The mRNA Moment

Nucleic acid-based therapies have been the subject of research and discovery for over three decades. After all, the prospect of fully harnessing the unique properties of DNA and RNA to instruct cells to produce or block the production of key proteins is tantalizing – potentially opening the door to personalized medicine. 

Several barriers to their viability as biopharmaceuticals have been overcome, including progress in formulation, stability, and drug-delivery – notably, with the introduction of lipid nanoparticles, as well as tissue targeting conjugate molecules. But it was the rapid yet successful development and deployment of mRNA vaccines during the COVID-19 pandemic that really took these medicines to the next level.

“In the face of a tremendous public health crisis, we saw emergency use authorizations for mRNA vaccines which effectively ‘normalized’ RNA medicine overnight,” says Varun Gadkari, Assistant Professor, Department of Chemistry, University of Minnesota. “Four years later, these therapies are now a fixture in day-to-day life and far more easily accessible.” 

Bifan Chen, Principal Scientist at Genentech, adds, “I think people saw the tremendous potential of mRNA vaccines over the pandemic years and realized how rapidly science has been advancing.”

This increased interest and demand is now being reflected in the number of startup businesses working on the development of nucleic acid therapies – companies that, according to Bingchuan Wei, Senior Principal Scientist at Genentech, are “taking over San Francisco.” Interestingly, their focus extends beyond mRNA and its delivery to the development of antisense oligonucleotides and siRNAs.

“We have observed the success and growth of antibody therapies during the last few decades as they opened up more ways to treat disease. I think scientists will always be looking for creative ways to treat disease and reduce patient suffering – now these new therapeutics open up even more avenues to improve patient health,” says Christina Vanhinsbergh, Senior Scientist, AstraZeneca, UK.

According to Ken Cook, EU Bio-Separations Manager at Thermo Fisher Scientific, the success of the mRNA vaccines has – quite rapidly – shifted biopharma’s focus away from monoclonal antibodies and redirected it to nucleic acid therapeutics, and their daughter oligonucleotide therapies. In fact, he argues that the proven ability to “trick” the body into making a protein also opens up treatments for genetic diseases that result in the production of non-functional proteins. “This has opened up a new field of medicine,” he says.

With great promise comes great complexity…
 

What’s clear is that the demand is not going away –rather, it is only growing. “There’s now a huge demand from the chemistry side as these therapies move into clinical trials. We need to understand if we’re delivering the best materials to our patients, which is quite different from traditional small-molecule or protein drugs. It’s an entirely new beast,”says Wei. As momentum increases, it becomes clear that traditional approaches to drug development and quality control may not be fit for purpose when it comes to mRNA- and oligonucleotide-based therapies. 

“These therapeutics and vaccines are new modalities, which require techniques to characterize them to ensure their safety and efficacy,” explains Cook. “Chromatography on its own cannot deliver a full estimation of all the impurities present in the drug products. New methods are required; in fact, mass spectrometry-based methods are already in development. These methods will eventually have to filter into the QC department as well as the development laboratories.” 

Specifically, oligonucleotides are highly charged molecules, making traditional methods like HPLC and mass spectrometry more difficult. “The solid phase synthesis based manufacturing process for oligonucleotides is complex, with each step adding potential impurities. Modifications are also needed to make them stable, as enzymes in the body can quickly degrade them. Developing methods to monitor these chemical impurities and diastereomers is tough but exciting,” explains Wei. For mRNA therapies, the primary challenge is delivery. “The mRNA itself is already difficult to analyze, but the lipid nanoparticles used to encapsulate it add even more complexity. Characterizing the particles – ensuring the right particle size and encapsulation efficiency – is crucial. We’re still developing innovative ways to measure, analyze and deliver these therapies.”

For Gadkari, many of the analytical challenges stem from the chemical complexity and structural heterogeneity of nucleic acids themselves. For example, mRNA therapeutics have structured untranslated regions (UTRs) that are as important to the efficacy/stability of the mRNA molecule as the translated region of the molecule. “Characterizing the intact mRNA structure is an important step in ensuring that the molecule will function as intended,” says Gadkari. “This highlights one of the major challenges: RNA structure elucidation is typically a challenging endeavor, and it is especially challenging when the molecules of interest are thousands of bases in length, as most mRNA vaccine molecules are.” He believes that once these challenges are addressed, the next challenge would be adapting the analytical tools for different size nucleic acid therapeutics – which requires experience gained from analysis of other nucleic acid molecules (in other words, adapting a method designed for 20 nt oligos up to analyzing 100 nt single guide RNAs). 

