Taming the Wild West of Oligonucleotide Analytics

“Take him home; love him; take a lot of pictures,” the doctor said to Eric’s parents* (1). “There’s nothing you can do.” The couple’s infant son had been diagnosed with spinal muscular atrophy (SMA) type I. And, without treatment, Eric probably wouldn’t see his second birthday (2). Until five years ago, there was no cure for SMA – the most common genetic cause of death in infants. But a new therapy based on oligonucleotides (oligos) called Spinraza (nusinersen, approved by the FDA in December 2016) has profoundly changed the outcome for SMA patients (3,4). In addition, we have also seen widespread use of oligonucleotides – specifically the use of messenger ribonucleic acid (mRNA) in vaccines – for a far more common illness: COVID-19 (5).

Oligonucleotide therapeutics are a new frontier in drug development, pushing boundaries in the treatment of cancer and genetic diseases in particular. Yet despite more than two decades of pharmaceutical research, widespread usage of oligo therapeutics has typically been hindered by inefficient delivery, preclinical toxicology, or lack of clinical efficacy. All that said, the rapidly growing number of oligo drugs receiving regulatory approval – and a number of recent developments – have renewed interest in the field. 

Modifying oligos stabilizes them but also makes them more difficult to analyze

The field of oligonucleotide development is now expanding rapidly, with many new platforms and methodologies for targeted delivery. Within this range of new therapeutics is a wide array of modifications that enhance oligo stability. But these modifications also present additional obstacles in characterizing the oligonucleotides and their closely-related impurities. The rapid growth of the sector and the novel challenges posed by oligo therapeutics are creating a significant need for complementary technology and increasingly sensitive instrumentation that will help to address analytical obstacles.

In the past, only oligo-focused biotechnology companies were working on these issues. Today, most major pharmaceutical companies have an oligo group, and credible biologists and chemists are building out their experience with the aim of becoming oligo experts. In the last five years, at TIDES Oligonucleotide and Peptide Therapeutics – the main conference for oligo therapeutics and peptide development professionals – the number of attendees has gone from around 600 to more than 1,500. And whereas in previous years the majority of people represented specialty oligo start-ups, now attendees include more traditional pharma companies that are licensing or developing oligo drugs. What has really changed mindsets towards oligos was COVID-19 and the use of mRNA as the principal active ingredient in some vaccines.

Oligonucleotide therapeutics are moving into the mainstream

Vaccine development is shifting toward mRNA, given its incredibly short drug development timeline and relatively straightforward development process. Considering their ease of development and product stability, there is even some talk of moving toward mRNA therapeutics instead of protein therapeutics.

Yet the opportunity extends beyond vaccines. Many scientists have not lost sight of where oligo therapeutics have traditionally been a big player: untreatable diseases, such as SMA; indeed, oligos have the potential to reach previously undruggable targets. Better still, some companies are combining antibody therapeutics with oligonucleotide therapeutics by attaching a piece of oligonucleotide onto a monoclonal antibody. This method uses the monoclonal antibody as a delivery device to get the antisense RNA into a cell to then target and activate or disrupt it. Done well, similar methodologies could offer a new frontier with oligo therapeutics completely displacing some of the recombinant drugs already on the market. 

The novel challenges of oligo analysis

We continue to witness an influx in the oligo business; many of these players have been our customers for a long time and were previously protein therapeutics professionals or even small molecule pharmaceutical scientists. Many of them are asking for oligonucleotide separation applications that allow scientists to isolate oligonucleotides from biological fluids and biological tissues. One of the key challenges in drug discovery and drug development is determining the pharmacodynamics and pharmacokinetics profiles. Oligos are often difficult to isolate from biological tissues, but separation is critical for analytical methods based on MS or even molecular biology techniques.

