Influence of oligonucleotides structures for separation of diastereomers by capillary electrophoresis method using polyvinylpyrrolidone 1,300,000.

In the field of oligonucleotides drug discovery, phosphorothioate (PS) modification has been recognized as an effective tool to overcome the nuclease digestion, and generates 2n of possible diastereomers, where n equals the number of PS linkages. However, it is also well known that differences in drug efficacy and toxicity are caused by differences in stereochemistry of oligonucleotides. Therefore, the development of a high-resolution analytical method that enables stereo discrimination of oligonucleotides is desired. Under this circumstance, capillary electrophoresis (CE) using polyvinylpyrrolidone (PVP) is considered as one of the useful tools for the separation analysis of diastereomers. In this study, we evaluated the several oligonucleotides with the structural diversities in order to understand the separation mechanism of the diastereomers by CE. Especially, five kinds of 2'-moieties were deeply examined by CE with PVP 1,300,000 polymer solution. We found that different trend of the peak shapes and the peak resolution were observed among these oligonucleotides. For example, the better peak resolution was observed in 6 mer PS3-DNA compared to the rigid structure of 6 mer PS3-LNA. As for this reason, the computational simulation revealed that difference of accessible surface area caused by the steric structure of thiophosphate in each oligonucleotide is one of the key attributes to explain the separation of the diastereomers. In addition, we achieved the separation of sixteen peak tops of the diastereomers in 6 mer PS4-DNA, and the complete separation of fifteen diastereomers in 6 mer PS4-RNA. These knowledge for the separation of the diastereomers by CE will be expected to the quality control of the oligonucleotide drugs.

Optimization of elution conditions and comparison of emerging biocompatible columns on the resolving power and detection sensitivity of oligonucleotides by ion-pairing reversed-phase liquid chromatography mass spectrometry.

A generic performance comparison strategy has been developed to evaluate the impact of mobile-phase additives (ion-pairing agent / counter ion systems), distinct stationary phases on resulting resolving power, and MS detectability of oligonucleotides and their critical impurities in gradient IP-RPLC. Stationary-phase considerations included particle type (core-shell vs. fully porous particles), particle diameter, and pore size. Separations were carried out at 60°C to optimize mass transfer (C-term). The incorporation of an active column preheater mitigated thermal mismatches, leading to narrower peaks and overcoming peak splitting. Acetonitrile as organic modifier outweighed methanol in terms of peak-capacity generation and yielded a 30% lower back pressure. Performance screening experiments were conducted varying ion-pairing agents and counter ions, while adjusting gradient span achieved an equivalent effective retention window. Hexafluoromethylisopropanol yielded superior chromatographic resolution, whereas hexafluoroisopropanol yielded significantly higher MS detection sensitivity. The 1.7 µm core-shell particle columns with 100 Å pores provided maximum resolving power for small (15-35 mers) oligonucleotides. Sub-min analysis for 15-35 polyT ladders was achieved operating a 50 mm long column at the kinetic performance limits. High-resolution separations between a 21-mer modified RNA sequence oligonucleotides and its related (shortmer and phosphodiester) impurities and complementary strand were obtained using a coupled column set-up with a total length of 450 mm.

Integrated Electrochemical Aptasensor Array toward Monitoring Anticancer Drugs in Sweat.

In the realm of clinical practice, the concurrent utilization of anticancer medications can enhance their overall therapeutic efficacy. However, it is crucial to acknowledge that the interactions among these anticancer drugs can potentially yield detrimental consequences on their intended outcomes. Consequently, the assessment of both anticancer potency and potential toxic side effects is greatly refined when multiple anticancer drugs are simultaneously detected and evaluated. Here, we designed a wearable electrochemical aptasensor array for monitoring multiple anticancer drugs in sweat. The integrated sensor array consists of three working electrodes modified with three different aptamers (Apt1, Apt2, and Apt3), a Au counter electrode, and a Ag/AgCl reference electrode. Molecular docking simulations were performed to show the binding affinities between three anticancer drugs and their corresponding aptamers. Various eigenvalues were derived from the square-wave voltammetry electrochemical signals, and these data sets were subjected to rigorous analysis through multivariate data analysis techniques. This analytical approach demonstrated exceptional performance by achieving flawless 100% accuracy in the precise identification of nine anticancer drugs consistently at uniform concentrations. Furthermore, the integrated wearable sensor array exhibited impressive capabilities, correctly recognizing all nine anticancer drugs with 100% accuracy and successfully distinguishing between these drugs in artificial sweat samples. The proposed sensor array presents good stability for 15 days. Flexibility tests showed stable device performance after 500 twisting cycles. This innovative wearable sensing array represents a novel approach for achieving real-time monitoring and precise adjustment of drug dosages. It offers invaluable insights for tailoring the treatment of anticancer drugs to individual patients, predicting both drug efficacy and potential adverse reactions within the field of clinical medicine.

