Characterization of Antisense Oligonucleotide Impurities by Ion-Pairing Reversed-Phase and Anion Exchange Chromatography Coupled to Hydrophilic Interaction Liquid Chromatography/Mass Spectrometry Using a Versatile Two-Dimensional Liquid Chromatography Setup

Goyon, Alexandre; Zhang, Kelly

doi:10.1021/acs.analchem.0c00114

Cited by 57 publications

(27 citation statements)

References 27 publications

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“…As anticipated in a review published in 2020, 1 several research papers involving the HILIC analysis of oligonucleotides have been recently published. 21,24 The BEH amide column was heated to between 30 and 40 °C, and mobile phases used contained 15−25 mM ammonium acetate. 21,24 Phosphate can interact with stainless-steel surfaces via nonspecific interactions, causing oligonucleotide adsorption and peak shape issues.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…21,24 The BEH amide column was heated to between 30 and 40 °C, and mobile phases used contained 15−25 mM ammonium acetate. 21,24 Phosphate can interact with stainless-steel surfaces via nonspecific interactions, causing oligonucleotide adsorption and peak shape issues. A biocompatible BEH amide column was commercialized in 2021.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Persistent triethylamine contamination can hamper the ionization of compounds analyzed in the positive ion mode, whereas traces of hexafluoro-2-propanol (HFIP) can cause adducts. As anticipated in a review published in 2020, several research papers involving the HILIC analysis of oligonucleotides have been recently published. , The BEH amide column was heated to between 30 and 40 °C, and mobile phases used contained 15–25 mM ammonium acetate. , Phosphate can interact with stainless-steel surfaces via nonspecific interactions, causing oligonucleotide adsorption and peak shape issues. A biocompatible BEH amide column was commercialized in 2021.…”

Section: Resultsmentioning

confidence: 99%

“…Column temperature affects LC performance by modulating the selectivity and mass transfer. A possible oligonucleotide adsorption issue was reported at elevated temperature in two studies published in 2020 and 2021 with a stainless-steel BEH amide column. , Nonspecific interactions between the phosphate groups of oligonucleotide and stainless surfaces are more pronounced under some specific conditions such as acidic pH . However, the novel biocompatible BEH amide column may overcome the pH and temperature limitations observed with the stainless-steel material.…”

Section: Resultsmentioning

confidence: 99%

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Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography–High-Resolution Mass Spectrometry Analysis

Goyon¹,

Scott²,

Kurita³

et al. 2021

Anal. Chem.

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CRISPR/Cas9 is a powerful genome editing approach in which a Cas9 enzyme and a single guide RNA (sgRNA) form a ribonucleoprotein complex effectively targeting site-specific cleavages of DNA. Accurate sequencing of sgRNA is critical to patient safety and is the expectation by regulatory agencies. In this paper, we present the full sequencing of sgRNA via parallel ribonuclease (RNase) T1, A, and U2 digestions and the simultaneous separation and identification of the digestion products by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS). When using RNase T1 digestion alone, a maximal sequence coverage of 81% was obtained excluding the nonunique fragments. Full sgRNA sequencing was achieved using unique fragments generated by RNase T1, A, and U2 parallel digestions. Thorough optimization of sgRNA digestions was performed by varying the nuclease-to-sgRNA ratio, buffer conditions, and reaction times. A biocompatible ethylene-bridged hybrid amide column was evaluated for the separation of RNase digestion products. To our knowledge, it is the first time that (i) RNA digests are separated and identified by HILIC-HRMS and (ii) chemically modified sgRNAs are directly sequenced via a bottom-up approach.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography–High-Resolution Mass Spectrometry Analysis

Goyon¹,

Scott²,

Kurita³

et al. 2021

Anal. Chem.

