Abstract

As the world grasps that oligonucleotides, mRNA and related molecules are a critical group of therapeutics and vaccines, so too do analysts feel vindicated that mass spectrometry data should be used to its full potential. Often seen as overkill in recent years, mass spectrometry can come to the fore again. We demonstrate routine analysis of oligonucleotides showing that advanced techniques to confirm sequence have come of age. We use examples of 21 mer and 40 mer oligonucleotides to demonstrate straightforward mass spectrometry analysis that confirms mass and sequence. We also highlight that more complex species with unusual modifications are now also within the remit of routine analysis, as well as demonstrating that identifying modifications is not the burden it was heretofore.

Instrumentation, Sample Preparation

An Agilent 1290 Infinity II UHPLC was attached to an Agilent 6545 Q-TOF MSD with binary gradient pump, mulitsampler, column compartment, and Diode Array Detector. The two strands were prepared at a concentration of 0.5 mg/mL in ultra-pure water. These samples were then desalted using Amicon® Ultra 0.5 mL Centrifugal filters with 3KDa membranes. 

Conclusions

New efforts in making oligonucleotide analysis by mass spectrometry have led to a suite of new tools that reflect a maturing market, and a response to developers who have long asked for improvements. With a combination of chemistries, hardware, and new software, the burden of analysis of oligonucleotides is considerably reduced. The groundwork laid here also indicates that a burgeoning market has catapulted tools into faster adoption. Wider adoption of these tools has the evident potential to help cope with pressures on bringing down development timelines, reducing pressures from healthcare funding, and bring new therapeutics and vaccines to market with product quality improvements.