Anion exchange chromatography of oligonucleotides under denaturing conditions

Sun, Jinyu; Dhruval, Joshi; Betancourt, Frank; Solodinin, Andrei; Woodland, Breyer; Yan, Hongbin

doi:10.1080/15257770.2019.1706096

Cited by 7 publications

(6 citation statements)

References 14 publications

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“…With the aid of CD and melting experiments, different denaturing conditions were qualitatively investigated for their ability to destabilize the HOS at an oligonucleotide concentration of 80 mg/mL. An increase in organic modifiers, pH, temperature, and chaotropic salts has been applied successfully for denaturation purposes . However, the situation might be different here considering that additives used to destabilize duplexes might have an opposite stabilization effect on HOSs such as G-quadruplexes.…”

Section: Resultsmentioning

confidence: 99%

Identification and Impact of On-Column Higher-Order Structure Formation during Anion Exchange Purification of Oligonucleotides

Verheyen,

Carloni,

Deschrijver

et al. 2024

Org. Process Res. Dev.

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During large-scale anion exchange purification of a guanosine-rich oligonucleotide sequence, a 10% yield loss was observed. The elution profile showed an unwanted peak in the wash step of the gradient and was suspected as the cause of the specific loss. The primary oligonucleotide structure and the high-salt conditions hinted toward the formation of a higher-order structure, which, due to its higher charge, would be retained more than the oligonucleotide product. Higher-order structures comprise the secondary and tertiary structures that oligonucleotides can form and are driven by noncovalent interactions which depend on the oligonucleotide primary structure and various environmental factors. Analytical experiments focusing on secondary and tertiary structure characterization, including circular dichroism and melting temperature, identified an unstable higher-order structure which was specifically stabilized on the column and was not stable once eluted in solution. We herein describe how the anion exchange purification of this guanosine-rich oligonucleotide sequence was redeveloped with support of analytical characterizations to limit the higher-order structure formation and increase final product recovery. To this end, different denaturing conditions were screened by circular dichroism experiments, providing input for purification conditions at a semipreparative scale. Different factors including pH, organic modifiers, denaturing agents, and salt were hereby evaluated. A selection of conditions was further upscaled to additionally evaluate the impact of concentration on the higher-order structure formation. Two conditions were identified that resolved the on-column higher-order structure formation and solved the initial observed yield loss.

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Section: Resultsmentioning

confidence: 99%

Identification and Impact of On-Column Higher-Order Structure Formation during Anion Exchange Purification of Oligonucleotides

Verheyen,

Carloni,

Deschrijver

et al. 2024

Org. Process Res. Dev.

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“…This increases the selectivity but, in some cases, reduces the sensitivity [ 6 ]. Similar separation selectivity may be obtained in ion-exchange chromatography, where the retention is based on electrostatic interactions between the negatively charged oligonucleotide and positively charged stationary phase surface [ 7 , 8 , 9 , 10 , 11 ]. The utilization of anion-exchange chromatography allows an elevated temperature or pH to be employed to control oligonucleotides’ separation selectivity by producing fully or partially denaturing conditions [ 8 ].…”

Section: Introductionmentioning

confidence: 85%

“…Separation techniques are mainly used to separate and determine oligonucleotides [ 5 , 6 ]. The two most popular analytical tools are ion-pair liquid chromatography and ion-exchange chromatography [ 6 , 7 , 8 ]. In the first technique, the amines are used as mobile phase additives.…”

Section: Introductionmentioning

confidence: 99%

“…Adsorbents with quaternary ammonium ligands are widely used as stationary phases. Different lengths of alkyl chains can be bounded to the quaternary nitrogen atom—usually short ones such as trimethylamine or diethylmethylamine [ 8 , 9 , 10 , 11 ]. They were successfully applied to separate unmodified, single-stranded OGNs and modified oligonucleotides but some disadvantages were noticed—for example, a lengthy analysis time or insufficient separation [ 9 , 10 , 11 ].…”