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“…Generally, the RPLC×RPLC method prefers to separate metabolites with mid to low polarity rather than metabolites with high polarity because of inadequate orthogonality. Compared with RPLC, hydrophilic interaction liquid chromatography (HILIC) performs better separation and retention behavior on most polar metabolites , and is more suitable for analyzing the metabolome . Thus, compared with RPLC×RPLC, HILIC×RPLC provides higher orthogonality, peak capacity, and broader metabolite coverage.…”

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confidence: 99%

Simultaneous Analysis of the Metabolome and Lipidome Using Polarity Partition Two-Dimensional Liquid Chromatography–Mass Spectrometry

et al. 2021

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Comprehensive metabolic profiling is a considerable challenge for systems biology since the metabolites in biological samples have significant polarity differences. A heartcutting two-dimensional liquid chromatography−mass spectrometry (2D-LC−MS) method-based polarity partition was established to analyze both the metabolome and lipidome in a single run. Based on the polarity partition strategy, metabolites with high polarity were retained and separated by one-dimensional hydrophilic chromatography, while low-and medium-polarity lipids were collected into a sample loop and injected into two-dimensional reversed-phase chromatography for separation. A simple online dilution strategy realized the online coupling of the 2D-LC−MS, which effectively solved band broadening and peak distortion caused by solvent incompatibility. Moreover, a dual gradient elution procedure was introduced to further broaden the coverage of low-polarity lipids. The metabolites' log P values, which this 2D-LC−MS method could analyze, ranged from −8.79 to 26.86. The feasibility of the 2D-LC−MS system was demonstrated by simultaneous analysis of the metabolome and lipidome in rat plasma related to depression. A total of 319 metabolites were determined within 40 min, including organic acids, nucleosides, carbohydrate derivatives, amino acids, lipids, and other organic compounds. Finally, 44 depression-related differential metabolites were screened. Compared with conventional LC−MS-based methods, the 2D-LC method covered over 99% of features obtained by two conventional methods. In addition, the selectivity and resolution of the hydrophilic metabolites were improved, and the matrix effects of the hydrophobic metabolites were reduced in the developed method. The results indicated that the established 2D-LC system is a powerful tool for comprehensive metabolomics studies.

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Electrophoretic characterization of LNP/AAV‐encapsulated nucleic acids: Strengths and weaknesses

Hutanu,

Ferreiro,

van Haasteren

et al. 2023

Electrophoresis

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The use of nucleic acids (NAs) has revolutionized medical approaches and ushered in a new era of combating various diseases. Accordingly, there is an increasing demand for accurate identification, localization, quantification, and characterization of NAs encapsulated in nonviral or viral vectors. The vast spectrum of molecular dimensions and intra‐ and intermolecular interactions presents a formidable obstacle for NA analytical development. Typically, the comprehensive analysis of encapsulated NAs, free NAs, and their spatial distribution poses a challenge that is seldom tackled in its complete complexity. The identification of appropriate physicochemical methodologies for large nonencapsulated or encapsulated NAs is particularly intricate and necessitates an evaluation of the analytical outcomes and their appropriateness in addressing critical quality attributes. In this work, we examine the analytics of non‐encapsulated or encapsulated large NAs (>500 nucleotides) utilizing capillary electrophoresis (CE) and liquid chromatography (LC) methodologies such as free zone CE, gel CE, affinity CE, and ion pair high‐performance liquid chromatography (HPLC). These methodologies create a complete picture of the NA's critical quality attributes, including quantity, identity, purity, and content ratio.

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Characterization of Antisense Oligonucleotide Impurities by Ion-Pairing Reversed-Phase and Anion Exchange Chromatography Coupled to Hydrophilic Interaction Liquid Chromatography/Mass Spectrometry Using a Versatile Two-Dimensional Liquid Chromatography Setup

Cited by 57 publications

References 27 publications

Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography–High-Resolution Mass Spectrometry Analysis

Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography–High-Resolution Mass Spectrometry Analysis

Simultaneous Analysis of the Metabolome and Lipidome Using Polarity Partition Two-Dimensional Liquid Chromatography–Mass Spectrometry

Electrophoretic characterization of LNP/AAV‐encapsulated nucleic acids: Strengths and weaknesses

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