Vanhinsbergh agrees that knowledge from smaller therapeutics method development could be shared and applied to the analysis of mRNA. Moreover, she says, “Developments in tissue targeting techniques (conjugation and smarter drug product formulations) apply more ubiquitously so will hopefully benefit mRNA therapeutics in line with smaller oligonucleotides.” However, as the structural chemistry, size, and heterogeneity of mRNA is individual, Vanhinsbergh believes that therapeutics should be treated as an individual class of sample from an analytical perspective.

Meet the Experts

Bifan Chen: I am currently a Principal Scientist at the Synthetic Molecule Analytical Chemistry department in Genentech. Trained from Prof. Ying Ge’s lab (University of Wisconsin-Madison) as a mass spectrometrist, I have always wanted to leverage my large molecule mass spectrometry background on emerging new modalities. Sequencing the relatively large guide RNA presents some unique challenges and at the same time opportunities for me to show how useful top-down mass spectrometry approaches can be.

Christina Vanhinsbergh: I started off studying molecular biology and genetics for my undergraduate degree thinking I would become a clinical scientist of some sort, but I found I was more interested in the molecular biology and mechanistic nature of biomolecules than clinical science. To develop a more industrially focused understanding of biotechnology, I studied a Masters in biological engineering and bioprocess engineering at the University of Sheffield, where I was able to elect for a research project chromatographically separating glycoproteins. I found my passion growing for separation science during that research and jumped at the chance to follow on with doctorate studies in multidimensional separations of oligonucleotide therapeutics  Following academic research I became a senior scientist specializing in oligonucleotides at AstraZeneca, working within the oligonucleotide platform. My work involves developing analytical methods to support the CMC strategy within the drug development process. I have developed methods for both smaller oligonucleotide therapeutics and larger mRNA therapeutics but I still feel like every day I am learning something new!

Varun Gadkari :I earned my B.S. and Ph.D. in biochemistry at The Ohio State University where my thesis research focused on the characterization of DNA replication/repair proteins. Through this work, and the strong RNA research community around me at OSU, I was exposed to a broad variety of cutting edge nucleic acids research as a graduate student. After my graduate work, I moved to the University of Michigan to complete postdoctoral training in Brandon Ruotolo's group, where I trained in native mass spectrometry (native MS). While I focused primarily on developing native MS methods for protein characterization, I quickly realized that these methods might prove useful in nucleic acid analysis/characterization. Towards the end of my postdoctoral training, I began working on some noncoding RNAs (riboswitches, tRNAs) to evaluate whether we could learn important aspects of nucleic acid structure and stability using native mass spectrometry. In 2022, I established my independent research program in the Department of Chemistry at University of Minnesota. The primary focus of my research group is the development and application of native MS-based methods to enable the chemical, and structural characterization of nucleic acids, proteins and the complexes they form.

Bingjuan Wei: I’m an analytical chemist by training. I graduated from Purdue University in 2011. My PhD work focused on packing nanosilica particles into capillaries for protein separation. I joined Genentech in 2014 and spent a decade in analytical development and chemistry, manufacturing and controls (CMC) development spanning various drug modalities from small molecule, protein, peptides, conjugates and recently, nucleic acid based therapies. . I currently serve as a  Senior Principal Scientist at the Synthetic Molecule Analytical Chemistry Department at Genentech. 

Ken Cook: I’ve been working in the field of biotherapeutic proteins and oligonucleotides for some time, supporting the development of analytical methods for the biopharmaceutical industry. Until about four or five years ago, my work predominantly focused on monoclonal antibodies. However, the success of mRNA vaccines has dramatically shifted the landscape. Today, most of the application requests I handle involve oligonucleotides, from short synthetic molecules to large mRNA constructs. 

Daniel Meston: My background is originally in analytical chemistry applied to small molecule pharmaceuticals. I investigated the use of mixed mode (SCX/RP) columns in tuning resolution as well as chiral mobile phase additives to achieve chiral/achiral selectivity under UHPLC conditions. After this experience, I did a PhD in proteomics, which sparked my interest in the analysis of complex biological samples. I then moved to Brussels where I focused more on different bioanalytical techniques as well as modeling and simulation approaches before moving to Gustavus Adolphus College in the US to develop expertise in 2D-LC separation of oligonucleotides which is becoming an increasingly important part of the toolbox of the pharmaceutical industry to deal with these extremely complex and chemically similar samples from the synthetic process.

