High-Throughput Chromatographic Separation of Oligonucleotides: A Proof of Concept Using Ultra-Short Columns

Abstract

Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the reference separation technique for characterizing oligonucleotides (ONs) and their related impurities. The aim of this study was to better understand the retention mechanism of ONs, evaluate the applicability of the linear solvent strength (LSS) retention model, and explore the potential of ultra-short columns having a length of only 5 mm for the separation of model ONs. First, the validity of the LSS model was evaluated for ONs having sizes comprised between 3 and 30 kDa, and the accuracy of retention time predictions was assessed. It was found that ONs in IP-RPLC conditions follow an “on–off” elution behavior, despite a molecular weight lower than that of proteins. For most linear gradient separation conditions, a column length between 5 and 35 mm was found to be appropriate. Ultra-short columns of only 5 mm were therefore explored to speed up separations by considering the impact of the instrumentation on the efficiency. Interestingly, the impacts of injection volume and post-column connection tubing on peak capacity were found to be negligible. Finally, it was demonstrated that longer columns would not improve selectivity or separation efficiency, but baseline separation of three model ONs mixtures was enabled in as little as 30 s on the 5 mm column. This proof-of-concept work paves the way for future investigations using more complex therapeutic ONs and their related impurities.

Rapid Intact Mass Analysis and Evaluation of the Separation Potential of Microfluidic Capillary Electrophoresis Mass Spectrometry for Oligonucleotides

“…Throughout the literature, ion-pairing liquid chromatography (LC) methods for oligonucleotide analysis involve mobile phase additives such as triethylamine (TEA) and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) − as well as other alkylamines and fluorinated alcohols (which can offer some benefits over TEA and HFIP). ,− While these ion-pairing additives aid in increasing sensitivity of oligonucleotides for MS analysis and assisting in their transport to the surface of the electrospray droplets, their use is generally avoided because their use over time can contaminate the mass spectrometer ion source leading to ion signal suppression. , In trapping instruments, these molecules can easily accumulate, and their corresponding m / z ’s are observed long after their use  ultimately complicating the use of the same MS instrument for positive ion mode analysis. , For facilities that require multipurpose use of their mass spectrometers, this can pose a significant challenge and can ultimately lead to instrument commitment for oligonucleotide analysis. , Recently, some suggestions of post sample analysis decontamination via solvent washing have demonstrated decreased contamination, but this increases both instrument run time and solvent requirements and does not take into account pre-existing LC system and column contamination. The need for multiple blank or column clean up analyses to avoid potential carryover or contamination also adds time to analysis.…”

“…This can lead to long sample run times, particularly for large sample sets from activities such as single-strand purification monitoring. In recent times, some promising shorter LC–MS gradients have been developed for oligonucleotide analysis, , some using ultrashort columns . However, some of these methods still rely on ion-pairing reagents, ,, while some are proposed only with sequences of poly dT and not with sequences of mixed composition and modifications, which are more representative of therapeutically relevant oligonucleotides …”