Hydrophilic Interaction Liquid Chromatography for Oligonucleotide Therapeutics: Method Considerations

Hydrophilic interaction liquid chromatography (HILIC) has emerged as a promising alternative to traditional ion-pair reversed phase liquid chromatography (IP-RPLC) methods for separating oligonucleotides (ON). This work investigates the application of HILIC to the separation of ON sequence and length variants, duplexes, and single-stranded components. Method variables, including ionic strength, temperature, mobile phase composition, and gradient optimization, are investigated in detail. A sequential injection procedure is also proposed to eliminate breakthrough effects and peak splitting. Some considerations for generic (platform) methods are also suggested.

Recently, hydrophilic interaction liquid chromatography (HILIC) has gained increasing interest for oligonucleotide (ON) separations (1–3). HILIC-mode separations offer attractive, sensitive, and robust alternatives to the most commonly used ion-pairing reversed-phase liquid chromatography (IP-RPLC) methods. HILIC has shown particular promise for ON diastereomer separations. It has been shown that high ionic strength and low mobile phase temperature favor diastereomer selectivity (3). HILIC is also capable of separating duplex forms from their single-stranded components because it can be readily operated in either denaturing or non-denaturing (native) conditions depending on the mobile phase temperature (4). Another important advantage of HILIC is that it allows the use of ion-pair-free mobile phases and so is favored for more versatile mass spectrometry (MS) applications (5,6). HILIC has also been successfully applied for the bottom-up sequencing of single guide RNA (sgRNA) in liquid chromatographymass spectrometry (LC–MS) and LC–tandem MS (LC–MS/MS) setups (7).

Although robust methods can be developed in HILIC for ONs, the retention mechanism is not fully understood. Some authors mention that ionic interactions may be involved in the retention mechanism (note that in this case it should be repulsive effect) (1), while others conclude that HILIC analysis of ONs is mainly driven by hydrogen bonding interactions, either by adsorption or partitioning (3). It is thought that mobile phases with high ionic strength appear to stabilize the immobilized water layer on the surface of the polar (that is, amide) stationary phase and minimize ionic interactions (3). However, some chromatographic models suggest the opposite: when salts are added to aqueous mobile phases, water molecules will predominantly solvate salt ions such that the highly ordered structure of water is disrupted. This would reduce the number of water molecules available to interact with a hydrophilic surface, and there would be a corresponding reduction in the thickness of the immobilized water layer. It is also acknowledged that HILIC columns provide good peak shape and similar selectivity when a protic co-solvent (that is, methanol) is used instead of acetonitrile. This fact further complicates the understanding of the retention mechanism. Finally, it should be noted that the amount of water required to elute ONs from HILIC columns (20–60% water) is quite high, much higher than the amount required to elute glycans or glycoproteins (15–35% water) (8). Bartlett concluded that the mechanism of retention of ONs in HILIC is a complex and unique multi-modal mechanism involving partitioning, hydrogen bonding, and electrostatic interactions (9). Because of the higher water content in the mobile phase, HILIC of ONs occurs with mobile phase compositions that are quite unique when compared to “traditional” HILIC. Non-specific binding appears to be a greater challenge for HILIC when compared to other types of chromatography; this is probably because of its optimal pH being between 5 and 8, which causes greater metal surface charging for non-metal-free column hardware (9).

The character and effect of the polar functional groups of HILIC phases on ON retention and selectivity has recently been investigated (10). A modified Tanaka test (adapted to HILIC of ONs with specific test compounds) was proposed to characterize the HILIC phases, but it was found that small molecule probes do not necessarily translate to the behavior of larger complex molecules (ONs). The amide phase was found to make the greatest contribution to hydrophilic partitioning and is likely to form the thickest adsorbed (immobilized) water layer surrounding the stationary phase (10).

