Analytical techniques for characterizing diastereomers of phosphorothioated oligonucleotides

Chen, Tao; Tang, Shijia; Fu, Yige; Napolitano, José G.; Zhang, Kelly

doi:10.1016/j.chroma.2022.463349

Cited by 17 publications

(7 citation statements)

References 116 publications

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“…ASOs are an emerging class of oligonucleotide therapeutics, and to date, 9 ASO-based therapeutics have been approved by FDA with 5 approved in the last 4 years. 30 Chromatographic analysis of ASOs is critical to process development, product quality, and safety yet challenging given that an array of closely related impurities can form such as short-mers, long-mers, incomplete sulfurization, stereoisomers, and impurities from side reactions with the reagents. 31 Ion-pair reversed phase (IPRP), AEX, mixed-mode chromatography (MMC), and SEC are the chromatography technologies commonly used to analyze ASOs, each exploiting unique characteristics of these compounds.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Resolving Solvent Incompatibility in Two-Dimensional Liquid Chromatography with In-Line Mixing Modulation

Tang¹,

Venkatramani²

2022

Anal. Chem.

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Two-dimensional liquid chromatography (2D-LC) is a powerful technique used to characterize complex samples such as synthetic polymers, biomacromolecules, and hybrid modalities (conjugates, oligonucleotides, nanoparticles, etc., which fall between traditional small molecules and large molecules). Characterizing such molecules often requires a highly orthogonal 2D-LC workflow to resolve structurally similar impurities. However, it remains a challenge to achieve truly orthogonal 2D-LC coupling due to incompatibility of the chromatographic conditions used in each dimension. In this work, we present a facile strategy of connecting an in-line mixer, in-line mixing modulation (ILMM), to realize challenging 2D-LC workflows: (1) coupling gel permeation chromatography (GPC) with reversed-phase liquid chromatography (RPLC) for hydrophobic oligomer analysis and (2) coupling ion-pair reversed-phase (IPRP) with hydrophilic interaction liquid chromatography (HILIC) for polar antisense oligonucleotide (ASO) analysis. Compared with the state-of-the-art commercially available active solvent modulation (ASM), engaging the ILMM significantly reduces the peak distortion in GPC-RPLC, allowing an at least 67% higher transfer volume from the primary to secondary dimension, and resolves the ASO sample breakthrough in selective comprehensive IPRP×HILIC. Also remarkably, ILMM demonstrated superiority in comprehensive RPLC×RPLC analysis in comparison with ASM, suggesting its potential in broader 2D-LC applications. In addition to chromatography improvement, ILMM offers several advantages over benchmark modulation approaches in regard to alleviating the need of an additional dilution flow and a simple as well as flexible system configuration, opening many opportunities to establish innovative and versatile multidimensional workflows for characterizing compounds with increasing complexity.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Resolving Solvent Incompatibility in Two-Dimensional Liquid Chromatography with In-Line Mixing Modulation

Tang¹,

Venkatramani²

2022

Anal. Chem.

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“…However, challenges emerge when dealing with highly PS-modified ONs, such as ASOs, as chromatographic peak broadening becomes a complicating factor (Figure S5B). Therefore, as also implemented for IP-RPLC-based methods, 17 strategies to suppress the diaster- eomer selectivity of ONs in HILIC are needed, particularly because studies using this chromatographic mode for ON analysis are barely emerging. ACN is commonly used as the organic modifier in HILIC.…”

Section: On Sequences Favoring Rigid Hoss Provide Better Chromatograp...mentioning

confidence: 99%

“…Among the liquid chromatography (LC)-based techniques used for characterizing PS-modified ONs, anion-exchange chromatography and ion-pairing reversed-phase LC (IP-RPLC) have demonstrated significant potential for (partially) separating ON diastereomers. − Additional complementary methods for studying diastereomers in PS-modified ONs are capillary electrophoresis using chiral selectors, ion mobility spectrometry, and spectroscopic methods [e.g., 31 P nuclear magnetic resonance (NMR) and circular dichroism]. − …”

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confidence: 99%

Unravelling the Link between Oligonucleotide Structure and Diastereomer Separation in Hydrophilic Interaction Chromatography

Lardeux,

Stavenhagen,

Paris

et al. 2024

Anal. Chem.

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“…For that reason, new analytical techniques are needed for their separation and quality control. A survey of various analytical methods for characterizing diastereomers of phosphorothioated oligonucleotides has recently been published [6]. Diastereomers are molecules that contain at least two chiral centers and are not mirror images of each other.…”

Section: Introductionmentioning

confidence: 99%

The separation of cyclic diadenosine diphosphorothioate and the diastereomers of its difluorinated derivative and the estimation of the binding constants and ionic mobilities of their complexes with 2‐hydroxypropyl‐β‐cyclodextrin by affinity capillary electrophoresis

Štěpánová,

Břehová,

Kašička

2024

Electrophoresis

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The incorporation of phosphorothioate linkages has recently been extensively employed in therapeutic oligonucleotides. For their separation and quality control, new high‐efficient and high‐sensitive analytical methods are needed. In this work, a new affinity capillary electrophoresis method has been developed and applied for the separation of a potential anticancer drug, 2′,3′‐cyclic diadenosine diphosphorothioate (Rp, Rp) (ADU‐S100), and three recently newly synthesized diastereomers of its difluorinated derivative, 3′,3′‐cyclic di(2′‐fluoro, 2′‐deoxyadenosine phosphorothioate). The separation was performed in the various background electrolytes (BGEs) within a pH range 5–9 using several native and derivatized cyclodextrins (CDs) as chiral additives of the BGE. Relatively good separations were obtained with β‐, γ‐, and 2‐hydroxypropyl‐γ‐CDs in some of the BGEs tested. However, the best separation was achieved using the 2‐hydroxypropyl‐β‐CD chiral selector at 43.5 mM average concentration in the BGE composed of 40 mM Tris, 40 mM tricine, pH 8.1. Under these conditions, all the previous four cyclic dinucleotides (CDNs) were baseline separated within 4 min. Additionally, the average apparent binding constants and the average actual ionic mobilities of the complexes of all four CDNs with 2‐hydroxypropyl‐β‐CD in the above BGE were determined. The formed complexes were found to be relatively weak, with the average apparent binding constants in the range of 12.2–94.1 L mol−1 and with the actual ionic mobilities spanning the interval (−7.8 to −12.7) × 10−9 m2 V−1 s−1. The developed method can be applied for the separation, analysis, and characterization of the above and similar CDNs.

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Analytical techniques for characterizing diastereomers of phosphorothioated oligonucleotides

Cited by 17 publications

References 116 publications

Resolving Solvent Incompatibility in Two-Dimensional Liquid Chromatography with In-Line Mixing Modulation

Resolving Solvent Incompatibility in Two-Dimensional Liquid Chromatography with In-Line Mixing Modulation

Unravelling the Link between Oligonucleotide Structure and Diastereomer Separation in Hydrophilic Interaction Chromatography

The separation of cyclic diadenosine diphosphorothioate and the diastereomers of its difluorinated derivative and the estimation of the binding constants and ionic mobilities of their complexes with 2‐hydroxypropyl‐β‐cyclodextrin by affinity capillary electrophoresis

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