the oligonucleotide have shown to impact non-specific adsorption, and therefore, chemical modifications of the oligonucleotide structure may influence their recovery Indeed, it should be noted that therapeutic ONs have to be chemically-modified to ensure proper pharmacokinetic properties and sufficient activity in vivo [65] In this context, modifications often involve the phosphate linkage and the furanose sugar moiety (deoxyribose in DNA and ribose in RNA) Among the modifications involving the phosphodiester backbone, the most widely used is a phosphorothioate (PS) bond, in which a sulfur replaces one of the non-bridging oxygen atoms of the phosphate linkage (reported in blue in Fig 6) [66] In addition, modifications applied to the furanose sugar moiety (reported in red in Fig 6) include substitutions in the 2’-position, with 2’-O-methyl (OMe), 2’O-methoxyethyl (MOE), and locked nucleic acid (LNA) [66] Based on the performed chemical modifications and base composition of the ONs (U/T, C, A, G, reported in grey in Fig 6), a change in the general features of the ON occurs and potentially impacts nonspecific adsorption The influence of chemical modifications on the 20-mer ON adsorption in both IP-RPLC and HILIC modes was therefore investigated and the list of the evaluated ONs is reported in Table As a result of previous findings, the bioinert hybrid surface columns (Wpr and Wpr_H) and their stainless-steel counterparts (Wss and Wss_H) were used in combination with the best LC system configuration (consisting of the Premier instrument equipped with an analytical flow cell) to evaluate the impact of ON chemistry on adsorption Corresponding chromatograms are reported in Figs and The main chromatographic descriptors and percentage recoveries (calculated as the ratio between the stainless-steel reference column and the bioinert column areas) are summarized in Table First of all, and as reported in Table 3, a higher recovery of all the ONs was obtained with the bioinert HST columns (Wpr/Wpr_H) as compared to their stainless-steel counterparts (Wss/Wss_H) It is worth mentioning that the electropositive metal oxide layer on the surface of stainless-steel columns is generally thought to result in ionic interactions with the negatively-charged backbone of the ONs and therefore be the cause of non-specific adsorption [62,67] This metal oxide is masked in the case of the hybrid surface (Wpr/Wpr_H) columns Indeed, this hypothesis is supported by the chromatograms of all ONs shown in Figs and 8, that correspond to the first and second set of 20-mer ON samples, respectively As reported in Table 3, a better recovery can be observed, indicating that ionic interactions between column surfaces and the ONs are attenuated with the bioinert columns Concerning the detailed impact of modifications, differences in the ON sugar (deoxyribose/ribose) and base composition (U/T, C, A, G) were first evaluated The IP-RPLC-UV and HILIC-UV chromatograms of this set of samples were reported in Fig 7A and Fig 7B, respectively For this part of the work, 20-mer homomolecular oligodeoxyribonucleotides (ODNs) were considered, namely an oligodeoxyadenosine (dA20), an oligodeoxycytidine (dC20), and an oligodeoxyguanosine (dG20) to be compared with an oligodeoxythymidine (dT20) A change on the sugar composition was also applied and a 20-mer oligoribonucleotide (ORN), namely oligouridine (rU20), was analyzed By fixing the length of the ONs at 20-mer, and therefore the number of the ON phosphate groups, it was possible to examine the extent of adsorption 10 H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 Fig Chemical structures of nucleic acids and chemical modifications of the investigated oligonucleotides Abbreviations: 2’-O-MOE, 2’-O-methoxyethyl; 2’-O-Me, 2’-Omethyl; DNA, deoxyribonucleic acid; LNA, locked nucleic acid; RNA, ribonucleic acid (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.) 11 H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 Fig (A) IP-RPLC-UV and (B) HILIC-UV chromatograms of 20-mer non-modified oligonucleotides using the bioinert (Wpr/Wpr_H) vs stainless-steel reference (Wss/Wss_H) columns from Waters % recovery calculated as the ratio between the stainless-steel and the bioinert columns areas are reported in red Premier system was used For gradient conditions, please refer to Section 2.4 (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 12 H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 Fig (A) IP-RPLC-UV and (B) HILIC-UV chromatograms of 20-mer modified oligonucleotides using the bioinert (Wpr/Wpr_H) vs stainless-steel reference (Wss/Wss_H) columns from Waters % recovery calculated as the ratio between the stainless-steel and the bioinert columns areas are reported in red Premier system was used For gradient