There is little mention of systematic method development for ON separations in HILIC. Very often, the same or similar conditions are used. Ammonium acetate, formate, or a bicarbonate-modified aqueous solution is applied along with acetonitrile. The ionic strength of the mobile phase additive, temperature effects, and pH have been studied (1,3,11), but very often separately (one factor at a time [OFAT]) rather than in a combined design of experiments (DoE) or multidimensional retention model.

Here, a systematic approach was used to compare three ammonium salts. The effect of the mobile-phase temperature and gradient programming was evaluated by retention modeling. Breakthrough effects were studied and a sequential (bracketed) injection mode was proposed to avoid peak splitting. Some generic (platform) method conditions were also proposed.

Materials and Methods

Method Screening and Optimization

First, for screening purposes, a three-factor DoE was applied to study the effect of gradient steepness (gradient time, [tG ]), temperature (T), and ionic strength (C). The impact of t_G and T was studied at two levels while the effect of C was studied at three levels. Then, the gradient program and temperature were simultaneously further optimized (using chromatographic modeling software) to obtain a 10-min separation that can be generally applied to different oligonucleotide ladder samples (for a broad range of ON length variants).

Studying Breakthrough Effects and Sequential Injections

Breakthrough effects (or partial peak splitting) often occur in HILIC because of an inherent mismatch between the sample diluent (biomolecules such as proteins or oligonucleotides must be solubilized in aqueous media) and the mobile phase, which is mostly comprised of aprotic solvent (acetonitrile) (12). Practicing chromatographers often make a compromise to avoid breakthrough effects by diluting the sample in an acetonitrile—water mixture that can lead to sample precipitation or denaturation. This study proposes a more elegant solution: the addition of pre- and post-injection plugs of a weak solvent (acetonitrile) while the sample is injected in a pure aqueous solvent plug (”bracketed” injection). Direct injections of di erent sample volumes will be compared to the bracketed injections performed, considering di erent ratios of solvent to sample plug volume.

Mobile Phase Preparation

High performance liquid chromatography (HPLC)- and MS-grade solvents were used. For the LC study, pre-mixed mobile phases were considered to avoid precipitation and solubility problems. Mobile phase A was 30:70 water–acetonitrile, while mobile phase B was 90:10 water–acetonitrile (Honeywell). It was found that such a mobile phase composition could elute all oligonucleotide standards with adequate retention when running linear gradients of 0–100%B. To investigate the effect of additives in the mobile phase, three di erent ionic strengths (C₁ = 10 mM, C₂ = 25 mM, C₃ = 50 mM) were considered for the three ammonium salts (acetate/formate/bicarbonate [Sigma Aldrich]) in both A and B. For the final optimized LC–UV methods, 50 mM salts were added to both mobile phases A and B. For the LC–MS application, neat acetonitrile (Honeywell) and water were used (not premixed) to decrease leachate effects from the reservoir bottle. Mobile phase A was 100 mM ammonium acetate (Waters Corporation) in water and kept in an low density polyethylene (LDPE) bottle while B was neat acetonitrile (Honeywell) kept in a glass bottle.

Samples

Various ON standards and ladders were used (all Waters Corporation): an oligo dT ladder, single-stranded DNA (ssDNA) ladder, a lipid-conjugated antisense oligonucleotide (ASO), a small interfering RNA (siRNA), and an sgRNA. Samples were reconstituted in HPLC-grade water, except for the LCMS analysis; the lipid-conjugated ASO was reconstituted with 20% acetone and the sgRNA in nuclease-free water.

Column and Chromatographic System

Low adsorption columns in dimensions 50 × 2.1 mm were packed with a 300 Å 1.7-µm amide-bonded stationary phase (Waters Corporation). The experiments were performed on a low dispersion and low adsorption binary ultrahigh-pressure LC (UHPLC) system coupled to a UV and/or time-of-flight mass spectrometry (TOF-MS) detector (Waters Corporation).