Development of multiple heartcutting two-dimensional liquid chromatography with ion-pairing reversed-phase separations in both dimensions for analysis of impurities in therapeutic oligonucleotides.

Oligonucleotides constitute an emerging and highly complex bioanalytical challenge and it is becoming increasingly clear that 1D methodologies are unable to fully resolve all possible impurities present in these samples. 2D-LC therefore constitutes a perfect solution wherein critical pairs can be sampled from a steep gradient 1D and separated in a shallower 2D gradient. Herein, we provide a facile 2D-LC method development approach to quickly generate high selectivity gradients utilizing ion pairing reverse phase (IPRP-IPRP). In particular we demonstrate how to iteratively generate a 12 % gradient from two training runs and then to utilize that data to predict retentions of analytes with a 2 % gradient with retention prediction errors as low as 3 and 11 %, respectively. This iterative method development workflow was applied to impurity profiling down to 1:1000 for the full-length product and phosphorothioate modified impurities. Additionally, we demonstrated the elucidation of critical pairs in complex crude pharmaceutical oligonucleotide samples by applying tailored high selectivity gradients in the second dimension. It was found that the iterative retention modeling approach allows fast and facile 2D-LC method development for complex oligonucleotide separations.

Multivalent DNA Flowers for High-Performance Isolation, Detection, and Release of Tumor-Derived Extracellular Vesicles.

Tumor-derived extracellular vesicles (T-EVs) hold great promise for understanding cancer biology and improving cancer diagnostics and therapeutics. Herein, we developed multivalent DNA flowers (DFs) containing repeated and equidistant EpCAM aptamers for the efficient isolation of T-EVs. The multivalent aptamer chains in DFs had good flexibility to adapt to the surface morphology of T-EVs and achieved multivalent ligand-receptor interactions, thus showing enhanced isolation ability compared to monovalent aptamers. Compared with other materials for isolation of EVs, DFs were generated by rolling circle amplification (RCA) and self-assembled into microspheres in a one-pot reaction, and the recognition molecules (aptamers) were directly replicated and assembled during the RCA reaction instead of chemical modification and immobilization on the surface of solid materials. Moreover, as optically transparent biomaterials, the content of EpCAM+ EVs could be directly reflected via membrane-based hydrophobic assembly of signaling modules in DFs@EpCAM+ EVs complex, and we found that the amount of EpCAM+ EVs showed greater accuracy in cancer diagnosis than total EVs (88.3 vs 69.7%) and was also higher than the clinically commonly used marker carcinoembryonic antigen (CEA) (88.3 vs 76.7%). In addition, T-EVs could be released by lysis of DFs with the nuclease, gently and easily, keeping high intact and activity of EVs for downstream biological function studies. These results demonstrated that DFs are efficient and nondestructive tools for isolation, detection, and release of T-EVs.

Characterization of synthetic guide ribonucleic acids through hydrophilic interaction chromatography coupled with mass spectrometry.

In this study, we aimed to develop a hydrophilic interaction liquid chromatography (HILIC) method for the analysis of single guide ribonucleic acid (sgRNA), a critical reagent used in CRISPR genome editing. Our results showed that effective profiling of sgRNA can be achieved by suppressing the surface charge of the stationary phase in HILIC. We identified hydrogen bonding as the primary retention mechanism with potential weak partitioning in HILIC separation of large oligonucleotides like 100-mer sgRNA. Moreover, we demonstrated that direct coupling of HILIC with mass spectrometry (MS) allows the intact mass analysis of sgRNA and its impurities with minimal adduct present. Finally, we characterized the post peak shown in the low temperature HILIC and identified it as sgRNA aggregates. Our findings provide valuable insight into the characterization of sgRNA and highlight the potential of HILIC-MS as a powerful analytical tool for relatively large oligonucleotide analysis.