The drugs themselves also look different. It is never one chemistry – and the chemistries are constantly changing. Over the last 20 years, all traditional oligonucleotide companies have been constantly trying to improve oligo chemistry to increase the half-life and bioactivity of therapeutics. Part of that involves making the oligos more nuclease-resistant, which requires a great deal of chemical modifications. And other modifications are made to increase the uptake of the oligonucleotide into cells and the cytosol, where the oligos are bioactive. Oligo drug developers often need to increase the potency of their therapeutics with chemical modifications; for instance, we saw the addition of phosphorothioates, which involved adding a sulphur atom onto the phosphate backbone. But now we are seeing the use of modified nucleic acids. 

Even subtle modifications can make a difference – not only to the oligo but also to the way we need to approach analysis. Indeed, it would be a mistake to think that, as the chemistry changes, the molecule largely remains the same – especially in terms of the analytical methods needed for characterization. In fact, each time chemistry changes are made, the tools and methods we use to perform the oligo analysis must be re-tested and potentially modified or even swapped out for a new set of methods. 

In short, thanks to the ever-changing chemistry of oligos, there is always a method development step. And that’s why most companies developing oligos are likely to face multiple challenges, including the lack of practical expertise to perform the various complex separations required or the chemistry knowledge to even understand how to get to the right separation. Separation scientists are in demand!

The Wild West of analyzing oligos

We have seen that there can be fundamental knowledge gaps in the oligo space – biologists who struggle with the chemistry or chemists who struggle with the biology. And that’s because they sit somewhere between a small molecule and a protein therapeutic. Oligos for the most part are biological molecules that are chemically synthesized (as with antisense therapeutics) or are recombinantly synthesized (as with mRNA- or AAV-based therapies). Given that oligos live in a world where both chemistry and biology are involved, it stands to reason that associated analytical method development and application require a knowledge of both sciences. And there lies the problem: most pharmaceutical analytical scientists come from either small molecule or protein therapeutic backgrounds, and they tend to import not only knowledge but also biases from their previous field.

For instance, protein purification is so good these days that we consistently approach 99 percent purity – which wasn’t the case in the past. For protein therapeutics, such a difference is minor and results in low analytical variability. Thus, protein therapeutic analytics have evolved from a wide-open field to one with set expectations and standardized methods – including USP methods with set ways of performing them. Similarly, with the small molecule industry, there are set standards and standardized methods, with clear definitions of purity – what is good or acceptable, what is bad, what makes for a good molecule or a bad one, and so forth. 

It is a very different story with oligos. The nature of oligos – and how they are synthesized – means that obtaining 100 percent purity is nearly impossible. With oligos, we are back in the Wild West, where we’re still learning and figuring out what’s “pure” and “not pure” – what’s good enough and what’s not. This uncertainty is compounded by regulatory requirements that vary from country to country, agency to agency. The approach to oligo purity and activity requires a whole different mindset. 

Working together to develop bespoke analytical solutions for oligos

Chromatography and separation rules are profoundly different for oligonucleotides when compared with small molecule or protein separations. And for my global technical support group, education is part of the solution. We have first explain to our partner why their older methods cannot be applied to analyze oligos with sufficient robustness. We have to share data and research, as well as the benefits of our ever growing experience. With every case, there is always some (and often a significant) element of method development before we land on the right chromatographic solution. Sometimes the molecules we are working on are completely new, in which case we find ourselves in a true collaboration with our customers – one that relies on our chromatography knowledge and their knowledge of the molecule.

To summarise – and contrary to the section within which this article sits (Solutions!) – there is no one solution for oligo analysis. There is no set playbook or formula. Instead, there are many discussions – and some trial and error – on the way to the right solution for each molecule. It’s challenging, but when we get there – and we very often do get there – it’s incredibly rewarding. After all, both sides know they are working towards radical new therapies that may treat or even cure diseases for individuals like Eric – now that’s what I call a solution.

*Not patient’s real name; scenario adapted from caregiver answers to polling questions at a public meeting hosted by Cure SMA (1).