Results and Discussion

The Effect of Additive: Comparison of Three Ammonium Salts

The effect of ionic strength was found to be important when C ≤ 40 mM and retention decreased with decreasing ionic strength. Beyond 40 mM, retention was found to be robustly stable, suggesting minimal electrostatic effects (with all three ammonium salts). Note that this mobile phase ionic strength limit may depend on the column hardware material and the stationary phase. If the additive concentration was too low (C = 10–20 mM), then breakthrough and/ or peak splitting might be significant even if the retention of the first eluted peaks is set to be apparently high (that is, the apparent retention factor is k_app ~ 5).

From a chromatographic separation point of view, ammonium acetate and formate seem equivalent. The same gradient program can be used with both salts, resulting in almost identical retention, selectivity, and peak patterns. However, ammonium bicarbonate resulted in slightly lower retention than acetate or formate salts, so the gradient program must be adjusted (shifted by approximately -5% B) to achieve similar retention. Note that the selectivity was slightly di erent for bicarbonate as well. With bicarbonate, some early eluting peaks were more sensitive to peak splitting and breakthrough (particularly at elevated temperatures), which is a disadvantage compared to the other two salts. However, this effect can be eliminated by using a bracketed injection.

The Effect of Temperature

In general, temperature was found to be a very important variable in tuning the selectivity and even the elution order of di erent ON species; therefore, temperature is recommended to be optimized for a given sample. For all samples, retention decreased with temperature decrease, suggesting that ONs were present in extended form with multiple sites for adsorptive interaction and that retention was mostly controlled by enthalpy changes upon adsorption-desorption steps (transfer of the solutes between stationary and mobile phases) in HILIC. For all ON samples, van’t Ho plots (the logarithm of retention vs. 1/T) followed a linear relationship. The positive slope of the van’t Ho plots suggests that ON solubility was favored toward the stationary phase (note that an immobilized water layer was adsorbed onto the surface of the stationary phase) rather than the mobile phase. Figure 1 illustrates the effect of temperature on selectivity and resolution for a complex ON mixture (including both length and sequence variants). Peaks 1–5 are 15, 20, 25, 30, and 35 nucleotide (nt) long oligodeoxythymidines (o-dTs), while peaks a–i are 20, 30, 40, 50, 60, 70, 80, 90, and 100-mer ssDNAs. Peak pairs “a” – 3, and “b” – 5 change their elution order with the changing temperature.

Figure 1: Resolution map (left) and chromatograms at three different temperatures (right). The resolution map shows the critical resolution (R_s) as a function of temperature (T) and gradient time (t_G). Red colors correspond to high resolution conditions (R_s > 1.5), while the blue areas indicate low resolution conditions. On the chromatograms, t_G is fixed at 20 min, while T is set at 40, 55, and 75 °C. Mobile phase buffer: 50 mM ammonium acetate, fl ow rate F = 0.4 mL/min. Peaks: 15, 20, 25, 30, and 35 nt o-dTs (1–5) and 20, 30, 40, 50, 60, 70, 80, 90, and 100-mer ssDNAs (a–i).

Another important observation was that peak splitting (for early eluting species) and breakthrough became more pronounced at elevated temperatures (T ≥ 50 °C). As well as tuning the selectivity and elution order of di erent species, temperature is also critical in setting “less” denaturing or “more” denaturing separation conditions in HILIC. ON duplexes can be stabilized in non-denaturing conditions below the duplex melting temperature (T_m). Melting temperature is the property used to measure duplex stability; however, it is not fully understood how the chromatographic conditions a ect the duplex stability (4). Pressure, temperature, pH, and co-solvents can cause significant shifts in the apparent melting temperature.

An siRNA standard (a mixture of annealed 25mer and 27mer RNA strands) was used to study temperature effects on duplex stability. Below 55 °C, a single peak was observed. At 60 °C, a very broad zone appeared on the chromatogram and its retention time decreased significantly compared to the sharp single peak, and when the temperature was increased above 65 °C, two sharp peaks were observed on the chromatogram. These results suggest that this particular siRNA begins dissociating into its single strands at 60 °C. Figure 2 shows the temperature effects on duplex stability of a siRNA.