Employing the Anchor DSPE-PEG as a Redox Probe for Ratiometric Electrochemical Detection of Surface Proteins on Extracellular Vesicles with Aptamers.

Quantitative analysis of surface proteins on extracellular vesicles (EVs) has been considered to be a crucial approach for reflecting the status of diseases. Due to the diverse composition of surface proteins on EVs and the interference from nonvesicular proteins, accurately detecting the expression of surface proteins on EVs remains a challenging task. While membrane affinity molecules have been widely employed as EVs capture probes to address this issue, their inherent biochemical properties have not been effectively harnessed. In this paper, we found that the electrochemical redox activity of the DSPE-PEG molecule was diminished upon its insertion into the membrane of EVs. This observation establishes the DSPE-PEG molecule modified on the Au electrode surface as a capture and a redox probe for the electrochemical detection of EVs. By utilizing methylene blue-labeled aptamers, the targeted surface proteins of EVs can be detected by recording the ratio of the oxidation peak current of methylene blue and DSPE-PEG. Without complicated signal amplification, the detection limit for EVs is calculated to be 8.11 × 102 particles/mL. Using this platform, we directly analyzed the expression of CD63 and HER2 proteins on the surface of EVs in human clinical plasma samples, demonstrating its significant potential in distinguishing breast cancer patients from healthy individuals.

Strategies for predictive modeling of overloaded oligonucleotide elution profiles in ion-pair chromatography.

Due to their potential for gene regulation, oligonucleotides have moved into focus as one of the preferred modalities modulating currently undruggable disease-associated targets. In the course of synthesis and storage of oligonucleotides a significant number of compound-related impurities can be generated. Purification protocols and analytical methods have become crucial for the therapeutic application of any oligonucleotides, be they antisense oligonucleotides (ASOs), small interfering ribonucleic acids (siRNAs) or conjugates. Ion-pair chromatography is currently the standard method for separating and analyzing therapeutic oligonucleotides. Although mathematical modeling can improve the accuracy and efficiency of ion-pair chromatography, its application remains challenging. Simple models may not be suitable to treat advanced single molecules, while complex models are still inefficient for industrial oligonucleotide optimization processes. Therefore, fundamental research to improve the accuracy and simplicity of mathematical models in ion-pair chromatography is still a necessity. In this study, we predict overloaded concentration profiles of oligonucleotides in ion-pair chromatography and compare relatively simple and more advanced predictive models. The experimental system consists of a traditional C18 column using (dibutyl)amine as the ion-pair reagent and acetonitrile as organic modifier. The models were built and tested based on three crude 16-mer oligonucleotides with varying degrees of phosphorothioation, as well as their respective n - 1 and (P = O)1 impurities. In short, the proposed models were suitable to predict the overloaded concentration profiles for different slopes of the organic modifier gradient and column load.

Continuous molecular monitoring of human dermal interstitial fluid with microneedle-enabled electrochemical aptamer sensors.

The ability to continually collect diagnostic information from the body during daily activity has revolutionized the monitoring of health and disease. Much of this monitoring, however, has been of physical "vital signs", with the monitoring of molecular markers having been limited to glucose, primarily due to the lack of other medically relevant molecules for which continuous measurements are possible in bodily fluids. Electrochemical aptamer sensors, however, have a recent history of successful in vivo demonstrations in rat animal models. Herein, we present the first report of real-time human molecular data collected using such sensors, successfully demonstrating their ability to measure the concentration of phenylalanine in dermal interstitial fluid after an oral bolus. To achieve this, we used a device that employs three hollow microneedles to couple the interstitial fluid to an ex vivo, phenylalanine-detecting sensor. The resulting architecture achieves good precision over the physiological concentration range and clinically relevant, 20 min lag times. By also demonstrating 90 days dry room-temperature shelf storage, the reported work also reaches another important milestone in moving such sensors to the clinic. While the devices demonstrated are not without remaining challenges, the results at minimum provide a simple method by which aptamer sensors can be quickly moved into human subjects for testing.