conditions, please refer to Section 2.4 (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 13 H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 Table Chromatographic parameters of 20-mer oligonucleotides analyzed by IP-RPLC and HILIC using the bioinert column (Wpr/Wpr_H) vs stainless-steel reference column (Wss/Wss_H) from Waters A Premier LC system was used for these measurements Corresponding chromatograms are provided in Figs and tR ; retention time (min), As; asymmetry factor @10 % recovery calculated as the ratio between the stainless-steel reference column (Wss or Wss_H) and the bioinert column (Wpr or Wpr_H) areas Wpr column Oligos dT20 dA20 dG20 dC20 rU20 dT20-PS rU20-PS rU20-MOE rU20-OMe dT20-LNA tR 7.36 6.33 8.70 4.46 6.20 7.79 7.04 7.24 9.73 6.81 As 1.14 1.09 0.95 1.10 1.07 0.79 0.60 1.10 1.16 1.07 Oligos ∗ 91449 71736 23513 48107 35697 69116 101987 60582 102905 69846 Height tR 19707 16032 918 11417 8418 10850 7105 12575 22407 15944 7.90 7.00 9.62 5.18 6.80 7.72 7.04 7.94 9.94 7.65 As 1.10 1.10 0.56 1.06 0.99 0.99 0.60 1.11 1.12 1.13 Wpr_H column tR dT20 dA20 dG20 dC20 rU20 dT20-PS rU20-PS rU20-MOE rU20-OMe dT20-LNA ds Area Wss column 9.27 8.19 9.93 10.73 14.89 7.46 13.76 10.04 12.54 10.07 13.71 As 1.18 1.47 1.68 1.83 1.08 1.14 0.92 1.12 1.14 1.10 0.89 Area 112015 114668 1900 37691 56310 91023 134301 93994 146944 93498 113938 Area 63371 52172 10429 26655 20571 54682 54906 42929 66108 60342 Height 18903 15396 270 7974 5081 10777 3972 11045 15989 14314 Wss_H column Height tR 20133 18747 64 5134 8893 13538 17173 15159 24542 16525 16516 9.53 8.46 10.10 11.00 15.10 7.71 13.98 10.28 12.76 10.31 13.95 As 1.38 4.59 1.43 3.78 1.48 1.12 1.15 1.24 1.23 1.09 1.12 Area 53026 5705 576 4605 24741 86244 114032 82634 126573 87066 69807 Height 8088 493 27 224 3602 12127 14890 12207 20163 14268 10014 % recovery 69.3% 72.7% 44.4% 55.4% 57.6% 79.1% 53.8% 70.9% 64.2% 86.4% % recovery 47.3% 5.0% 30.3% 12.2% 43.9% 94.7% 84.9% 87.9% 86.1% 93.1% 61.3% The columns shared the same history in terms of usage before the injections of the three model oligonucleotide samples were carried out phenomena in correlation with the other ON functional groups, namely the extracyclic functional groups on the different bases and the sugar hydroxyl groups Interpreting the chromatographic behavior of the 20-mer ONs analyzed in IP-RPLC mode (Fig 7A), ORN rU20 was among the species displaying low recovery Specifically, ORNs are more hydrophilic than ODNs because it contains ribose and not deoxyribose A ribose sugar has a hydrophilic hydroxyl that largely defines the RNA macromolecule and that might be responsible for the enhanced adsorption of the ORN by interactions involving the hydroxyl groups themselves [68] Same conclusion was drawn in HILIC mode (Fig 7B) [43] In addition, synergistic electrostatic interactions might be taking place between the rU20 functional groups (i.e phosphate groups, sugar hydroxyl groups, and the extracyclic functional groups on the U) and the column oxide layer surface Decreased recovery (44%) was therefore also observed on the Wss_H vs the Wpr_H column Based on the HILIC chromatographic profiles (Fig 7B), it was also possible to correlate the extent of non-specific adsorption with various ON functional groups (i.e extracyclic functional groups on the different bases and the sugar hydroxyl groups) Specifically, differences were seen for dA20, dG20 and dC20, which must be indicative of extracyclic nucleobases or the possible formation of intramolecular secondary structures For dA20 and dC20 (both containing exocyclic amino groups), very low % recoveries were observed on the Wss_H column in comparison to the Wpr_H column (5% for dA20 and 12% for dC20, respectively) This could be traced back to the involvement of the nucleobases in potential surface interactions However, it should be considered that stainlesssteel columns (Wss_H) required longer initial conditioning times than bioinert columns (Wpr_H) and it might be possible that a like-for-like conditioning process might be needed for some samples In this specific case, the conditioning was carried out with a mixture of poly-dT (see Section 3.1.1) Thymidine (T), compared to A, C, and G, is the nucleobase with the most limited capacity for hydrogen bonding, and it contains two oxide groups and a methyl as heterocycle functional groups [48,68] Indeed, it could be possible that extended column conditioning might be required for ONs containing exocyclic amino groups, like in the case of dA20 and dC20, that are capable of even more extensive hydrogen bonding and electron sharing This might better mitigate the non-specific adsorption encountered with the stainless-steel column in HILIC mode As shown in Fig S3, a significant increase in the recovery of dA20 was achieved after an