July 19, 2022 PAO-07-022-CL-06
Susan Darling (SD): SCIEX had been discussing mRNA analysis with many companies and scientists well before the emergence of the COVID-19 pandemic. Even before the pandemic, mRNA was being developed as therapeutics for cancer and as vaccines targeting a number of different viruses. The main challenge for mRNA was delivery to the cell before it was degraded. This was solved by formulation of mRNA into lipid nanoparticles. mRNA developers and their biopharma partners were seeking analytical solutions that could accelerate their efforts and provide more and better information faster and at earlier stages in the development process. The COVID-19 vaccines demonstrated the legitimacy of mRNA technology and that it is safe and efficacious in humans. That drove a significant amount of investment in startups developing novel mRNA therapies, as well as those creating new lipid nanoparticle (LNP) solutions. Big pharma companies are also very active in the mRNA space now, with most beginning to establish programs if they don’t already have them.
So, today there is a great deal of activity in RNA, but until recently there remained a lack of fit-for-purpose analytical tools needed to ensure that optimal manufacturing processes that yield the highest-quality mRNA product can be developed. mRNA is inherently unstable and prone to degradation during preparation, processing, formulation, and long-term storage. Rapid, robust analytical methods with high sensitivity and resolution for a wide range of mRNA molecules that can be readily transferred from R&D to QA/QC and can also be used for in-process monitoring are essential for maintaining this momentum and further accelerating mRNA vaccine and therapy development.
SD: It is important to determine the purity and structure — length and sequence, secondary structures that can form due to the size of mRNA, and the nature of the 5′ cap and poly-A tail needed for in vivo stability and proper function — of mRNA molecules. If the therapy is formulated using LNP or another delivery vehicle, the confirmation of mRNA encapsulation is also desired.
Many historical research tools, such as gel-based techniques, are fairly widely used. Conventional slab-gel electrophoresis (SGE) for structural analysis is generally variable and more qualitative, because it is based on staining and interpretation of lighter and darker bands. Northern blot analysis and real-time PCR are also used, but they can be troublesome, because they are labor-intensive and lack precision. Microchip-based gel techniques are also not sufficiently robust, reproducible, or sensitive for use in the QC environment.
Mass spectrometry (MS) methods can be used for rapid and reproducible detection, separation, and sizing of mRNA, 5′ cap analysis, and confirmation of LNP composition. However, these methods require highly skilled technicians to perform the analyses and to evaluate the generated data. Mass spectrometry is also limited in the size range of RNA products it can analyze.
As a result, various capillary electrophoresis (CE) methods are widely used for RNA analysis. CE is an automated version of gel electrophoresis technology that is much simpler, faster, and more quantitative while offering high resolution and precision, making it useful for confirming the purity, heterogeneity, and glycan association of biologic drugs of all types. It is well-suited for the analysis of large molecules, such as proteins and nucleic acids, including RNA. Specialized and standardized reagents and kits optimized for specific CE methods, such as capillary isoelectric focusing (CIEF), capillary zone electrophoresis (CZE), and CE-sodium dodecyl sulfate (CE-SDS) with multiple detection options, such as UV and fluorescence detection, provide complete workflow solutions that are sensitive and precise but also simple and flexible enough for QC applications.
What is particularly advantageous about CE with LIF detection is the ability to provide rapid, reproducible, and sensitive detection, separation, and sizing of RNA molecules with single-base resolution down to 15 nucleotides. In addition, CE was built on established CGE technology for protein analysis and is widely used in the biopharma industry to fully measure and characterize many different types of drug substances at every step in the discovery, development, and manufacture of drug products. It therefore serves as an important analytical tool for better understanding RNA products.
Quincy Mehta (QM): There is definitely a lot of interest right now in the development of analytical tools that work specifically for RNA analysis. Historically, a lot of bridging has been done to apply existing analytical solutions, but the growth of the sector really calls for more fit-for-purpose tools.
SD: RNA therapy and vaccine developers want high-resolution solutions that are “ready for primetime,” which means they have sufficient data reproducibility and robustness such that they can be utilized early in development and then moved into QC. That was the challenge SCIEX was tackling when coming up with the new targeted RNA analysis kit: the ability to rapidly and reproducibly separate a wide range of sizes of RNA molecules.
QM: The new RNA 9000 Purity and Integrity kit is one of the first analytical tools specifically targeted for RNA analysis, and it really allows us to harness the power of SCIEX CE and enable RNA product developers to leverage that technology.
SD: I also want to stress that it is important not to take for granted that we developers can simply fill a capillary full of what we call an “entangled polymer network” and do a separation. The ability to take what used to be a manual gel-based by-hand method and put it in a capillary would not be possible without technology developed and patented by SCIEX 30 years ago. The replaceable gel patent allowed 96-capillary DNA sequencers to be built and essentially enabled the sequencing of the human genome in an automated fashion. When SCIEX developed the kit for RNA analysis using CE, we built on our decades of experience filling capillaries with replaceable gel networks.
SD: Our PA800 Plus instrument was first introduced in 2004, and it has become an industry standard. It is used in the development of almost every protein-based biologic, including in the QC environment. I think that is why customers come to us with requests for new solutions that they can use on this instrument — they know that we offer kits for monoclonal antibody and single- and double-stranded DNA analyses, and they want other fit-for-purpose solutions that can also be used in QC, such as for RNA products. Recently, due to customer need for high throughput, we released a multi-capillary CE system so our customers can use these standard kits in a high-throughput mode.