Optimization of orthogonal separations for the analysis of oligonucleotides using 2D-LC.

Oligonucleotides are commonly analysed using one dimensional chromatography (1D-LC) to resolve and characterise manufacturing impurities, structural isomers and (in respect to emerging oligonucleotide therapeutics) drug substance and drug product. Due to low selectivity and co-elution of closely related oligonucleotides using 1D-LC, analyte resolution is challenged. This leads to the requirement for improved analytical methods. Multidimensional chromatography has demonstrated utility in a range of applications as it increases peak capacity using orthogonal separations, however there are limited studies demonstrating the 2D-LC analysis of closely related oligonucleotides. In this study we optimised OGN size and sequence based separations using a variety of 1D-LC methods and coupled these orthogonal modes of chromatography within a 2D-LC workflow. Theoretical 2D-LC workflows were evaluated for optimal orthogonality using the minimum convex hull metric. The most orthogonal workflow identified in this study was ion-pair reversed phase using tributylammonium acetate (IP-RP-TBuAA) coupled with strong anion exchange in conjunction with sodium perchlorate (SAX-NaClO4) at high mobile phase pH. We developed a heart-cut (IP-RP-TBuAA)-(SAX-NaClO4) 2D-LC method for analysis of closely related size and sequence variant OGNs and OGN manufacturing impurities. The 2D-LC method resulted in an increased orthogonality and a reduction in co-elution (or close elution). Application of a UV based reference mapping strategy in conjunction with the 2D-LC method demonstrated a reduction in analytical complexity by reducing the reliance on mass based detection methods.

Evaluating orthogonality between ion-pair reversed phase, anion exchange, and hydrophilic interaction liquid chromatography for the separation of synthetic oligonucleotides.

The orthogonality of separation between ion-pair reversed phase (IP-RP), anion exchange (AEX), and hydrophilic interaction liquid chromatography (HILIC) was evaluated for oligonucleotides. A polythymidine standard ladder was first used to evaluate the three methods and showed zero orthogonality, where retention and selectivity were based on oligonucleotide charge/size under all three conditions. Next, a model 23-mer synthetic oligonucleotide containing 4 phosphorothioate bonds with 2' fluoro and 2'-O-methyl ribose modifications typical of small interfering RNA was used for evaluating orthogonality. The resolution and orthogonality were evaluated between the three modes of chromatography in terms of selectivity differences for nine common impurities, including truncations (n-1, n-2), addition (n + 1), oxidation, and de-fluorination. We first evaluated different ion-pairing reagents that provided the best separation of the key impurities while suppressing diastereomer separation due to phosphorothioate linkages. Although different ion-pairing reagents affected resolution, very little orthogonality was observed. We then compared the retention times between IP-RP, HILIC, and AEX for each impurity of the model oligonucleotide and observed various selectivity changes. The results suggest that coupling HILIC with either AEX or IP-RP provide the highest degree of orthogonality due to the differences in retention for hydrophilic nucleobases and modifications under HILIC conditions. IP-RP provided the highest overall resolution for the impurity mixture, whereas more co-elution was observed with HILIC and AEX. The unique selectivity patterns offered by HILIC provides an interesting alternative to IP-RP or AEX, in addition to the potential for coupling with multidimensional separations. Future work should explore orthogonality for oligonucleotides with subtle sequence differences such as nucleobase modifications and base flip isomers, longer strands such as guide RNA and messenger RNA, and other biotherapeutic modalities such as peptides, antibodies, and antibody-drug-conjugates.

Polybutylene terephthalate-based stationary phase for ion-pair-free reversed-phase liquid chromatography of small interfering RNA. Part 2: Use for selective comprehensive two-dimensional liquid chromatography.