additional column conditioning was performed in the form of 15 consecutive injections of dA20 In short, analysts must be careful if they are to rely on sample-based conditioning of stainless-steel columns Interestingly, in both modes, dG20 showed a low recovery no matter the column used (lower area and height values as compared to all other ONs, as reported in Table 3) Despite not being supported by the data, a possible explanation for this behavior might be the formation of long and stable supramolecular structures characterized by the cooperative binding of interlocked slipped strands forming stable G-quadruplexes [69,70] These supramolecular structures can have a remarkably increased size as compared to a linear 20-mer strand, while showing an extremely high thermal stability (utterly resistant above temperature of 100°C) [70] Therefore, when analyzing dG20, the overly high peak adsorption (as compared to the other 20-mer ONs) might be eventually traced back to the formation of these heterogeneous intramolecularly hydrogen-bonded complexes Concerning the DNA duplex that was analyzed in HILIC mode (Fig 7B), it should be considered twice as many phosphate groups per length versus a single-stranded molecule In addition, the extracyclic functional groups on the bases are involved in the hydrogen bonding between the two complementary strands and therefore not available for further interactions The higher percentage recovery of the duplex DNA (61%) in comparison with the singlestranded DNA sequences (dT20, dA20, dG20, and dC20) shows that the duplex without the exposed functional groups on the nucle14 H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 obases results in improved recovery despite the greater number of phosphate groups and a completely different secondary structure The DNA duplex in this study was predicted to have a melting temperature (Tm ) of 66°C (based on the provider specifications) Interestingly, under the applied HILIC conditions, no traces of single strands were found, confirming that HILIC was exceptionally well suited to the analysis of this duplex ON When analyzed by high temperature IP-RPLC, this ON was detected only in its melted state (Fig S4) To this point, more pronounced adsorption behavior is observed in HILIC vs IP-RPLC mode (Fig 7, Table 3), and may be explained by differences in pH conditions It has been reported that the oxide layer on the metal surface of stainless-steel columns has an isoelectric point (pI) of approximately [62] Therefore, at mobile phase pH values equal to this pI, as in the case of the HILIC conditions used in this work, the surface oxide layer should be 50% positively-charged and therefore interfering with the HILIC mechanism and actively participating in the binding of negativelycharged ONs On the contrary, in IP-RPLC mode, the mobile phase pH (7.9) being higher than pI, net positive surface charge is reduced and therefore negatively-charged analytes are slightly less prone to adsorption According to this hypothesis, these additional electrostatic interactions in HILIC mode explain the more pronounced adsorption observed on stainless-steel HILIC columns (Wss_H) compared to stainless-steel RPLC columns (Wss) Additional modifications involving the phosphate backbone (phosphorothioate PS linkages) and the sugar moiety (OMe, MOE, and LNA residues) were also evaluated in IP-RPLC and HILIC modes Chromatographic parameters and chromatograms are reported in Table and Fig For these analyses, dT20 and rU20 were kept as reference ONs and the modifications were applied to three nucleotides at both the 5’- and 3’-positions (as reported in Table 1) As reported in Table 3, ONs containing PS linkages showed increased adsorption in comparison to their non-modified counterparts in IP-RPLC mode (Fig 8A) This effect could have been predicted by the fact that the sulfur atom is less electronegative than oxygen, leading to weaker ion-pairing with the components of the mobile phase and therefore additional risk of undesired ionic interactions with the column surfaces Contrary to IP-RPLC, in HILIC mode the backbone of a PScontaining ON is not involved in additional interactions with an ion-pairing agent (as in IP-RPLC) Differences in electronegativity between PO and PS moieties therefore explain much better recovery and peak resolution of PS ONs under HILIC conditions as compared to IP-RPLC mode, especially concerning rU20-PS Regarding modifications involving the sugar moiety, it should be noted that they might change the lipophilicity of the ONs (which relates to favorable pharmacokinetic properties) [71] Specifically, the absence of the hydrophilic hydroxyl that characterizes the RNA is responsible for diminishing ionic interactions and therefore obtaining a better recovery In agreement