Figure 2: Illustration of temperature effects in HILIC on duplex stability. Column: 50 × 2.1 mm, 1.7-µm 300 Å amide-bonded. Mobile phase buffer: 50 mM ammonium acetate, flow rate F = 0.4 mL/min, t_G = 10 min. Sample: siRNA standard (mixture of annealed 25mer and 27mer RNA strands).

The Effect of Gradient Sharpness and Shape

When optimizing the separation of complex ON mixtures, the two most important method variables are probably gradient steepness (t_G) and mobile phase temperature (T). First, an appropriate mobile phase system (that is, 50 mM ammonium acetate and acetonitrile as co-solvent) must be selected to allow elution of all species in the sample mixture. This may require some screening experiments. Then, the best practice is to apply a retention model combining t_G and T to optimize the peak resolution (R_s). The entire optimization process requires only four experiments (2 × t_G + 2 × T). By adjusting t_G, the entire elution window of all peaks and the Rs between short and long ONs (size variants) can be adjusted. Meanwhile, by changing the temperature, the selectivity between sequence variants of similar ON lengths can be improved. The retention of length variants is primarily sensitive to gradient steepness, while the relative retention of sequence variants is mainly determined by temperature.

The empirical observation in LC that the retention of compounds of a homolog series (that is, the length variants of ONs) increases in logarithmic fashion with an increasing number of homolog units is called the homolog rule (13,14). As a result of the homolog rule, the selectivity between n-1-mers decreases significantly for longer ONs when running linear mobile phase gradients. Very often, unnecessarily high selectivity is obtained for the short ONs and very low (limited) selectivity for the long ONs. Therefore, a more uniform selectivity (retention pattern) should be established for the separation of length variants to approach uniform selectivity across the entire elution window of ON homologs. An elegant approach to set up a generic (platform) method is based on an “inverse function” or “inverse gradient” method (15,16). If a linear mobile phase gradient (linear input function) results in a logarithmic (“concave”) retention time distribution (logarithmic response function) for a set of compounds and the analytes elute by on-off mechanism (like ONs do), then programing a logarithmic concave gradient program is predicted to yield a linear retention time distribution across the entire elution window (inverting the input and response functions) (17). This inverse gradient approach has recently been applied for ONs in IP-RPLC (18). The same considerations apply in HILIC. Figure 3 compares peak patterns obtained by running a linear vs. a concave gradient program for different ONs.

Figure 3: Comparison of linear (top) and concave multi-linear (bottom) gradients for ONs HILIC separation. Column: 50 × 2.1 mm, 1.7-µm 300 Å amide-bonded. Mobile phase A: 50 mM ammonium acetate in 30:70 water–acetonitrile. Mobile phase B: 50 mM ammonium acetate in 90:10 wateracetonitrile, gradient: 10–60%B in 10 min, F = 0.4 mL/min, T = 50 °C. Samples: lipid conjugated ASO oligonucleotide (orange trace), oligo dT ladder (red trace), and ssDNA ladder (black trace).

It is worth noting that for ON separations, HILIC and IP-RPLC modes may be alternative separation techniques but they are not entirely orthogonal. The strength of the interaction increases with the length of the ON, whether it’s a hydrophobic (RP) or polar (HILIC) retention mechanism. If the nucleotide sequence is uniform (homopolymer or homolog), the ONs will elute according to their length. If the sequences vary, retention and selectivity can change significantly.