With the increasing numbers of nucleic acid-based pharmaceuticals like antisense oligonucleotides (ASO), small interfering ribonucleic acid (siRNA) entering the market, research facilities, pharmaceutical industries and also regulatory authorities have been looking for efficient analytical methods for these synthetic oligonucleotides (ON). Besides of conventional one-dimensional (1D) reversed-phase liquid chromatography with or without ion-pairing (IP-RP-LC, RP-LC), hydrophilic liquid chromatography (HILIC) and mixed-mode chromatography (MMC), two-dimensional (2D) approaches combining two orthogonal chromatographic techniques also become more relevant due to the high structural complexity of oligonucleotides. Recently, we tested a polybutylene terephthalate(PBT)-based stationary phase under ion-pairing free RP mode for the liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) analysis of siRNA (Patisiran). In this study, retention profile and chromatographic orthogonality, respectively, were compared to other LC-modes like HILIC, IP-RPLC, another ion-pair free cholesterol-bonded RPLC and MMC considering their normalized retention times. Finally, because of higher orthogonality, the ion-pairing free PBT-bonded RPLC as first dimension (1D) was hyphenated with HILIC in the second dimension (2D) in a selective comprehensive 2D-LC setup leading to an enhanced resolution for peak purity evaluation of the main ON entities.

A wearable electrochemical fabric for cytokine monitoring.

Wearable biosensors monitoring various biomarkers in sweat provide comprehensive and prompt profiling of health states at molecular levels. Cytokines existed in sweat with trace amounts play an important role in cellular activity modulation. Unfortunately, flexible and wearable biosensors for cytokine monitoring have not yet been achieved due to the limitation of membrane-based structure and sensing strategy. Herein, we develop a novel electrochemical fabric based on aptamer-functionalized carbon nanotube/graphene fibers for real-time and in situ monitoring of IL-6, a paramount cytokine biomarker for inflammation and cancer. This fabric system possesses flexibility, anti-fatigue ability and breathability for wearable applications and can apply to different body parts in various forms. Moreover, the electrochemical fabric can track other biomarkers by replacing the coupling aptamer, serving as a universal platform for sweat analysis. This fabric-based platform holds the potential to facilitate an intelligent and personalized health monitoring approach.

Microflow liquid chromatography - multi-emitter nanoelectrospray mass spectrometry of oligonucleotides.

While the most sensitive LC-MS methods for oligonucleotide analysis contain ion-pairs in the mobile phase, these modifiers have been associated with instrument contamination and ion suppression. Typically, entire LC-MS systems are reserved for oligonucleotide LC-MS when using ion-pairing buffers. To overcome these limitations, numerous HILIC methods, liberated from ion-pairs, have been recently developed. Since ion-pairs play a role in analyte desorption from ESI droplets, their removal from mobile phases tend to impact method sensitivity. An effective way to recover MS sensitivity is to reduce the LC flow rate and therefore reduce ESI droplet size. With a focus on MS sensitivity, this study investigates the applicability of a microflow LC- nanoelectrospray MS platform in oligonucleotide ion-pair RP and HILIC LC-MS methods. The platform is effective and substantially increased the MS sensitivity of HILIC methods. Furthermore, LC method development for both types of separations provide insight into microflow chromatography of oligonucleotides, an under investigated chromatographic scale.

Exploring the use of the desirability function to optimize the separation of oligonucleotide impurities by ion pair-RP LCMS.

The use of small alkyl amines as ion pair reagents permits enhanced separation of impurities of phosphate diester oligonucleotides, which can be beneficial to quality control applications, and aid elucidation of the mechanisms of impurity formation. In general, however, separation of the individual components that comprise the majority of oligonucleotide impurities requires development of several independent chromatographic methods. Ideally, a single method capable of separating the individual components of all impurity classes would be developed. The mathematical concept of the desirability function has been explored here for its ability to determine the combination of experimental factors that result in a single, globally optimized chromatographic method. The optimized mobile phase, consisting of 1 mM propylamine (PA), 30 mM ammonium bicarbonate (ABC), and 1 mM octanoic acid (C8A), produced excellent agreement between measured and predicted peak resolution values for a set of n - 1 impurities. The relative importance of the mobile phase constituents on the mechanism of separation has been discussed. The approach holds great promise for the improved separation of components in complex chromatographic systems.

Polybutylene terephthalate-based stationary phase for ion-pair-free reversed-phase liquid chromatography of small interfering RNA. Part 1: Direct coupling with mass spectrometry.