with this hypothesis, sugar-modified ONs (rU20-MOE, rU20-OMe, and dT20LNA) showed a better recovery as compared to rU20 in both chromatographic modes, with remarkably higher area and height values, as reported in Table Interestingly, in HILIC mode (Fig 8B, Table 3), a consistent and even better recovery (in the range 85% – 95%) than in IP-RPLC was observed for all the sugar-modified ONs on the Wss_H in comparison to the Wpr_H column In addition to what was previously described in IP-RPLC, rU20-MOE, rU20-OMe, and dT20-LNA each contains a modification that masks the hydrophilic hydroxyl of the RNA This might impede hydrogen-bonding between the HILIC stationary phase and the hydroxyl groups of ORNs (substituted in sugar-modified ONs) The combination of all these effects would explain the improvements in recovery and resolution as compared to rU20 in HILIC mode Conclusions The goal of this study was to understand the contribution of each step of the chromatographic process in the problematic of non-specific adsorption of DNA and RNA oligonucleotides ranging from 15- to 100-mer residues in length Under the conditions used in this paper (concentrations in the μM range), column hardware is of significant impact because it is generally made of stainlesssteel and represents more than 70% of the flow path surface that a sample will encounter throughout an analysis Columns made of different bioinert hardware (i.e titanium-lined, PEEK-lined and hybrid organic/inorganic surface columns) were compared to their stainless-steel counterparts in both IP-RPLC and HILIC modes In this context, successive injections of a mixture of ONs (conditioning sample) were carried out to saturate any potential adsorption sites of the column hardware For the IP-RPLC stainless-steel columns, the peak areas gradually increased and a plateau was reached after about 15 injections It is noteworthy that full recovery of all oligonucleotides from the conditioning sample was achieved upon first injection when using the IP-RPLC bioinert columns Nevertheless, the injections of DNA and RNA mixtures of ONs that followed showed that the sample conditioning procedure is not longstanding, and that IP-RPLC bioinert columns should be preferred Standard stainless-steel HILIC columns showed some utility but only after long sample conditioning However, it was found that this strategy does not completely solve the problem and specific protocols might be required based on the nature of the samples used for conditioning versus the samples to be analyzed Bioinert HILIC columns should also be preferentially used when possible To the best of our knowledge, commercially available bioinert HILIC columns are currently limited to hybrid organic/inorganic surface and PEEK technologies Both these hardware surfaces were tested in comparison to their stainless-steel counterparts and remarkable improvements in peak recoveries were found, across a wide range of different ONs including dT15-35 as short DNA, dT40-100 as long DNA, and rU15-30 as short RNA Next, three different chromatographic systems were compared, and it was found that non-specific adsorption of ONs on instrumentation materials was not significant Recoveries from an LC with bioinert flow paths were, at most, only improved by 10% However, the conditions applied here correspond to relatively high mass loads that would be suited to characterizing a drug substance The analysis of trace samples, like is encountered with pharmacokinetic studies, might show more meaningful differences Particular care should instead be taken with the UV flow cell, since strong adsorption might occur on UV flow cells that have not been passivated through sample-based conditioning Conditioning of the UV flow cell is therefore highly recommended to ensure consistent results Analysts might also need to be equally scrupulous whenever a new flow path component is installed into their LC In addition, the material used in the UV flow cell (titanium or Teflon) was found to significantly affect the peak shapes of the largest ONs (60- to 100-mer) analyzed by IP-RPLC, for which titanium should be preferentially used Finally, the evaluation of a larger set of ONs was performed, including a DNA duplex and ONs having different base compositions (U/T, C, A, G), furanose sugars (DNA/RNA), and modifications occurring at the phosphate linkage (PS) or at the sugar moiety (OMe, MOE, and LNA) Based on our data, the choice of the column hardware had the most relevant impact on the extent of nonspecific adsorption In addition, HILIC separations have proven to be extremely attractive for the analysis of RNA-based ONs, duplex, PS- and sugar-modified ONs However, bioinert HILIC columns are mandatory for successful HILIC operation, especially with this particular type of oligonucleotide