Section: Introductionmentioning

confidence: 99%

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Dendrimer Anion-Exchange Stationary Phase for Separation of Oligonucleotides

et al. 2022

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Oligonucleotides are used in many research areas. Thus, there is a need for their successful separation methods. Ion-exchange chromatography is the most popular separation technique, but it has limitations for these compounds. For this reason, new stationary phases are developed in order to increase separation selectivity. This study aimed to apply a series of dendrimer anion exchangers with various bonded layers to separate oligonucleotides by using different mobile phases to find conditions that allow sufficient separation. The number of anion-exchange layers, type of salt, and pH significantly impacted the oligonucleotide analysis. The developed chromatographic method was characterized by adequate selectivity for oligonucleotides differing in sequence length. It is essential to underline that the number of bonded layers appeared to have a significant influence, and the three layers appeared optimal. Based on our results, it may be concluded that the dendrimer stationary phases can be successfully used as an alternative to commonly applied packing materials in ion-exchange chromatography.

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“…Strong AEX (Mono Q) FPLC for transcript purification and in the studies of transcription reactions has also been demonstrated ( Koubek et al, 2013 ). Analysis of oligonucleotides (OGNs) and RNA under denaturing conditions in conjunction with AEX HPLC has been performed in a variety of different systems in an approach to remove secondary/tertiary structures in OGNs and RNA ( Thayer et al, 2005 ; Cook and Thayer, 2011 ; Kanavarioti, 2019 ; Sun et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Anion exchange HPLC monitoring of mRNA in vitro transcription reactions to support mRNA manufacturing process development

Welbourne,

Loveday,

Nair

et al. 2024

Front. Mol. Biosci.

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mRNA technology has recently demonstrated the ability to significantly change the timeline for developing and delivering a new vaccine from years to months. The potential of mRNA technology for rapid vaccine development has recently been highlighted by the successful development and approval of two mRNA vaccines for COVID-19. Importantly, this RNA-based approach holds promise for treatments beyond vaccines and infectious diseases, e.g., treatments for cancer, metabolic disorders, cardiovascular conditions, and autoimmune diseases. There is currently significant demand for the development of improved manufacturing processes for the production of mRNA therapeutics in an effort to increase their yield and quality. The development of suitable analytical methods for the analysis of mRNA therapeutics is critical to underpin manufacturing development and the characterisation of the drug product and drug substance. In this study we have developed a high-throughput, high-performance liquid chromatography (HPLC) workflow for the rapid analysis of mRNA generated using in vitro transcription (IVT). We have optimised anion exchange (AEX) HPLC for the analysis of mRNA directly from IVT. Chromatography was performed in under 6 min enabling separation of all of the key components in the IVT, including nucleoside triphosphates (NTPs), Cap analogue, plasmid DNA and mRNA product. Moreover, baseline separation of the NTPs was achieved, which facilitates accurate quantification of each NTP such that their consumption may be determined during IVT reactions. Furthermore, the HPLC method was used to rapidly assess the purification of the mRNA product, including removal of NTPs/Cap analogue and other contaminants after downstream purification, including solid phase extraction (SPE), oligo deoxythymidine (oligo-dT) affinity chromatography and tangential flow filtration (TFF). Using the developed method excellent precision was obtained with calibration curves for an external mRNA standard and NTPs giving correlation coefficients of 0.98 and 1.0 respectively. Intra- and inter-day studies on retention time stability of NTPs, showed a relative standard deviation ≤ 0.3% and ≤1.5% respectively. The mRNA retention time variability was ≤0.13%. This method was then utilised to monitor the progress of an IVT reaction for the production of Covid spike protein (C-Spike) mRNA to measure the increasing yield of mRNA alongside the consumption of NTPs during the reaction.

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Anion exchange chromatography of oligonucleotides under denaturing conditions

Cited by 7 publications

References 14 publications

Identification and Impact of On-Column Higher-Order Structure Formation during Anion Exchange Purification of Oligonucleotides

Identification and Impact of On-Column Higher-Order Structure Formation during Anion Exchange Purification of Oligonucleotides

Dendrimer Anion-Exchange Stationary Phase for Separation of Oligonucleotides

Anion exchange HPLC monitoring of mRNA in vitro transcription reactions to support mRNA manufacturing process development

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