Oligonucleotide separation techniques for purification and analysis: What can we learn for today's tasks?

Abstract

Nucleic acids are the blueprint of life. They are not only the construction plan of the single cell or higher associations of them, but also necessary for function, communication and regulation. Due to the pandemic, the attention shifted in particular to their therapeutic potential as a vaccine. As pharmaceutical oligonucleotides are unique in terms of their stability and application, special delivery systems were also considered. Oligonucleotide production systems can vary and depend on the feasibility, availability, price and intended application. To achieve good purity, reliable results and match the strict specifications in the pharmaceutical industry, the separation of oligonucleotides is always essential. Besides the separation required for production, additional and specifically different separation techniques are needed for analysis to determine if the product complies with the designated specifications. After a short introduction to ribonucleic acids (RNAs), messenger RNA vaccines, and their production and delivery systems, an overview regarding separation techniques will be provided. This not only emphasises electrophoretic separations but also includes spin columns, extractions, precipitations, magnetic nanoparticles and several chromatographic separation principles, such as ion exchange chromatography, ion-pair reversed-phase, size exclusion and affinity.

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

“…or oligonucleotides [446]). Beyond the use of non-specific hydrophobic (C 18 [447] or carbon-based materials [165,448]) or electrostatic interactions [449,450], perhaps the most significant advancements reported involve the use of much smaller volumes (typically just a few µL) of both sample and elution [451] and the application of custom materials [452] that can give some selectivity prior to the separation.…”

“…One of the simplest (and most widely used) sample pretreatment procedures (for clean‐up and preconcentration) prior to CE is off‐line solid‐phase extraction (SPE). This concept has been extensively reviewed considering the material used [436–440] or the target analytes (i.e., parabens [441], peptides [442], natural products [443], drugs of abuse [444], pollutants [445], organic acids [435], or oligonucleotides [446]). Beyond the use of non‐specific hydrophobic (C 18 [447] or carbon‐based materials [165, 448]) or electrostatic interactions [449, 450], perhaps the most significant advancements reported involve the use of much smaller volumes (typically just a few µL) of both sample and elution [451] and the application of custom materials [452] that can give some selectivity prior to the separation.…”

Improvement of oligonucleotide separation using a repetto high‐performance liquid chromatography recycling approach

“…Separation of biologically important substances such as proteins, peptides, and nucleic acids, has become an important area of application for various modes of highperformance liquid chromatography (HPLC) in recent years [1]. One of the key applications in nucleic acid analysis has recently emerged as chromatography has taken a dominant position not only for preparative isolation but also for characterization in synthetic oligonucleotide quality control (QC) [2,3]. Oligonucleotides play a critical role in a variety of molecular methods including poly-Article Related Abbreviations: TEAA, Triethylammonium acetate; RNAi, RNA interference.…”

Two quality and stability indicating imaged CIEF methods for mRNA vaccines

“…Separation methods for the analysis of mRNA in various contexts have been recently reviewed by Minkner et al. [20]. Several chromatographic separation principles, such as ion exchange, ion‐pair reversed‐phase, size exclusion and affinity, and capillary electrophoretic approaches such as capillary zone electrophoresis, pulsed‐field capillary electrophoresis, capillary gel electrophoresis, and capillary isoelectric focusing (CIEF), have been highlighted, furthermore discussing spin columns, extractions, precipitations, and magnetic nanoparticles [20].…”