Bioanalysis Advances for the Booming Oligonucleotide Market

Advantages of LC-MS/MS in oligonucleotide analysis: selective and versatile 

Oligonucleotide therapies are short, single- or double-stranded DNA or RNA molecules that use Watson-Crick base pairing to affect the expression of target RNA. Their potential therapeutic applications are consequently enormous, and, in fact, are  being investigated as treatments for everything from genetic disorders like muscular dystrophy to neurodegenerative disorders and cardiovascular disease. Recent developments in oligonucleotide drug delivery, stability and immune response control have enabled this therapeutic space to grow dramatically, with further sustained growth expected over the next decade. The market research firm Roots Analysis estimates the oligonucleotide synthesis market will expand from US$4.8 billion in 2024 to $13.5 billion in 2035.

To support this growth, efficient bioanalytical solutions are required for every step of drug development. These types of molecules however provide unique challenges compared to traditional small molecule and protein-based drugs, and therefore require bespoke analytical solutions. 

Historically ligand binding assays (LBAs) have been used for oligo bioanalysis, due to the high sensitivity the technique offers. However, with recent developments in mass spectrometry (MS) technology and approaches, liquid chromatography with tandem mass spectrometry (LC-MS/MS) has become not just a viable alternative, but in many situations a better option. The additional selectivity and versatility offered by LC-MS/MS affords many practical and scientific benefits, that can add significant value at a variety of stages throughout drug development.

Efficient early-stage preclinical analysis

In the early-stages of drug development, the main advantages of LC-MS/MS are time and cost. This is because LC-MS/MS bioanalytical approaches do not require the custom probe synthesis that LBA techniques do. At later stages of drug development this may not be a significant limiting factor, but in early drug development, and particularly candidate selection, the time and cost of synthesising the required probes can be prohibitive. LC-MS/MS offers the opportunity to utilise more generic methodologies to generate fit-for-purpose data. It can allow similar extraction and LC-MS/MS conditions for a variety of oligonucleotides, with only minor adaptations to the methodology required between candidates. This utilisation of fit-for-purpose generic methods allows for fast and efficient data generation to support early non-GLP decision making. 

The availability of appropriate internal standards can be a limiting factor in the applicability of LC MS/MS for oligonucleotide bioanalysis. Stable label isotope versions of the analyte are always the preferred option for an LC-MS/MS internal standard, as they compensate best for minor variations from sample to sample. However, these require specialised custom synthesis, which is often expensive and time- consuming. Analogue internal standards are then usually considered the next best solution. These are structurally similar versions of the analyte that have a suitably different mass to ensure differentiation from the analyte, for example replacing all the cytosines in a sequence with thymines.

The commercial synthesis of analogues is much more widely available, and quicker and less expensive to produce. Despite this, the time and cost associated is still not significantly less than for LBA probes. It is instead the use of generic internal standards that provides the compatibility with these early workflows. By utilising a cocktail of commercially available oligonucleotides and selecting the most appropriate one to use as an internal standard for quantitation during data processing, the issue of the time and cost associated with custom consumables can be overcome. By designing the cocktail to include a mix of oligonucleotides with various nucleotide compositions and oligomer lengths, it is possible to ensure their compatibility with most common therapeutic oligonucleotides. Whilst these internal standards do not provide the optimum behaviours of stable label internal standards, they do allow sufficiently reliable data generation for early stage non-GLP studies. Thus, enabling fast and cost-effective data generation.

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Metabolite Selectivity and Quantitation

LC-MS/MS is not just beneficial in early-stage bioanalysis. It’s biggest selling point in any type of analysis, compared to LBA techniques, is its selectivity. In oligonucleotide bioanalysis this can be particularly advantageous. Oligonucleotides are normally metabolised in vivo by cleaving nucleotides from the end of the sequence (n-1, n-2 etc). As LBA techniques generally utilise probes which correspond to only part of the oligonucleotide sequence, they often don’t have the specificity to distinguish between these metabolites and the parent analyte. The advantage LC-MS/MS can provide is that by utilising both mass and chromatographic separation, it is generally possible to ensure resolution of these metabolites from the parent. This therefore ensures that their presence doesn’t impact parent quantitation. This ability to differentiate also provides the opportunity for the metabolites themselves to be quantified in the same assay as the dosed oligonucleotide. This consequently provides the opportunity for increased understanding of the metabolism in parallel to the parent PK/TK data generation.

Clinical Support

Although LC-MS/MS is becoming more common place in preclinical oligonucleotide workflows due to its flexibility, in the clinical space where high sensitivity is the main driver for analytical technique selection LBA still reigns. This has generally led to a requirement to change analytical techniques late into a programme, which in turn has often resulted in a reluctance to use LC MS/MS at all, due to doubts about whether you can bridge the data between techniques. 

Recent advancements in MS technology, and growing industry experience, are changing this situation and leading to LC-MS/MS becoming a truly viable option throughout drug development. Recent clinical bioanalysis work conducted at CRL utilised the latest Sciex MS technology (Sciex 7500) and thorough sample clean up steps, to produce an assay that simultaneously quantifies an antisense oligonucleotide and it’s GalNac conjugated precursor at levels as low as 50pmol/L in human plasma.

The development of hybrid LBA-MS approaches also offer further potential benefits for later stage studies, by combining the advantages of both techniques. The hybrid approach uses an LBA capture step to efficiently remove matrix components that could impact quantitation and limit sensitivity. The LC-MS/MS is then used to provide selectivity from metabolites.

Overall, this means LC-MS/MS can be a highly flexible and efficient analytical technique for oligonucleotide bioanalysis. As LC-MS/MS technology continues to develop, and the sensitivity limitations continue to decrease, the flexibility and selectivity advantages of the technique mean that it is likely that it will play an ever-increasing role in oligonucleotide bioanalysis.

Rebecca Paterson leads the method development team, within the Chromatographic Bioanalysis department at Charles River Laboratories Edinburgh.