analysis 15 H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 Declaration of Competing Interest [10] Jennifer Nguyen and Matthew Lauber are employees of Waters Corporation (Milford, MA, USA) that has provided the Waters columns and the Acquity Premier system used in this work [11] CRediT authorship contribution statement [12] Honorine Lardeux: Conceptualization, Investigation, Writing – original draft, Visualization Alexandre Goyon: Resources, Writing – review & editing Kelly Zhang: Resources, Writing – review & editing Jennifer M Nguyen: Resources, Writing – review & editing Matthew A Lauber: Resources, Writing – review & editing Davy Guillarme: Conceptualization, Funding acquisition, Resources, Project administration, Writing – original draft, Writing – review & editing, Supervision Valentina D’Atri: Conceptualization, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration [13] [14] [15] [16] Acknowledgements [17] The authors wish to thank Jean-Luc Veuthey from the University of Geneva for his fruitful comments and discussions, Daniel Eßer (YMC Europe GmbH, Dinslaken, Germany) for providing the YMC columns used in this work, Brian Rivera (Phenomenex Inc, Torrance, CA, USA) for providing the Phenomenex columns, Szabolcs Fekete (Waters) for sharing insights on potential ON retention effects, and Sebastien Besner (Waters) for sharing considerations about flow cell materials The authors also wish to thank Waters Corporation (Milford, MA, USA) for the loan of the Acquity Premier system and for the gift of the Waters columns used in this work [18] [19] [20] [21] Supplementary materials [22] Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2022.463324 [23] References [1] K Dhuri, C Bechtold, E Quijano, H Pham, A Gupta, A Vikram, R Bahal, Antisense oligonucleotides: an emerging area in drug discovery and development, J Clin Med (2020) 2004, doi:10.3390/jcm9062004 [2] A.M Quemener, L Bachelot, A Forestier, E Donnou-Fournet, D Gilot, M Galibert, The powerful world of antisense oligonucleotides: from bench to bedside, WIREs RNA 11 (2020), doi:10.1002/wrna.1594 [3] J Talap, J Zhao, M Shen, Z Song, H Zhou, Y Kang, L Sun, L Yu, S Zeng, S Cai, Recent advances in therapeutic nucleic acids and their analytical methods, J Pharm Biomed Anal 206 (2021) 114368, doi:10.1016/j.jpba.2021.114368 [4] J Kim, C Hu, C Moufawad El Achkar, L.E Black, J Douville, A Larson, M.K Pendergast, S.F Goldkind, E.A Lee, A Kuniholm, A Soucy, J Vaze, N.R Belur, K Fredriksen, I Stojkovska, A Tsytsykova, M Armant, R.L DiDonato, J Choi, L Cornelissen, L.M Pereira, E.F Augustine, C.A Genetti, K Dies, B Barton, L Williams, B.D Goodlett, B.L Riley, A Pasternak, E.R Berry, K.A Pflock, S Chu, C Reed, K Tyndall, P.B Agrawal, A.H Beggs, P.E Grant, D.K Urion, R.O Snyder, S.E Waisbren, A Poduri, P.J Park, A Patterson, A Biffi, J.R Mazzulli, O Bodamer, C.B Berde, T.W Yu, Patient-customized oligonucleotide therapy for a rare genetic disease, N Engl J Med 381 (2019) 1644–1652, doi:10 1056/NEJMoa1813279 [5] A Bateman-House, L Kearns, Individualized therapeutics development for rare diseases: the current ethical landscape and policy responses, Nucleic Acid Ther (2021), doi:10.1089/nat.2021.0035 [6] N.M El Zahar, N Magdy, A.M El-Kosasy, M.G Bartlett, Chromatographic approaches for the characterization and quality control of therapeutic oligonucleotide impurities, Biomed Chromatogr 32 (2018) e4088, doi:10.1002/bmc 4088 [7] A Goyon, P Yehl, K Zhang, Characterization of therapeutic oligonucleotides by liquid chromatography, J Pharm Biomed Anal 182 (2020) 113105, doi:10 1016/j.jpba.2020.113105 [8] C.G Huber, P.J Oefner, G.K Bonn, High-resolution liquid chromatography of oligonucleotides on nonporous alkylated styrene-divinylbenzene copolymers, Anal Biochem 212 (1993) 351–358, doi:10.1006/abio.1993.1340 [9] I.C Santos, J.S Brodbelt, Recent developments in the characterization of nucleic acids by liquid chromatography, capillary electrophoresis, ion mobility, [24] [25] [26] [27] [28] [29] [30] [31] [32] 16 and mass spectrometry (2010–2020), J Sep Sci 44 (2021) 340–372, doi:10 10 02/jssc.2020 0833 M Gilar, K.J Fountain, Y Budman, U.D Neue, K.R Yardley, P.D Rainville, R.J Russell, J.C Gebler, Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides, J Chromatogr A 958 (2002) 167–182, doi:10.1016/S0 021-9673(02)0 0306-0 H Cramer, K Finn, E Girindus, Purity analysis and impurities determination by reversed-phase high- performance liquid chromatography, in: Handbook of Analysis of Oligonucleotides and Related Products, CRC Press, 2011, pp 1–46, doi:10.1201/b10714-2 T Cecchi, Ion pairing chromatography, Crit Rev Anal Chem 38 (2008) 161– 213, doi:10.1080/10408340802038882 T Cecchi, Theoretical models of ion pair chromatography: a close up of recent literature production, J Liq