Breakthrough Effects and Bracketed Injections

Sample injection in HILIC can cause problems because of the higher eluent strength of the injection solvent compared to the mobile phase. In this case, where there is a solvent mismatch between the mobile phase composition and the injection solvent, the sample is not su iciently focused at the column inlet (12). Fine adjustment of the injection diluent could minimize this mismatch (that is, increasing the percentage of acetonitrile in the sample diluent). However, this adjustment is not always possible because of the nature of the sample, which may cause denaturation, sample artifacts, or even precipitation. This solvent mismatch usually results in undesirable effects on the chromatographic separation, ranging from slight broadening to severe peak deformation or even splitting and analyte breakthrough. Breakthrough is a phenomenon where part of the sample migrates through the column with little interaction with the stationary phase and therefore elutes close to the column dead time, whereas the other part of the peak elutes at the normal retention time.

In our examples, the oligo dT ladder was the most sensitive to peak splitting. When the injection volume was set to 0.5 µL, the first compound (15 nt) eluted in a split peak. When the injected volume was increased, the later eluting ONs also showed significant splitting and some of the solutes eluted at the column void (Figure 4), which limits the column load and sensitivity.

Figure 4: Illustration of injection breakthrough effect in HILIC. Column: 50 × 2.1 mm, 1.7-µm 300 Å amide-bonded. Mobile phase A: 50 mM ammonium bicarbonate in 30:70 water–acetonitrile. Mobile phase B: 50 mM ammonium bicarbonate in 90:10 water–acetonitrile, gradient: 5–55%B in 10 min (linear gradient), F = 0.4 mL/min, T = 50 °C. Sample: oligo dT ladder.

A bracketed injection sequence can help eliminate breakthrough effects. In a bracketed injection, the sample is injected into the column head together with a pre- and post-plug of weak solvent (in this case acetonitrile). Using this bracketed injection, the sample injection volume could be increased up to 2 µL for the dT ladder without splitting. Figure 5 shows a comparison between bracketed injections performed with various solvent-to-sample plug volume ratios when 1 and 2 µL of sample plug is introduced into the flow path. Note that with a normal injection mode, only a 0.3 µL sample could be safely injected, while the bracketed injection allowed the sample load to be increased to 2 µL (a factor ~7 improvement).

Figure 5: Bracketed injections (acetonitrile solvent pre-plug + sample plug + acetonitrile solvent post-plug) performed with diff erent ratios of solvent-to-sample plugs. Conditions and sample: the same as described in Figure 4.

HILIC–MS Application

With insights collected on the tunability of ON HILIC methods, the direct hyphenation of these separations with mass spectrometric detection was studied. Two unique methods were optimized to analyze a CRISPR sgRNA molecule as well as a lipid-conjugated ASO. For the sgRNA, an aqueous eluent modified with 100 mM ammonium acetate was applied. In the case of the lipid-conjugated ASO, a lower, 25 mM concentration of ammonium salt was employed. Both cases produced effective levels of ion signal that could be directly interpreted with an accurate mass measurement derived from a monoisotopic peak or from MaxEnt deconvolution of the raw mass spectra.

For the CRISPR sgRNA synthesized with a human HPRT sequence complementarity, an intact molecular weight of 32,276.97 Da was obtained, which is in close agreement to the sequence of this sgRNA—inclusive of its 5’ and 3’ modified termini. Results obtained for the lipid-conjugated ASO are shown in Figure 6. In contrast to the sgRNA, the lipid ASO was not strongly retained on the HILIC column. This can be attributed to it being a significantly smaller oligonucleotide that is conjugated to a C16 lipid. This lipid ASO contains a fully phosphorothioated backbone, along with several other modifications. A high temperature separation was found to reduce peak splitting (partial separation of diastereomers). With a final method using a 25 mM ammonium acetate eluent, two isobaric species were separated (Peaks 1 and 2 in Figure 6). In addition, a sample impurity lighter in mass by 419 Da was resolved.

Figure 6: HILIC–MS analysis of a lipid conjugated ASO. Column: 50 × 2.1 mm, 1.7-µm 300 Å amide-bonded. (a) Total ion chromatogram from a 50 pmol injection. Mass spectra acquired for (b) Peak 1, (c) Peak 2, and (d) Peak 3. A monoisotopic mass of 6046.11 Da is determined for the main species. An isobaric species is chromatographically resolved as Peak 2, and Peak 3 is determined to be predominately comprised of a 419 Da lighter impurity. Peak 3 Mi = 5,627.09 Da.

