Mobile Phase Aging and Its Impact on Electrospray Ionization of Oligonucleotides.

The implementation of fluoroalcohol/alkylamine mobile phase systems in oligonucleotide LC-MS provides a good balance between chromatographic separations and MS sensitivity. Since its introduction, several parameters including mobile phase composition, additive concentration, alkylamine hydrophobicity, and different fluoroalcohols have been carefully evaluated and optimized. While our understanding of this mobile phase system has increased over the years, there are challenges that continue to hinder method performance and remain poorly understood. One of these challenges is the constant loss of MS sensitivity over time, commonly termed mobile phase aging. This study investigates two aging mechanisms associated with loss of MS sensitivity: alkylamine oxidation and aggregate formation. The relationship between pH, organic solvent, oxygen, and mobile phase aging is characterized, and mitigation strategies to extend mobile phase lifetime are discussed.

Characterization of mRNA therapeutics.

Therapeutic messenger RNAs (mRNAs) have emerged as powerful tools in the treatment of complex diseases, especially for conditions that lack efficacious treatment. The successful application of this modality can be attributed to its ability to encode entire proteins. While the large nature of these molecules has supported their success as therapeutics, its extended size creates several analytical challenges. To further support therapeutic mRNA development and its deployment in clinical trials, appropriate methods to support their characterization must be developed. In this review, we describe current analytical methods that have been used in the characterization of RNA quality, identity, and integrity. Advantages and limitations from several analytical techniques ranging from gel electrophoresis to liquid chromatography-mass spectrometry and from shotgun sequencing to intact mass measurements are discussed. We comprehensively describe the application of analytical methods in the measurements of capping efficiency, poly A tail analysis, as well as their applicability in stability studies.

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.

Managing nonspecific adsorption to liquid chromatography hardware: A review.

The choice of alternative materials over stainless steel hardware in the construction of liquid chromatography systems has unveiled the degree to which nonspecific adsorption impacts the reproducibility of LC methods. Some of the major contributors to nonspecific adsorption losses are charged metallic surfaces and leached metallic impurities, that may interact with the analyte and result in analyte loss and overall poor chromatographic performance. In this review, we describe several mitigation strategies available to chromatographers to minimize nonspecific adsorption to chromatographic systems. Alternative surfaces to stainless steel such as titanium, PEEK, and hybrid surface technologies are discussed. Furthermore, mobile phase additives used to prevent metal ion-analyte interactions are reviewed. Nonspecific adsorption of analytes is not reserved to metallic surfaces, as analytes may adsorb to the surfaces of filters, tubes, and pipette tips during sample preparation. Identifying the source of nonspecific interactions is paramount, as mitigation strategies may differ depending on what stage nonspecific losses are taking place. With this in mind, we discuss diagnostic methods that may help the chromatographer to differentiate losses resulting from sample preparation, and losses during LC runs.

Impact of Nonspecific Adsorption to Metal Surfaces in Ion Pair-RP LC-MS Impurity Analysis of Oligonucleotides.

Nonspecific adsorption has been a consistent challenge in the analysis of oligonucleotides. Nonspecific adsorption is a result of interactions between charged acidic analytes and adsorption sites present in metallic surfaces located in the fluidic path of chromatography systems. Due to their high surface area, adsorption to column frits is especially concerning. Poor peak shape, low recovery and compromised LOQ have been associated with this phenomenon. Alternative methods including substitution of stainless steel for different hardware materials and mobile phase additives have been explored in an attempt to minimize this issue. Chemical modification of metal surfaces using hybrid surface technology (HST) by-passes the limitation of stainless steel construction material by forming a hybrid organic/inorganic layer that acts as a barrier and limits nonspecific interactions. In this study we explore the implications of this new technology in sensitive analysis and determination of relative impurity levels of oligonucleotides. Higher relative impurity levels and better reproducibility were obtained with columns using HST.

Current State of Oligonucleotide Characterization Using Liquid Chromatography-Mass Spectrometry: Insight into Critical Issues.

As interests increase in oligonucleotide therapeutics, there has been a greater need for analytical techniques to properly analyze and quantitate these biomolecules. This article looks into some of the existing chromatographic approaches for oligonucleotide analysis, including anion exchange, hydrophilic interaction liquid chromatography, and ion pair chromatography. Some of the key advantages and challenges of these chromatographic techniques are discussed. Colloid formation in mobile phases of alkylamines and fluorinated alcohols, a recently discovered analytical challenge, is discussed. Mass spectrometry is the method of choice to directly obtain structural information about oligonucleotide therapeutics. Mass spectrometry sensitivity challenges are reviewed, including comparison to other oligonucleotide techniques, salt adduction, and the multiple charge state envelope. Ionization of oligonucleotides through the charge residue model, ion evaporation model, and chain ejection model are analyzed. Therapeutic oligonucleotides have to undergo approval from major regulatory agencies, and the impurities and degradation products must be well-characterized to be approved. Current accepted thresholds for oligonucleotide impurities are reported. Aspects of the impurities and degradation products from these types of molecules are discussed as well as optimal analytical strategies to determine oligonucleotide related substances. Finally, ideas are proposed on how the field of oligonucleotide therapeutics may improve to aid in future analysis.