Nowadays, ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the dominating generic method for the analysis of nucleic acid related compounds, such as antisense-oligonucleotides (ASO), small-interfering ribonucleic acid (siRNA) or other DNA or RNA type molecules and their conjugates. Despite of its effective performance, the usage of a high concentration of ion-pairing reagent in the eluent in IP-RPLC is unfavorable for the hyphenation with mass spectrometry (MS) which is required for a detailed structural characterization of the analytes and their structurally related impurities. In this work, we tested a polybutylene terephthalate (PBT)-bonded silica-based stationary phase for the separation of generically synthesized Patisiran as siRNA (antisense and sense single strands as well as their annealed double strand) giving some unexpected selectivity without any presence of ion-pairing reagents. Important chromatographic conditions affecting the separation have been investigated and evaluated. Furthermore, MS and tandem MS (MS/MS) characterization was possible without contamination of the MS system with ion-pair agent and related problems.

Method development for the acquisition of adsorption isotherm of ion pair reagents Tributylamine and Triethylamine in ion pair chromatography.

Tributylamine (TBuA) and triethylamine (TEtA) are the most commonly used ion pair reagents in ion pair chromatography especially for the analysis of oligonucleotides. In order to improve the understanding of the retention and separation mechanism of oligonucleotides in ion pair chromatography, it is important to understand the retention mechanism and the nature of interaction of these ion pair reagents with the stationary phase in the chromatographic column. Adsorption isotherm is helpful in evaluating such interactions, and subsequently predicting the retention mechanism. Alkylamines are very polar molecules which lack suitable chromophore and are commonly present in charged forms. Therefore, their determination and the subsequent acquisition of their adsorption isotherms using traditional liquid chromatography is very difficult. In this study, we first developed an analytical method for the determination of TBuA and TEtA in a typical chromatographic mobile phase (acetonitrile-water) and then used the same method to acquire the adsorption isotherms for tributylammonium acetate (TBuAA) and triethylammonium acetate (TEtAA). This method started with the conversion of the alkylammonium ions to free neutral forms by treating the sample with a strong base, followed by pentane-mediated extraction and finally the analysis of the extracts using gas chromatography-flame ionization detector (GC-FID). This three-step method was validated for parameters like range, linearity, intra-day and inter-day precision and accuracy, limit of detection and limit of quantitation. For the adsorption isotherms, the C18 column was first equilibrated with the solutions having different concentrations of alkylammonium ions and then stripped with eluent devoid of alkylammonium ions. Several stripping eluents were investigated and it was discovered that the eluent requirement could be decreased by the addition of sodium chloride. The effluents from the stripping phase were collected and analyzed using the developed analytical method to acquire the adsorption data. Under the investigated conditions, adsorption of TBuAA and TEtAA showed type III and type I isotherm behavior respectively.

Surface Functionalization of Extracellular Vesicles with Nucleic Acids towards Biomedical Applications.

Extracellular vesicles (EVs) are lipid bilayer-delimited particles secreted by cells and are regarded as a promising class of nanocarriers for biomedical applications such as disease diagnosis, drug delivery, and immunomodulation, as they carry biomarkers from the parental cells and can also transport diverse cargo molecules between cells. Surface functionalization of EVs can help obtain detectable signals for their quantification and also add various properties for EV-based delivery. Aptamers are specific oligonucleotides selected as artificial antibodies that could serve as 'cruise missiles' to target EVs for diagnosis or as navigators to bring EVs to lesions for treatment. DNA logic devices or nanostructures based on aptamers are intelligent designs to endow EVs with additional features, such as multi-target disease diagnosis in one pot and promoting retention of EVs in complex disease microenvironments. Oligonucleotides or DNA nanostructures composed of natural nucleic acids can be easily degraded by nuclease in the biological sample which limits their applications. Thus, the oligonucleotides composed of artificial nucleic acids which are synthesized against degradation would be a potential strategy to improve their stability in vitro or in vivo. Herein, we review the methods for surface functionalization of EVs by nucleic acids and highlight their applications in quantification and targeted delivery towards disease diagnosis and therapy.

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

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.