Analytical solutions emerge
 

The inherent complexity and heterogeneity of nucleic acids have energized the search for improved separations and mass spectrometry-based methods. 

“I am seeing broader adoption of photodissociation methods, such as ultraviolet photodissociation (UVPD) for tandem MS of nucleic acids, substantially improving sequence coverage of larger molecules,” says Gadkari. “I am also excited to see the emergence of top-down and native mass spectrometry in this space as two MS based methods that yield not only sequence information, but also provide structural context, which is just as important as sequence for therapeutic molecules.”

According to Daniel Meston, Research Associate Professor in Chemistry, Gustavus Adolphus College, multidimensional chromatography is becoming increasingly important in the elucidation of highly structural specific impurities in early phase drug discovery. “Heartcutting 2D-LC is emerging as an extremely useful part of the analytical scientist’s toolbox; we have seen a number of pharmaceutical companies begin to install at least one 2D-LC system in their upstream research labs,” he says. “We have been largely interested in pushing the performance of chromatographic methods to improve the separation power as well as investigating the unique challenges that 2D-LC faces when transferring these biomolecules onto complementary modes of LC.”

Aware of these challenges in oligonucleotide analysis, Chen and his team have developed a specialized LC-MS technology for oligonucleotide analysis – focusing on guide RNAs used in CRISPR-Cas9 gene editing systems, which are necessary for developing targeted cancer therapies. “We are using a top-down mass spectrometry strategy for the 100-mer guide RNA without any sample preparation and digestion. The key feature is the introduction of large oligonucleotide samples to the mass spectrometer through a small pore, high-pH resistant reversed phase column in a size exclusion mode,” Chen explains. “In a short 10 min LC-MS run, we were achieving nearly 70 using size exclusion mode,” Chen explains. “In a short 10 min LC-MS run, we were achieving nearly 70 percent sequence coverage in our previous work for the 100-mer guide RNA; now, we are nearly at 100 percent sequence coverage with the improved dissociation strategy.”

For Cook, high resolution mass spectrometry (HRMS) is at the heart of these advancements – thanks to its added sensitivity and accuracy. “Using a novel magnetic bound nuclease for controlled partial digestion with high-resolution MS/MS for the analysis and new software tools for the data analysis, we can now directly sequence the mRNA vaccines to confirm the correct sequence in the product,” he says. “These tools are also being used with the smaller synthetic RNA drug candidates.” 

Data analysis is another improving aspect of oligonucleotide/mRNA analysis, with most of the major vendors tackling challenges across arduous and complex processing, high numbers of large data files, and multiple signals to extract and integrate. “I have worked with numerous vendors to give feedback on processing improvements to help design these tools for the analytical community,” says Vanhinsbergh. “By making the data more accessible, analysis turnaround times will reduce and will hopefully become more cost effective.” 

Vanhinsbergh also emphasizes sustainable and environmentally friendly analytical approaches for the developments of these therapeutics – an aspect often neglected. “Simplifying the mobile phase and using less toxic reagents will be a fantastic move towards sustainability improvements in analysis,” she says. “It will also assist with the introduction and transfer of analytical methods internationally, where additional safety controls may be placed on existing mobile phase constituents.” Cross collaboration with both industry and academia experts has proven central to her efforts towards achieving her sustainability goals – using ion-pair free separations, such as hydrophilic liquid chromatography (HILIC). 

Meanwhile, Chen underlines miniaturization and high-throughput screening as some of the biggest emerging trends in the field that help researchers overcome technical challenges. Chen also notes that machine learning and AI may well come into play – aiding data analysis and helping us make faster, more informed decisions. Wei agrees, RNA-based therapies are “perfect candidates” for this approach because they have well-defined sequences and building blocks, which makes it easier to apply AI and machine learning techniques

But as one complexity challenge is addressed, another pops up in its place; for example, conjugate technologies that enable drug targeting add an additional dimension to the analytics, according to Cook. The development of lipid nanoparticles and conjugation of GalNAc to target liver hepatocytes has enabled better drug delivery and recent registration of therapeutics with these formulations or conjugations demonstrates regulatory acceptance. “I think we will also likely see other types of conjugations to oligonucleotides, which will further complicate the analytical strategy as these conjugates will exhibit their own impurity profiles. To ensure these drugs are safe for patients, analysts will need to continue to develop methods that cope with more complicated samples and impurities,” suggests Vanhinsbergh.  