Conclusion

It has been demonstrated that HILIC presents a viable and effective alternative to traditional IP-RPLC methods for separating ONs. Through a systematic investigation of various method parameters such as ionic strength, temperature, and gradient steepness/program, optimized conditions have been established that improve selectivity and resolution of ON sequence and length variants. The importance of mobile phase temperature to help analysts prepare for both denaturing and non-denaturing conditions must be highlighted. The use of a bracketed injection effectively mitigates breakthrough effects, allowing for increased sample load without compromising separation quality.

Generic (platform) method conditions are also proposed that can be broadly applied to diverse ON samples. These results emphasize the potential of HILIC for applications in mass spectrometry, where the molecular weights of ONs such as CRISPR sgRNAs and lipid-conjugated ASOs can be quickly determined along with the masses of their impurities.

Continued exploration into the retention mechanisms of ONs in HILIC will further enhance the robustness and applicability of this technique in the rapidly evolving field of oligonucleotide therapeutics.

Acknowledgment

The authors would like to acknowledge the help and support of colleagues from the Consumables and Laboratory Automation business unit at Waters Corporation.

References

(1) Gilar, M.; Koshel, B. M.; Birdsall, R. E. Ion-Pair Reversed-Phase and Hydrophilic Interaction Chromatography Methods for Analysis of Phosphorothioate Oligonucleotides. J. Chromatogr. A 2023, 1712, 464475. DOI: 10.1016/j.chroma.2023.464475

(2) Goyon, A.; Blevins, M. S.; Napolitano, J. G.; et al. Characterization of Antisense Oligonucleotide and Guide Ribonucleic Acid Diastereomers by Hydrophilic Interaction Liquid Chromatography Coupled to Mass Spectrometry. J. Chromatogr. A 2023, 1708, 464327. DOI: 10.1016/j.chroma.2023.464327

(3) Lardeux, H.; Stavenhagen, K.; Paris, C. Unravelling the Link between Oligonucleotide Structure and Diastereomer Separation in Hydrophilic Interaction Chromatography. Anal. Chem. 2024, 96, 9994–10002. DOI: 10.1021/acs.analchem.4c01384

(4) Gilar, M.; Redstone, S.; Gomes, A. Impact of Mobile and Stationary Phases on siRNA Duplex Stability in Liquid Chromatography. J. Chromatogr. A 2024, 1733, 465285. DOI: 10.1016/j.chroma.2024.465285

(5) Lobue, P. A.; Jora, M.; Addepalli, B.; Limbach, P. A. Oligonucleotide Analysis by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry in the Absence of Ion-Pair Reagents. J. Chromatogr. A 2019, 1595, 39–48. DOI: 10.1016/j.chroma.2019.02.016

(6) Anand, P.; Koleto, M.; Kandula, D. R.; Xiong, L.; MacNeill, R. Novel Hydrophilic-Phase Extraction, HILIC and High-Resolution MS Quantification of an RNA Oligonucleotide in Plasma. Bioanal. 2022, 14, 47–62. DOI: 10.4155/bio-2021-0216

(7) Goyon, A.; Scott, B.; Kurita, K.; et al. Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography–High-Resolution Mass Spectrometry Analysis. Anal. Chem. 2021, 93, 14792−14801. DOI: 10.1021/acs.analchem.1c03533

(8) Bobaly, B.; D’Atri, V.; Goyon, A.; et al. Protocols for the Analytical Characterization of Therapeutic Monoclonal Antibodies. II – Enzymatic and Chemical Sample Preparation. J. Chromatogr. B 2017, 1060, 325–335. DOI: 10.1016/j.jchromb.2017.06.036

