Investigating Total Breakthrough Behavior to Improve HILIC×IP-RPLC Analysis of Oligonucleotides

Oligonucleotide therapeutics demand highly resolving analytical methods capable of characterizing complex impurity profiles—something conventional one-dimensional liquid chromatography (1D-LC) often cannot achieve on its own. A study conducted by members of the School of Pharmaceutical Sciences at the University of Geneva (Switzerland) and the University of Lyon (Villeurbanne, France) demonstrated that oligonucleotides can exhibit total breakthrough (TB) behavior under ion-pair reversed-phase liquid chromatography (IP-RPLC) conditions—independent of sequence or size. Their study explores this phenomenon to overcome severe solvent-mismatch issues in comprehensive hydrophilic interaction liquid chromatography (HILIC)×IP-RPLC.

By applying a Design of Experiments (DoE)—guided optimization approach, the authors (1) identified the key factors that control TB when ion-pairing reagents and transferred salts are present, leading to the development of ahigh-resolution two-dimensional liquid chromatography (2D-LC) method for characterizing oligonucleotide impurities. LCGC International spoke to Megane Aebischer of the Institute of Pharmaceutical Sciences of Western Switzerland, lead author of the paper, about this work.

What led you to study TB in oligonucleotide separations, and why is it important?

Our motivation to investigate TB behavior of oligonucleotides came from challenges in impurity profiling of oligonucleotide products. Traditional 1D-LC methods are widely used, but they often lack sufficient separation power for complex impurity profiles. To address this, 2D-LC offers clear advantages by combining orthogonal separation modes, which greatly improve peak capacity. However, comprehensive 2D-LC method development can be difficult, mainly due to solvent compatibility issues between dimensions. While several strategies have been proposed to address these challenges, they often rely on complex instrumental setups involving additional tubing, valves, columns, pumps, or flow splits - which can discourage method development.

By studying the TB behavior of oligonucleotides, we aimed to find a simpler and more practical solution avoiding incompatibility effects, reducing instrumental complexity and making 2D-LC implementation for oligonucleotide analysis more accessible.

Why does ion-pair reversed-phase liquid chromatography (IP-RPLC) remain the gold standard for oligonucleotides characterization, despite the availability of other modes like HILIC andanion-exchange chromatography(AEX)?

IP-RPLC remains the gold standard for oligonucleotides characterization, because it is historically the most established and well-studied separation mode. It provides robust performance and, despite the use of ion-pairing reagent, remains compatible with mass spectrometry (MS), which is essential for oligonucleotide analysis.

AEX is used less frequently, mainly due to its incompatibility with MS, while HILIC represents, from my point of view, a promising alternative for impurity profiling. Although its separation mechanisms are not yet fully understood, it offers good performance and MS compatibility.

From my perspective, each mode has its own strengths, but 2D-LC provides a powerful way to combine the advantages of multiple separation modes within a single method, offering improved resolution for complex oligonucleotide samples.

What challenges arise when combining highly orthogonal modes such as HILIC × IP-RPLC in 2D-LC ?

When combining orthogonal modes such as HILIC × IP-RPLC, differences in elution strength between the ¹D effluent (used as the ²D injection solvent) and the ²D initial mobile phase can cause undesirable breakthrough effects (ranging from slight peak broadening and fronting to severe deformation or even peak splitting with additional peaks). In the most extreme case, a portion of the analyte travels through the column with minimal interaction with the stationary phase, eluting close to the column dead time. These effects are usually amplified with large injection volumes—as typically encountered in 2D-LC—and need to be avoided to preserve optimal separation performance.

Can you briefly explain the concept of total breakthrough (TB) and how it differs from conventional chromatographic retention behavior? How does TB help overcome incompatibility issues?

Typically, when a small injection volume is injected, a symmetrical retained peak is obtained. As the injection volume increases, undesirable breakthrough effects begin to appear. However, for certain compounds like oligonucleotides, further increasing the injection volume leads to the onset of TB behavior. It refers to a chromatographic phenomenon observed when a sufficiently large sample volume is injected. It results in the appearance of a breakthrough peak corresponding to a non-retained analyte, followed by a fully symmetrical retained peak without any distortion, fronting, or tailing.

However, TB is analyte-dependent. For instance, it has been shown that negatively charged small molecules do not exhibit TB but instead show peak distortion or splitting, even at high injection volumes (2). In contrast, for oligonucleotides, the occurrence of TB represents a clear advantage, allowing us to inject large volumes in the second dimension and achieve improved performance in 2D separations.