Chromatogr Relat Technol 38 (2015) 404– 414, doi:10.1080/10826076.2014.941267 H.J Fritz, R Belagaje, E.L Brown, R.H Fritz, R.A Jones, R.G Lees, H.G Khorana, Studies on polynucleotides 146 High-pressure liquid chromatography in polynucleotide synthesis, Biochemistry 17 (1978) 1257–1267, doi:10.1021/ bi0 060 0a020 C.G Huber, P.J Oefner, G.K Bonn, High-performance liquid chromatographic separation of detritylated oligonucleotides on highly cross-linked poly-(styrene-divinylbenzene) particles, J Chromatogr A 599 (1992) 113–118, doi:10.1016/0021- 9673(92)85463- M Gilar, K.J Fountain, Y Budman, J.L Holyoke, H Davoudi, J.C Gebler, Characterization of therapeutic oligonucleotides using liquid chromatography with on-line mass spectrometry detection, Oligonucleotides 13 (2003) 229–243, doi:10.1089/154545703322460612 K.J Fountain, M Gilar, J.C Gebler, Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry, Rapid Commun Mass Spectrom 17 (2003) 646–653, doi:10.1002/rcm.959 K Bleicher, E Bayer, Analysis of oligonucleotides using coupled high performance liquid chromatography-electrospray mass spectrometry, Chromatographia 39 (1994) 405–408, doi:10.1007/BF02278754 A Apffel, J.A Chakel, S Fischer, K Lichtenwalter, W.S Hancock, New procedure for the use of high-performance liquid chromatography–electrospray ionization mass spectrometry for the analysis of nucleotides and oligonucleotides, J Chromatogr A 777 (1997) 3–21, doi:10.1016/S0 021-9673(97)0 0256-2 A Apffel, J.A Chakel, S Fischer, K Lichtenwalter, W.S Hancock, Analysis of oligonucleotides by HPLC−electrospray ionization mass spectrometry, Anal Chem 69 (1997) 1320–1325, doi:10.1021/ac960916h C.G Huber, A Krajete, Analysis of nucleic acids by capillary ion-pair reversedphase HPLC coupled to negative-ion electrospray ionization mass spectrometry, Anal Chem 71 (1999) 3730–3739, doi:10.1021/ac990378j B Chen, M.G Bartlett, Evaluation of mobile phase composition for enhancing sensitivity of targeted quantification of oligonucleotides using ultra-high performance liquid chromatography and mass spectrometry: application to phosphorothioate deoxyribonucleic acid, J Chromatogr A 1288 (2013) 73–81, doi:10.1016/j.chroma.2013.03.003 L Gong, J.S.O McCullagh, Comparing ion-pairing reagents and sample dissolution solvents for ion-pairing reversed-phase liquid chromatography/electrospray ionization mass spectrometry analysis of oligonucleotides, Rapid Commun Mass Spectrom 28 (2014) 339–350, doi:10.1002/rcm.6773 A.C McGinnis, E.C Grubb, M.G Bartlett, Systematic optimization of ion-pairing agents and hexafluoroisopropanol for enhanced electrospray ionization mass spectrometry of oligonucleotides, Rapid Commun Mass Spectrom 27 (2013) 2655–2664, doi:10.1002/rcm.6733 R Erb, H Oberacher, Comparison of mobile-phase systems commonly applied in liquid chromatography-mass spectrometry of nucleic acids, Electrophoresis 35 (2014) 1226–1235, doi:10.10 02/elps.20130 0269 L Gong, Comparing ion-pairing reagents and counter anions for ion-pair reversed-phase liquid chromatography/electrospray ionization mass spectrometry analysis of synthetic oligonucleotides, Rapid Commun Mass Spectrom 29 (2015) 2402–2410, doi:10.1002/rcm.7409 B Basiri, H van Hattum, W.D van Dongen, M.M Murph, M.G Bartlett, The role of fluorinated alcohols as mobile phase modifiers for LC-MS analysis of oligonucleotides, J Am Soc Mass Spectrom 28 (2017) 190–199, doi:10.1007/ s13361- 016- 1500- R Liu, Y Ruan, Z Liu, L Gong, The role of fluoroalcohols as counter anions for ion-pairing reversed-phase liquid chromatography/high-resolution electrospray ionization mass spectrometry analysis of oligonucleotides, Rapid Commun Mass Spectrom 33 (2019) 697–709, doi:10.1002/rcm.8386 M.G Bartlett, S Omuro, Evaluation of alkylamines and stationary phases to improve LC–MS of oligonucleotides, Biomed Chromatogr 35 (2021), doi:10.1002/ bmc.5045 S.G Roussis, M Pearce, C Rentel, Small alkyl amines as ion-pair reagents for the separation of positional isomers of impurities in phosphate diester oligonucleotides, J Chromatogr A 1594 (2019) 105–111, doi:10.1016/j.chroma 2019.02.026 M Donegan, J.M Nguyen, M Gilar, Effect of ion-pairing reagent hydrophobicity on liquid chromatography and mass spectrometry analysis of oligonucleotides, J Chromatogr A 1666 (2022) 462860, doi:10.1016/j.chroma.2022.462860 B Basiri, M.M Murph, M.G Bartlett, Assessing the interplay between the physicochemical parameters of ion-pairing reagents and the analyte sequence on the electrospray desorption process for oligonucleotides, J Am Soc Mass Spectrom 28 (2017) 1647–1656, doi:10.1007/s13361- 017- 1671- H Lardeux, A Goyon, K Zhang et al Journal of Chromatography A 1677 (2022) 463324 [33] D.V McCalley, Understanding and manipulating the separation in hydrophilic interaction liquid chromatography, J