Separation of oligonucleotide phosphorothioate diastereoisomers by pellicular anion-exchange chromatography

Thayer, James R.; Wu, Yansheng; Hansen, Erik W.; Angelino, Mark D.; Rao, S. Narasimha

doi:10.1016/j.chroma.2010.12.051

Cited by 47 publications

(27 citation statements)

References 32 publications

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“…Comparison of IP-RP-HPLC to CGE and IEC Three major modes with high resolution capability were widely used for separation of phosphorothioate ODNs by CGE [29,30], IEC [31,32] and IP-RP-HPLC [33,34]. In this study, there was not any improvement in the separation of crude 5′C 22 -Flu by CGE and IEC methods compared with previous optimal IP-RP-HPLC (Fig.…”

Section: Adjustment Of Gradient and Application Of Ip-rp-hplccontrasting

confidence: 51%

The analysis of major impurities of lipophilic-conjugated phosphorothioate oligonucleotides by ion-pair reversed-phase HPLC combined with MALDI-TOF-MS

Liu

Deng

et al. 2012

Anal Bioanal Chem

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A simple and rapid ion-pair reversed phase high-performance liquid chromatography (IP-RP-HPLC) method was developed to analyse the major impurities of lipophilic-conjugated phosphorothioate oligonucleotides (ODNs), which provided better separation performance than capillary gel electrophoresis and ion exchange chromatograph methods. The study showed that covalent conjugations of lipophilic group (docosanyl, C(22)) to ODN at 5'-termini (denoted as 5'C(22)-Flu) or 3'-termini (denoted as 3'C(22)-Flu) exhibited similar chromatographic retention behavior. Some important analytical conditions of IP-RP-HPLC, including column type, ion-pairing buffer composition, and separation temperature, were investigated for the effects on the separation of crude 5'C(22)-Flu. As expected, the method developed was successfully applied to the analysis of crude 3'C(22)-Flu and both purified products. Furthermore, the related impurities derived from the synthetic process were identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrum. These MS results are of benefit to understanding the major process-related impurities in lipophilic-ODN conjugates synthesis, thereby elevating the quality of target products.

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Section: Adjustment Of Gradient and Application Of Ip-rp-hplccontrasting

confidence: 51%

The analysis of major impurities of lipophilic-conjugated phosphorothioate oligonucleotides by ion-pair reversed-phase HPLC combined with MALDI-TOF-MS

Liu

Deng

et al. 2012

Anal Bioanal Chem

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“…Further chromatographic analyses were performed after thermal denaturation (unfolding) followed by a slow cooling to room temperature (refolding), in order to assess if the two peaks were distinct folding conformations. 4,17 Indeed, if the native structures are different conformers, the same initial pattern must be observed after a denaturation/refolding process. Elution in denaturing conditions showed a total disappearance of native elution profile with a single structure detected at longer retention time (red line in Figure 1a ) in keeping with the increased number of charges exposed by unfolded oligonucleotide.…”

Section: Resultsmentioning

confidence: 99%

Two Interconvertible Folds Modulate the Activity of a DNA Aptamer Against Transferrin Receptor

Porciani

Signore

Marchetti

et al. 2014

Molecular Therapy - Nucleic Acids

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Thanks to their ability to recognize biomolecular targets with high affinity and specificity, nucleic acid aptamers are increasingly investigated as diagnostic and therapeutic tools, particularly when their targets are cell-surface receptors. Here, we investigate the relationship between the folding of an anti-mouse transferrin receptor DNA aptamer and its interaction with the transferrin receptor both in vitro and in living cells. We identified and purified two aptamer conformers by means of chromatographic techniques. Fluorescence-anisotropy measurements showed that only one fold is able to bind mouse transferrin receptor. Besides displaying enhanced endocytosis in living mouse fibroblasts, the purified active fold is internalized also in human pancreatic cancer cells. Starting from these observations, we rationally designed variations of the parent sequence aimed at stabilizing the active fold, and consequently increase aptamer activity. A truncated version and full-length mutants with higher affinity than the parent sequence are shown.

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“…The mixture of diastereoisomers is in form of Sp and Rp (Figure a), which have the same length, sequence, mass and charge (Eckstein, ; Thayer, Wu, Hansen, Angelino, & Rao, ) and are formed generally with a constant ratio (Almer, Stawinski, Stroemberg, & Thelin, ). Consequently, PS n ‐mer oligonucleotides may be present as a mixture of 2 ( n − 1) diastereomers (where n is the length of the oligonucleotide).…”

Section: Chirality Of Ps Oligonucleotidesmentioning

confidence: 99%

Chromatographic approaches for the characterization and quality control of therapeutic oligonucleotide impurities

Zahar

Magdy

El-Kosasy

et al. 2017

Biomedical Chromatography

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Phosphorothioate (PS) oligonucleotides are a rapidly rising class of drugs with significant therapeutic applications. However, owing to their complex structure and multistep synthesis and purification processes, generation of low-level impurities and degradation products are common. Therefore, they require significant investment in quality control and impurity identification. This requires the development of advanced methods for analysis, characterization and quantitation. In addition, the presence of the PS linkage leads to the formation of chiral centers which can affect their biological properties and therapeutic efficiency. In this review, the different types of oligonucleotide impurities and degradation products, with an emphasis on their origin, mechanism of formation and methods to reduce, prevent or even eliminate their production, will be extensively discussed. This review will focus mainly on the application of chromatographic techniques to determine these impurities but will also discuss other approaches such as mass spectrometry, capillary electrophoresis and nuclear magnetic resonance spectroscopy. Finally, the chirality and formation of diastereomer mixtures of PS oligonucleotides will be covered as well as approaches used for their characterization and the application for the development of stereochemically-controlled PS oligonucleotides.

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Separation of oligonucleotide phosphorothioate diastereoisomers by pellicular anion-exchange chromatography

Cited by 47 publications

References 32 publications

The analysis of major impurities of lipophilic-conjugated phosphorothioate oligonucleotides by ion-pair reversed-phase HPLC combined with MALDI-TOF-MS

The analysis of major impurities of lipophilic-conjugated phosphorothioate oligonucleotides by ion-pair reversed-phase HPLC combined with MALDI-TOF-MS

Two Interconvertible Folds Modulate the Activity of a DNA Aptamer Against Transferrin Receptor

Chromatographic approaches for the characterization and quality control of therapeutic oligonucleotide impurities

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