Oligonucleotide Analyses
Purity Analysis by LCMS
Synthetic oligonucleotides can have several impurities carry over from synthesis that affect downstream analysis and subsequent synthetic steps. Ion-paired reversed-phase LC-MS is a reliable and high-resolution system for purity assessment, impurity characterization, and purification. We implement numerous methods to analyze and purify oligonucleotides of any size, from single base pairs to full, intact mRNAs.
Purity Analysis without Ion Pairing
At Rilas, we specialize in separating the hard-to-separate. Whether it’s by flow injection analysis (FIA) or hydrophilic interaction chromatography (HILIC) methods, we have multiple methods to analyze oligonucleotides at high resolution without introducing problematic ion-pairing agents. Our customers often request the development of methods that are compatible with other workflows on the same instrument, necessitating the removal of ion pairing agents from the methods to ensure compatibility.
Exact Mass Determination
For larger oligonucleotides, Rilas provides the same exact mass determination services as for proteins and conjugates. This includes intact molecule deconvolution, as well as enzymatic digestion followed by MS2 fragmentation analysis.
Sequence Confirmation
Oligonucleotides work through specific sequence interactions between the oligo and a target molecule. To ensure this interaction is performed properly, sequence confirmation of the oligos are often required. By separating the oligo by LC, then ionizing, fragmenting, and reading the fragments by MS2 the sequence of an oligonucleotide can be confirmed against the theoretical sequence.
Conjugate Characterization
In recent years, oligonucleotides have become a cargo of choice for many advanced therapeutics owing to their high potency and specificity when compared to small molecule cargoes. Platforms for oligonucleotide cargo molecules include proteins (mAbs), peptides, and polymers. Rilas has experience purifying and characterizing these conjugates, including loading levels, site specific conjugation mapping, and more!
Instrumentation:
Agilent 6545XT AdvanceBio LC/Q-TOF
Agilent 1260 Infinity II Bio-inert
Agilent 1290 Infinity II HPLC/MS
Agilent 1260 HPLC/UV/ELSD