Chromatogr A 1523 (2017) 49–71, doi:10 1016/j.chroma.2017.06.026 [34] A.J Alpert, Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds, J Chromatogr A 499 (1990) 177–196, doi:10.1016/S0 021-9673(0 0)96972-3 [35] R.N Easter, K.K Kröning, J.A Caruso, P.A Limbach, Separation and identification of oligonucleotides by hydrophilic interaction liquid chromatography (HILIC)—inductively coupled plasma mass spectrometry (ICPMS), Analyst 135 (2010) 2560, doi:10.1039/c0an00399a [36] Q Li, F Lynen, J Wang, H Li, G Xu, P Sandra, Comprehensive hydrophilic interaction and ion-pair reversed-phase liquid chromatography for analysis of dito deca-oligonucleotides, J Chromatogr A 1255 (2012) 237–243, doi:10.1016/j chroma.2011.11.062 [37] R Easter, C Barry, J Caruso, P Limbach, Separation and identification of phosphorothioate oligonucleotides by HILIC-ESIMS, Anal Methods (2013) 2657, doi:10.1039/c3ay26519f ´ ´ [38] S Studzinska, F Łobodzinski, B Buszewski, Application of hydrophilic interaction liquid chromatography coupled with mass spectrometry in the analysis of phosphorothioate oligonucleotides in serum, J Chromatogr B 1040 (2017) 282–288, doi:10.1016/j.jchromb.2016.11.001 [39] P.A Lobue, M Jora, B Addepalli, P.A Limbach, Oligonucleotide analysis by hydrophilic interaction liquid chromatography-mass spectrometry in the absence of ion-pair reagents, J Chromatogr A 1595 (2019) 39–48, doi:10.1016/j.chroma 2019.02.016 ´ [40] A Kilanowska, B Buszewski, S Studzinska, Application of hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the retention and sensitivity studies of antisense oligonucleotides, J Chromatogr A 1622 (2020) 461100, doi:10.1016/j.chroma.2020.461100 [41] A Demelenne, M.-J.J Gou, G Nys, C Parulski, J Crommen, A.-C.C Servais, M Fillet, Evaluation of hydrophilic interaction liquid chromatography, capillary zone electrophoresis and drift tube ion-mobility quadrupole time of flight mass spectrometry for the characterization of phosphodiester and phosphorothioate oligonucleotides, J Chromatogr A 1614 (2020) 460716, doi:10.1016/j chroma.2019.460716 [42] A Goyon, K Zhang, Characterization of antisense oligonucleotide impurities by ion-pairing reversed-phase and anion exchange chromatography coupled to hydrophilic interaction liquid chromatography/mass spectrometry using a versatile two-dimensional liquid chromatography set, Anal Chem 92 (2020) 5944–5951, doi:10.1021/acs.analchem.0c00114 [43] M Huang, X Xu, H Qiu, N Li, Analytical characterization of DNA and RNA oligonucleotides by hydrophilic interaction liquid chromatography-tandem mass spectrometry, J Chromatogr A 1648 (2021) 462184, doi:10.1016/j.chroma 2021.462184 [44] P Holdšvendová, J Suchánková, M Buncˇ ek, V Bacˇ kovská, P Coufal, Hydroxymethyl methacrylate-based monolithic columns designed for separation of oligonucleotides in hydrophilic-interaction capillary liquid chromatography, J Biochem Biophys Methods 70 (2007) 23–29, doi:10.1016/j.jbbm.2006.11.003 [45] R.A Mowery, The corrosion of 316 stainless steel in process liquid chromatography with acetonitrile or methanol carriers, J Chromatogr Sci 23 (1985) 22– 29, doi:10.1093/chromsci/23.1.22 [46] K.E Collins, C.H Collins, C.A Bertran, Stainless steel surfaces in LC systems, Part I—corrosion and erosion, LCGC N Am 18 (20 0) 60 0–608 [47] D.R Stoll, J.J Hsiao, G.O Staples, Troubleshooting LC separations of biomolecules, part I: background, and the meaning of inertness, LCGC N Am 38 (2020) [48] L Gong, Analysis of oligonucleotides by ion-pairing hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry, Rapid Commun Mass Spectrom 31 (2017) 2125–2134, doi:10.10 02/rcm.80 04 [49] G.J Guimaraes, M.G Bartlett, The critical role of mobile phase pH in the performance of oligonucleotide ion-pair liquid chromatography–mass spectrometry methods, Futur Sci OA (2021), doi:10.2144/fsoa- 2021- 0084 [50] T Nagayasu, C Yoshioka, K Imamura, K Nakanishi, Effects of carboxyl groups on the adsorption behavior of low-molecular-weight substances on a stainless steel surface, J Colloid Interface Sci 279 (2004) 296–306, doi:10.1016/j jcis.2004.06.081 [51] R Tuytten, F Lemière, E Witters, W Van Dongen, H Slegers, R.P Newton, H Van Onckelen, E.L Esmans, Stainless steel electrospray probe: a dead end for phosphorylated organic compounds? J Chromatogr A 1104 (2006) 209– 221, doi:10.1016/j.chroma.20 05.12.0 04 [52] A Wakamatsu, K Morimoto, M Shimizu, S Kudoh, A severe peak tailing of phosphate compounds caused by interaction with stainless steel used for liquid chromatography and electrospray mass spectrometry, J Sep Sci 28 (2005) 1823–1830, doi:10.10 02/jssc.20 040 027 [53] J.M Nguyen, M Gilar, B Koshel, M Donegan, J MacLean, Z Li, M.A Lauber, Assessing the impact of nonspecific binding on oligonucleotide bioanalysis, Bioanalysis 13 (2021) 1233–1244, doi:10.4155/bio- 