In recent years, the development of next-generation oligonucleotide therapies with high target specificity and stability has greatly increased, yet the precise and productive analytical testing of these molecules has remained a significant challenge.
In this SelectScience webinar, now available on demand, Mary Trudeau, principal applications scientist at Waters Corporation, describes how to address the analytical challenges posed by therapeutic oligonucleotides and outlines approaches to optimize sample prep and LC-MS acquisition for maximum sensitivity and selectivity.
Think you’d benefit but missed the live event? Register now to watch the webinar at a time that suits you and read on to find highlights from the live Q&A session.
MT: We use small molecule LC-MS method validation guidance, which includes dynamic range, linearity, precision, accuracy, inter/intra assay performance and minimal matrix assessment.
MT: This would be a balance of LC performance and MS detection choice. Multiple charged precursors allow us to use tandem quadrupole with ease-of-use benefit. However, high-resolution mass spectrometry (HRMS) allows quantitative and qualitative analysis. Intact analysis still can be limited on HRMS though. If size is an issue, oligonucleotide digestion would be the next plausible analysis technique.
MT: Absolutely. Much like peptide analysis, due to their charged ionic backbone, we can predict the multiply charged precursors, as I showed with the MassPrep ODT standard (15-35T). In this example, the 4- and 5- precursors were in the mass range of our tandem MS quadrupole m/z 2050 on both quadrupoles.
MT: One can theoretically assume the charge states feasible for tandem quadrupole m/z range 2050 would be most likely -9,-10, -11 and up. The trade-off is the larger oligonucleotides will often have less MS sensitivity due to dilution of signal across all charge states.
MT: For oligonucleotide analysis, we are focused on negative ion mode. But in general, yes. Ion pairing reagents often cause MS suppression. For this reason, we often try to balance the amount of IP reagent required for adequate retention and separation of the analytes of interest.
MT: There are many reported in the literature and often the choice will depend on the oligonucleotide and whether the detection is optical or MS. Common reagents are hexylamine, triethylamine, triethylammonium acetate, acetic acid, hexafluoroisopropanol, with methanol or acetonitrile. In our labs, it is common for us to use TEA/HFIP, HA/HFIP, or DIEA/HFIP.
MT: HFIP is the buffering reagent with an effective pH 7.9-8.5. It improves the selectivity of LC separation and often can be used in very low concentration (10-50 mM ) and causes much less MS suppression compared to other buffering reagents like acetic acid.
MT: Non-specific binding associated with metal adsorption will vary with oligo properties such as size and phosphorylation, and LC conditions. With lower mass loads, oligo loss will be more pronounced. We must also be mindful of size, larger oligonucleotides may also run into solubility issues and hydrophobic losses. For these reasons, we often profile adsorptive loss potential at many mass loads, various sample diluents, and collection/sample vessels with low bind and glass vs. polypropylene. We advise avoiding glass, and we use QuanRecovery Vials and Plates for our oligonucleotide analysis.
MT: We use a 50 mM MES solution (pH 5.5-6.7), 100 mM Ammonium Sulfate, 6M guanidine solution, and 1% Triton X.
MT: There are various approaches to mitigate adsorption mechanisms that are due to ionic interaction with metal surfaces that are similar to that of oligonucleotides: paying attention to pH, using additives like phosphoric acid or phosphate buffer, or chelating reagents like EDTA in the sample or the system. These are not permanent, making it hard to achieve robust LC methods. Thus, ACQUITY Premier Systems and Columns with MaxPeak HPS Technology ensure consistent performance for a diversity of analytes such as organic acids, phosphate steroids, nucleosides, nucleotides, acidic and/or phosphorylated peptides.
MT: Try wider pore columns like BEH 300Å, you may need to digest to smaller oligonucleotide fragments.
MT: The advantage of solid-phase extraction (SPE) is that it is the most universal technique for oligonucleotides and whether you use reversed-phase (Oasis HLB) or anion-exchange (Oasis WAX), they are both effectively desalting the sample. It is typical to need more wash steps or volumes to improve the removal of surfactants and detergents often used in sample preparation of oligonucleotides.
MT: The micro elution SPE plate is fundamentally the same as our SPE cartridges and plates, but with much smaller sorbent beds. A micro elution plate holds 2 mg of sorbent. We have the ability to load 750µL undiluted sample and recover in only 25µL (3 column volumes). This is up to 15 X concentration. Capacity would only be of concern for urine, and this would require larger macro plates (10, 30 or 60 mg sorbent bed size).
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