“I think it’s safe to say we’re squarely into the era of nucleic acid medicine,” states Gadkari. To him, the possibilities of nucleic acids as therapeutic molecules are tremendously promising and analytical science will continue to play a strong role in ensuring that these therapeutics reach their potential. “This question about ‘the role of analytical science’ always surprises me when I see it. There is not a lot of science which can be done without analytical measurement. Everything has to be ultimately measured in some way, whether it’s simply for the sake of quantification or whether it’s for characterization,” he says. “Because of this, I feel like I can never think of a scenario where analytical science would not play a role in the advancement of any promising science.”

Through the Eyes of a Biopharma Pioneer

Koen Sandra shares his thoughts on the future of mRNA and oligonucleotide analysis
 

The approval of mRNA vaccines did not only help us confine the COVID pandemic, but has also urged the development of proper analytical methods to study various critical structural properties associated with mRNA – which compared to protein and antibody analytics are far less mature. I believe we are now at the point with mRNA and oligonucleotides that we were with antibody analysis 15 years ago, which creates plenty of opportunities – and challenges – for us analytical scientists! 

For example, intact mRNA is currently tricky to measure with MS, although some early attempts seem promising. LC-MS following ribonuclease digestion is gaining a lot of traction to assess various attributes of mRNA, such as 3’ poly tail length and post-transcriptional modifications. Measurements of intact mRNA are on the rise too. Capillary electrophoresis and chromatographic modes like SEC, AEX, IP-RPLC and HILIC provide information on mRNA integrity and fragmentation; as well as SEC on covalent and non-covalent aggregates. MALS, mass photometry and CD-MS are also relevant to obtain MW information on these molecular giants.

In addition, mRNA is typically formulated in LNPs – creating an additional analytical need. Techniques like SEC(-MALS), field flow fractionation (FFF) and DLS come in the picture here. 

With regard to oligonucleotides, these are generated using ribonucleases – which are amenable to LC-MS using either IP-RPLC or HILIC as chromatographic mode. The use of low adsorption flow paths is critical, hence, instrument and column vendors have introduced various solutions, e.g. biocompatible or bio-inert instrumentation, PEEK-lined or deactivated stainless steel columns.

This is especially important for HILIC but to a certain extent also IP-RPLC. HILIC is heavily explored as an alternative for IP-RPLC because the latter suffers from the use of sticky (contaminating) alkylamines and the use of HFIP, which is categorized as a PFAS (I’m not sure if there will be restrictions in the near future on the use of HFIP). MS sensitivity is, however, much higher when using IP-RPLC compared to HILIC. 

For synthetic oligonucleotides, phosphorothioate diastereomers are particularly troublesome. Do we want to reveal these or push them in one chromatographic peak – bearing in mind that they might have different pharmacological properties. Nowadays, oligonucleotides are coupled to antibodies or antibody fragments. Here knowledge of protein and oligonucleotide analysis is key – and completely new analytical challenges arise.

In conclusion, these nucleic acid-based medicines present a lot of fun for us analytical scientists! 

Koen Sandra is CEO & Co-owner, RIC Group, Kortrijk, Belgium; and Visiting Professor, Ghent University, Belgium

The stakes – and potential reward – have never been higher…
 

Evidently, the growing demand for nucleotide-based therapeutics, especially mRNA and oligonucleotide therapies, has spurred a wave of analytical innovation, with researchers developing advanced methods to meet characterization challenges – at all stages of the biopharma pipeline. “Their influence goes far beyond the bench,” says Meston.

Regulatory bodies must also keep pace with rapid scientific progress to ensure that safe and effective treatments reach patients quickly. Indeed, the balance between innovation and regulation will be crucial to embracing the era of nucleic acid medicine. “To reach full potential quickly, the analytics and [regulatory] approval processes need to be in place quickly,” advises Cook. “Many companies and regulatory bodies are working together to ensure these products reach the market faster than traditionally expected.”

Vanhinsbergh notes recent progress in this regard; for example, the EMA has released a draft of the “Guideline on the development and manufacture of oligonucleotides” – which can help analysts understand what regulators expect to see during submissions.

As analytical scientists across the world continue to rise to meet new challenges, our community must not be shy in celebrating the essential role we play in shaping and delivering next generation therapies. The future is exciting, but the path forward demands continued collaboration, creativity, and agility.