2021- 0115 [54] G.J Guimaraes, J.M Sutton, M Gilar, M Donegan, M.G Bartlett, Impact of nonspecific adsorption to metal surfaces in ion pair-RP LC-MS impurity analysis of oligonucleotides, J Pharm Biomed Anal 208 (2022) 114439, doi:10.1016/j jpba.2021.114439 [55] S Rao, B Rivera, J.A Anspach, Bioinert versus biocompatible: the benefits of different column materials in liquid chromatography separations, LCGC Supplies 36 (2018) 24–29 [56] , Appendix 3: RNA chromatographic system cleaning and passivation treatment, in: RNA Purification and Analysis: Sample Preparation, Extraction, Chromatography, Wiley-VCH Verlag GmbH & Co KGaA, Weinheim, Germany, 2022, pp 185–190, doi:10.1002/9783527627196.app3 n.d [57] S Liu, C Zhang, J.L Campbell, H Zhang, K.K.C Yeung, V.K.M Han, G.A Lajoie, Formation of phosphopeptide-metal ion complexes in liquid chromatography/electrospray mass spectrometry and their influence on phosphopeptide detection, Rapid Commun Mass Spectrom 19 (2005) 2747–2756, doi:10.1002/ rcm.2105 [58] J.C Heaton, D.V McCalley, Some factors that can lead to poor peak shape in hydrophilic interaction chromatography, and possibilities for their remediation, J Chromatogr A 1427 (2016) 37–44, doi:10.1016/j.chroma.2015.10.056 [59] K.T Myint, T Uehara, K Aoshima, Y Oda, Polar anionic metabolome analysis by nano-LC/MS with a metal chelating agent, Anal Chem 81 (2009) 7766–7772, doi:10.1021/ac901269h [60] M Gilar, M DeLano, F Gritti, Mitigation of analyte loss on metal surfaces in liquid chromatography, J Chromatogr A 1650 (2021) 462247, doi:10.1016/ j.chroma.2021.462247 [61] T Fehrenbach, S Wiese, Materials in HPLC and UHPLC - what to use for which purpose, in: The HPLC Expert II: Find and Optimize the Benefits of your HPLC/UHPLC, Wiley-VCH Verlag GmbH & Co KGaA, Weinheim, Germany, 2017, pp 223–267, doi:10.1002/9783527694945.ch7 [62] M DeLano, T.H Walter, M.A Lauber, M Gilar, M.C Jung, J.M Nguyen, C Boissel, A.V Patel, A Bates-Harrison, K.D Wyndham, Using hybrid organic– inorganic surface technology to mitigate analyte interactions with metal surfaces in UHPLC, Anal Chem 93 (2021) 5773–5781, doi:10.1021/acs.analchem 0c05203 [63] T.H Walter, B.J Murphy, M.C Jung, M Gilar, R.E Birdsall, J Kellett, Faster time to results for ultra-performance liquid chromatographic separations of metalsensitive analytes, (2021) https://www.chromatographytoday.com/article/ liquid-chromatography/65/waters-corporation/faster-time-to-results-for-ultra - performance- liquid- chromatographic-separations-of-metal-sensitive-analytes /2903 (accessed January 3, 2022) [64] S Fekete, Defining material used in biopharmaceutical analysis, LCGC Eur 34 (2021) 245–248 [65] A Khvorova, J.K Watts, The chemical evolution of oligonucleotide therapies of clinical utility, Nat Biotechnol 35 (2017) 238–248, doi:10.1038/nbt.3765 [66] C.I.E Smith, R Zain, Therapeutic oligonucleotides: state of the art, Annu Rev Pharmacol Toxicol 59 (2019) 605–630, doi:10.1146/ annurev-pharmtox-010818-021050 [67] R.E Birdsall, J Kellett, S Ippoliti, N Ranbaduge, M.A Lauber, Y.Q Yu, W Chen, Reducing metal-ion mediated adsorption of acidic peptides in RPLC-based assays using hybrid silica chromatographic surfaces, J Chromatogr B 1179 (2021) 122700, doi:10.1016/j.jchromb.2021.122700 [68] H.J Cleaves, C.M Jonsson, C.L Jonsson, D.A Sverjensky, R.M Hazen, Adsorption of nucleic acid components on rutile (TiO2 ) surfaces, Astrobiology 10 (2010) 311–323, doi:10.1089/ast.2009.0397 [69] M Marzano, A.P Falanga, P Dardano, S D’Errico, I Rea, M Terracciano, L De Stefano, G Piccialli, N Borbone, G Oliviero, π –π stacked DNA G-wire nanostructures formed by a short G-rich oligonucleotide containing a –3 inversion of polarity site, Org Chem Front (2020) 2187–2195, doi:10.1039/ D0QO00561D [70] A.B Kotlyar, N Borovok, T Molotsky, H Cohen, E Shapir, D Porath, Long, monomolecular guanine-based nanowires, Adv Mater 17 (2005) 1901–1905, doi:10.10 02/adma.20 0401997 [71] M Manoharan, -Carbohydrate modifications in antisense oligonucleotide therapy: importance of conformation, configuration and conjugation, Biochim Biophys Acta Gene Struct Expr 1489 (1999) 117–130, doi:10.1016/S0167-4781(99)00138-4 17 ... fixing the length of the ONs at 20-mer, and therefore the number of the ON phosphate groups, it was possible to examine the extent of adsorption 10 H Lardeux, A Goyon, K Zhang et al Journal of Chromatography... Application to the analysis of unmodified and modified 20-mer oligonucleotides Differences in the general features of the oligonucleotide have shown to impact non-specific adsorption, and therefore, chemical... was observed for all the columns for the small ODNs ORN relative peak areas were measured to be 50–80% for the Wss_H, 50–60% for the Yss_H and 80–95% for the Ypk_H column Values for the small ODNs