Investigating the total breakthrough behavior of oligonucleotides in ion-pairing chromatography and comprehensive
Megane K Aebischer1, Yusraa Abdurahman1, Sabine Heinisch2
1School of Pharmaceutical Sciences, University of Geneva, CMU - Rue Michel Servet 1, 1211 Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel Servet 1, 1211 Geneva 4, Switzerland.
Abstract:
Oligonucleotides (ONs) have gained significant attention as therapeutic agents, due to their ability to selectively target genetic sequences. As ON-based drug development expands, analytical methods are essential to ensure quality and purity. While ion pairing reversed phase liquid chromatography (IP-RPLC) is the gold standard for ON impurities characterization, alternative chromatographic modes are increasingly used. However, fully resolving all impurities with a single one-dimensional method remains challenging. Comprehensive two-dimensional liquid chromatography offers improved impurity profiling by combining orthogonal separations. However, differences in elution strength between the fraction collected from the first dimension (1D) and the mobile phase used in the second dimension (2D) can cause peak distortion and breakthrough phenomena. Interestingly, under certain conditions, these distortion effects can disappear, yielding a breakthrough peak accompanied by a symmetrical retained peak. This phenomenon, referred to as "total breakthrough," was recently described in 2D-LC separations of small molecules and peptides. Although the underlying mechanisms are not yet fully elucidated, the total breakthrough (TB) strategy provides an advantage in 2D-LC, by enabling the use of large injection volumes, effectively overcoming injection-related issues commonly caused by solvent incompatibility between dimensions. In this study, we first demonstrated that ONs exhibit TB behavior in IP-RPLC. Upon confirming this behavior, we selected a 2D HILIC × IP-RPLC configuration (a combination often considered challenging due to limited solvent compatibility) and performed an in-depth investigation of both 1D HILIC collected fraction and 2D IP-RPLC conditions affecting breakthrough behavior. We found that understanding TB of ONs in IP-RPLC is more complex than with classical small molecules, primarily due to the interplay between ion-pairing agents in the 2D -mobile phase and residual salts originating from 1D Through careful optimization, we successfully established a comprehensive HILIC × IP-RPLC method that enabled analysis of ONs mixture, taking advantage of the TB behavior of ONs, while preserving sufficient sensitivity.
Insights
Oligonucleotide (ON) analysis benefits from total breakthrough (TB) in 2D-LC. This study demonstrates TB in HILIC × IP-RPLC, enabling sensitive impurity profiling of ON mixtures.
Area of Science:
- Analytical Chemistry
- Chromatography
- Oligonucleotide Therapeutics
Background:
- Oligonucleotides (ONs) are crucial therapeutic agents requiring robust analytical methods for quality control.
- Ion pairing reversed-phase liquid chromatography (IP-RPLC) is standard for ON impurity analysis, but one-dimensional methods face resolution challenges.
- Comprehensive two-dimensional liquid chromatography (2D-LC) enhances impurity profiling but faces solvent incompatibility issues between dimensions.
Purpose of the Study:
- To investigate the phenomenon of total breakthrough (TB) in oligonucleotide separations using 2D-LC.
- To establish a HILIC × IP-RPLC method optimized for ON impurity analysis by leveraging TB behavior.
- To overcome injection volume limitations and solvent incompatibility challenges in ON analysis.
Main Methods:
- Demonstration of TB behavior in Oligonucleotides (ONs) within IP-RPLC.
- Development and optimization of a two-dimensional HILIC × IP-RPLC method.
- In-depth investigation of 1D HILIC fraction and 2D IP-RPLC conditions influencing breakthrough.
Main Results:
- Oligonucleotides exhibit total breakthrough (TB) behavior in IP-RPLC, a phenomenon previously observed in small molecules and peptides.
- The HILIC × IP-RPLC method successfully utilized TB for ON analysis, overcoming common solvent incompatibility issues.
- Understanding TB in ONs is complex due to ion-pairing agents and residual salts, but optimization yielded a sensitive method.
Conclusions:
- Total breakthrough (TB) is a viable strategy for enhancing oligonucleotide analysis in 2D-LC, specifically within HILIC × IP-RPLC configurations.
- The developed method allows for sensitive impurity profiling of oligonucleotide mixtures by exploiting TB.
- This approach addresses key challenges in ON analysis, improving quality control for therapeutic development.
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