(9) Bartlett, M. G. Current State of Hydrophilic Interaction Liquid Chromatography of Oligonucleotides. J. Chromatogr. A 2024, 1736, 465378. DOI: 10.1016/j.chroma.2024.465378

(10) Hsiao, J. J.; Bertram, L. J. Systematic Evaluation of HILIC Stationary Phases for MS Characterization of Oligonucleotides. LCGC Intern. 2024, 1 (9), 10–20. DOI: 10.56530/lcgc.int.ij3573k8

(11) Lardeux, H.; D’Atri, V.; Guillarme, D. Recent Advances and Current Challenges in Hydrophilic Interaction Chromatography for the Analysis of Therapeutic Oligonucleotides. TrACs 2024, 176, 117758. DOI: 10.1016/j.trac.2024.117758

(12) Perez-Robles, R.; Fekete, S.; Kormány, R.; Navas, N.; Guillarme, D. Improved Sample Introduction Approach in Hydrophilic Interaction Liquid Chromatography to Avoid Breakthrough of Proteins. J. Chromatogr. A 2024, 1713, 464498. DOI: 10.1016/j.chroma.2023.464498

(13) Jandera, P. Simultaneous Optimisation of Gradient Time, Gradient Shape and Initial Composition of the Mobile Phase in the High-Performance Liquid Chromatography of Homologous and Oligomeric Series. J. Chromatogr. A 1999, 845, 133–144. DOI: 10.1016/S0021-9673(99)00331-3

(14) Wei, Y.; Yao, C.; Zhao, J.; Geng, X. Influences of the Mobile Phase Composition and Temperature on the Retention Behavior of Aromatic Alcohol Homologues in Hydrophobic Interaction Chromatography. Chromatographia 2002, 55, 659–665. DOI: 10.1007/BF02491779

(15) Cusumano, A.; Guillarme, D.; Beck, A.; Fekete, S. Practical Method Development for the Separation of Monoclonal AntiBodies and Antibody-Drug-Conjugate Species in Hydrophobic Interaction Chromatoraphy, Part 2: Optimization of the Phase System. J. Pharm. Biomed. Anal. 2016, 121, 161–173. DOI: 10.1016/j.jpba.2016.01.037

(16) Fekete, S.; Murisier, A.; Lauber, M.; Guillarme, D. Empirical Correction of Non-Linear pH Gradients and a Tool for Application to Protein Ion Exchange Chromatography. J. Chromatogr. A 2021, 1651, 462320. DOI: 10.1016/j.chroma.2021.462320

(17) Bobály, B.; Randazzo, G. M.; Rudaz, S.; Guillarme, D.; Fekete, S. Optimization of Non-Linear Gradient in Hydrophobic Interaction Chromatography for the Analytical Characterization of Antibody-Drug Conjugates. J. Chromatogr. A 2017, 1481, 82–91. DOI: 10.1016/j.chroma.2016.12.047

(18) Fekete, S.; Imiolek, M.; Lauber, M. Waters Application Note, 720008460, August 2024.

About the Authors

Szabolcs Fekete worked in the pharmaceutical industry at analytical R&D for 10 years, then moved to the University of Geneva in Switzerland and worked as a scientific collaborator for a decade. In April 2021, he joined Waters Corporation and now works as a consulting scientist.

Makda Araya is a senior scientist at Waters Corporation. She helps develop tools for separation and characterization of new modalities and sample preparation techniques for bioanalytical DMPK measurements.

Balasubrahmanyam Addepalli is a director at Waters Corporation. He is engaged in the development, evaluation, and application aspects of chromatography columns, enzymes, and other reagents for the characterization of large biomolecules.

Matthew Lauber is a senior director within Waters, where he oversees the development and support of new columns, reagents and enzymes for the analysis of biomolecules.

Koen Sandra is the editor of “Biopharmaceutical Perspectives”. He is the CEO of RIC group (Kortrijk, Belgium) and Visiting Professor at Ghent University (Ghent, Belgium). He is also a member of LCGC International’s editorial advisory board.