What role did salts and the ion-pairing reagent play in governing ON retention and the onset of TB behavior?

In a HILIC × IP-RPLC setup, when the ²D injection solvent contains salts (from the HILIC effluent), oligonucleotides tend to associate with these salts. This interaction limits their ability to form ion pairs with the ion-pairing reagent in the IP-RPLC mobile phase, thereby reducing their interaction with the IP-RPLC column. As a result, oligonucleotides are less retained, and the TB phenomenon occurs at smaller injection volumes compared to conditions without salts.

Several other parameters affect the TB occurrence. For this reason, a major part of our study focused on evaluating how key parameters (such as salt or ion-pairing reagent concentrations) influence the occurrence of TB, to identify the conditions that promote this behavior.

How did the DoE approach contribute to understanding and optimizing TB conditions in your 2D-LC setup?

The DoE approach provided a deeper understanding of TB behavior and allow to minimize the number of experiments required to explore an unknown phenomenon. For example, it revealed how injected fraction parameters influence this phenomenon and enable the identification of optimal conditions to promote TB. The method development was greatly facilitated by this systematic approach as it guided the optimization of the 2D-LC method.

What were the most surprising findings regarding TB in oligonucleotides compared to previously studied small molecules and peptides?

In previous studies, negatively charged molecules did not exhibit TB. They only showed peak distortion, even at high injection volumes. The ability of a compound to display TB behavior was therefore hypothesized to be related to its charge. Surprisingly, oligonucleotides, which also carry a net negative charge, displayed clear TB behavior. Initially, we considered whether this could be due to the presence of the ion-pairing reagent, but our results suggested something else. We ultimately hypothesized that the TB behavior of oligonucleotides is related to their specific on-off retention mechanism, which differs fundamentally from the retention mechanisms observed for smaller molecules.

How does exploiting TB improve the resolution, peak shape, and sensitivity of impurity profiling in oligonucleotide analysis?

Exploiting TB behavior removes solvent compatibility constraints in 2D-LC, allowing large ²D-injection volumes. For example, when developing a comprehensive 2D-LC method, the sampling time can be extended (up to the loop capacity), enabling longer 2D gradients and consequently improving resolution without compromising peak shape.

However, the main limitation is sensitivity, as a portion of the oligonucleotide elutes in the breakthrough peak near the column dead time and is therefore not detected in the retained peak. Thus, careful method optimization is required to fully exploit TB, while maintaining sufficient analytical sensitivity.

What practical advantages does the TB strategy offer over traditional approaches like solvent modulation or trapping columns for 2D-LC compatibility?

Traditional strategies to improve interdimensional compatibility in 2D-LC often require modifications of the instrumentation, such as active solvent modulation valve, trapping columns, or additional pumps. In contrast, the TB strategy offers a simpler and more practical alternative, as it eliminates the need for such specialized hardware. This makes method development easier, faster, and more accessible.

How do you envision TB-based 2D-LC methods influencing future analytical workflows for oligonucleotide and biotherapeutic development?

I believe that the TB phenomenon offers a major advantage for oligonucleotide impurity profiling. It is particularly valuable for comprehensive 2D-LC, which remains one of the most challenging approaches due to the strong interdependence of optimization parameters leading to numerous constraints. Since only a few studies have reported comprehensive 2D-LC methods for oligonucleotides, reducing compatibility constraints using TB strategy represents an important step toward making 2D-LC method development more accessible and practical for oligonucleotides analysis. Given its strong potential to substantially increase resolution, this approach could facilitate the characterization of newly developed biopharmaceuticals of increasing structural complexity.

References

1. Aebischer, M. K.; Abdurahman, Y.; Heinisch, S. et al. Investigating the Total Breakthrough Behavior of Oligonucleotides in Ion-Pairing Chromatography and Comprehensive HILIC × IP-RPLC. J. Chromatogr. A 2025, 1765, 466486. DOI: 10.1016/j.chroma.2025.466486
2. Chapel, S.; Rouvière, F.; Peppermans, V.; Desmet, G.; Heinisch, S. A Comprehensive Study on the Phenomenon of Total Breakthrough in Liquid Chromatography. J. Chromatogr. A 2021, 1653, 462399. https://doi.org/10.1016/j.chroma.2021.462399