Two-dimensional sequential selective comprehensive chiral×reversed-phase liquid chromatography of synthetic phosphorothioate oligonucleotide diastereomers

• Published in: Journal of chromatography. A Vol. 1730; p. 465076
• Main Authors: Li, Feiyang, Knappe, Cornelius, Carstensen, Niklas, Favorat, Enrico, Gao, Mimi, Holkenjans, Wiebke, Hetzel, Terence, Pell, Reinhard, Lämmerhofer, Michael
• Format: Journal Article
• Language: English
• Published: Netherlands 16.08.2024
• Subjects: Chromatography, Reverse-Phase - methods; Phosphorothioate Oligonucleotides - chemistry; Reproducibility of Results; Stereoisomerism; Phosphorothioate oligonucleotides; Chiral stationary phase; Diastereomer separation; Two-dimensional liquid chromatography; Amylose tris(α-methylbenzylcarbamate)
• ISSN: 1873-3778

In recent years, many nucleic acid-based pharmaceuticals have been approved and entered the market, and even a larger number are in late stage clinical trials. Conventional oligonucleotides are facing issues in vivo like fast renal clearance and nuclease degradation. Therefore, to increase their stability, phosphorothioation is a frequent modification of therapeutic oligonucleotides (ONs) which also leads to improved binding affinity facilitating cell internalization and intracellular distribution. At the same time, by replacing a phosphodiester linkage with a phosphorothioate group, a phosphorous stereogenic center is generated which causes the formation of R - and S -diastereomers. It increases the structural diversity. For example, with 15 of those phosphorothioate (PS) linkages, 32,768 different diastereomers are expected. Since the phosphorothioate is introduced non-stereoselectively, the molecular complexity of the resultant phosphorothioate ON products is tremendously increased impeding the chromatographic separation in the course of quality control. Since distinct phosphorothioate diastereomers have different bioactivities and pharmacological properties, there is increasing interest in implications of stereoisomerism of phosphorothiate oligonucleotides. From a quality and regulatory viewpoint, batch-to-batch reproducibility of the diastereomer profile may be of significant concern. In order to address this issue, this study investigates the stereoselectivity of LC methods for two phosphorothioate oligonucleotide (PSO) compounds differing in their molecular size and numbers of PS linkages. Diastereoselectivity of ion-pairing reversed-phase liquid chromatography (IP-RPLC), RPLC without ion-pairing agents and LC with chiral polysaccharide-based column were evaluated for model PSOs and an active pharmaceutical ingredient (API) of PSO with trivalent N-acetylgalactosamine (GalNAc) conjugate. Due to the structural complexity of PSOs, the separation power for the diastereomer mixture was increased by using sequential selective comprehensive two-dimensional chromatography with an amylose tris(α-methylbenzylcarbamate)-immobilized chiral stationary phase (CSP) in the first dimension and ion-pair RPLC with ethylammonium acetate in the second dimension. Improved diastereomer selectivity was obtained and a larger number of peaks could be separated.