QM: There are, actually, many different highly sensitive, automated, and robust workflow solutions leveraging the PA800 plus for nucleic acids. Standardized reagents and methods make it possible to implement consistent analyses anywhere in the world, which helps streamline process development and technology transfer within and between companies. Importantly, compared with chip-based CE systems, the PA800 Plus provides flexibility for method modification and optimization to generate optimal results for each specific project. When combined with such optimized methods, the PA800 Plus has the potential to be used as a platform technology spanning many different areas of research.
SD: SCIEX is part of Danaher. One of the five core Danaher values is “Customers talk, we listen.” This really drives our behavior. One year, we spent over 3,000 hours talking with customers. What we learned was that our customers are investing not just in mRNA but in many different modalities and are struggling because the techniques that are available are more research-grade and not suitable for supporting projects as they move from preclinical to clinical development stages.
That was the genesis of the project focusing on RNA. We started by developing a solution for the PA800 Plus that required customers to do a fair bit of preparatory work. Those discussions also led SCIEX to work on a new multi-capillary, multi-channel CE system. Our R&D chemistry, applications, hardware, and software teams worked to develop that system — the BioPhase 8000.
QM: The new RNA kit is a significant improvement over what was initially offered as a technical note that was developed for the PA800 Plus. This new kit also really helps round out what we offer in terms of different CE kits and applications. As Susan said, when we saw that initial interest in oligonucleotide therapies early on, we had existing kits for double-stranded oligo (DNA) analysis. While that is still a very hot topic, and those kits are readily being used, our kit-based offering for single-stranded analysis was missing a wider-range solution. The RNA 9000 Purity and Integrity kit focuses on the larger end of single-stranded oligo analysis and further rounds out our gene therapy and nucleic acid analysis capabilities.
SD: The key thing about the new RNA kit is that it is supported on both the BioPhase 8800 and the PA800 Plus. RNA therapy and vaccine developers can use this kit during early-phase work on the multi-capillary system to optimize methods more quickly and then analyze more candidates with much higher throughput from discovery through process development. Because validation and verification have been performed for both platforms, for which results are highly correlated, seamless transfer of optimized methods to the QA/QC environment is possible without the need for any bridging studies, which dramatically streamlines the process and lowers costs.
QM: The BioPhase 8800 system leverages a new cartridge that allows parallel processing of eight different CE samples simultaneously, providing all the advantages of CE-SDS and CIEF with UV or LIF detection while retaining sample integrity and delivering consistent, accurate results.
For mRNA characterization, the multi-capillary BioPhase 8800 system offers the ability to determine the size, integrity, and purity of eight samples in parallel with the necessary reproducibility, accuracy, and resolution required. It provides the next level of automation and data analysis necessary to simplify analyses, reduce analysis times, and increase consistency and accuracy, thereby accelerating early-development–stage screening activities.
QM: The new RNA analysis kit is purposely designed to enable the evaluation of RNAs with widely varying sizes. Use of the kit streamlines the work that pharma companies must do to develop robust analytical methods in the RNA space. It has been validated and verified for use on both the BioPhase 8800 and the PA800 Plus and simply requires dilution in water followed by injection.
The kit comes with everything typically required to analyze RNA samples: high-purity, CE-grade water, a commonly used dye for tagging RNA, and a wash solution for the uncoated capillary, which provides longer injection life compared to coated capillaries. The analysis takes approximately 20 minutes per sample.
SD: One of the really interesting capabilities of this kit that is really resonating with our customers is the ability to analyze RNA molecules of many different sizes. With other kits on the market, it is not possible to separate molecules ranging from 50 to 9,000 bases and to resolve small impurities and changes, which is the case with SCIEX’s new RNA kit. That makes it possible to analyze small guide RNA molecules used for CRISPR gene editing and large mRNA molecules in the same assay — you don’t have to run multiple assays.
QM: Beyond mRNA vaccines, there are other types of RNA products receiving a lot of attention these days. One is the guide RNA required for CRISPR Cas-9 gene editing, to which Susan alluded. The complexity is much greater in this case, because there is not only the mRNA payload encased in an LNP as in the COVID-19 vaccines, but an mRNA payload plus a guide RNA that directs that mRNA where to go. Guide RNAs are generally quite a bit smaller than mRNA molecules. Both are active ingredients in these therapies and therefore need to be well understood. Having the ability to analyze both in a single run significantly reduces the time and effort required to gain that important knowledge.
Another key feature of SCIEX CE technology is its highly reproducible, very smooth baseline. That matters because, with the new RNA kit, it is possible to easily detect impurities, even if they are very small and present in very low quantities. Being able to quantify those impurities gives our customers the super robust solutions they need to show that they have controlled methods and can confirm that every production batch results in a controlled and robust product, which is essential for gaining regulatory approval. Essentially, therefore, this new kit enables our customers to get to market faster.
In fact, it is the unknown about molecules in development that creates risk in the development of robust, reproducible, large-scale processes that are consistent. The extremely high resolving power and sensitivity of the RNA kit for CE analysis give pharmaceutical scientists the information that they need to more rapidly identify early mRNA candidates that have a much higher likelihood of becoming marketed medicines.
When the new analysis kit is used with the BioPhase 8800 — with assured data reproducibility within the same run on the eight capillaries — more candidates can be screened more quickly and at a higher quality level than could be done in the past at early development stages. In addition, RNA drug developers can be assured of good assay reproducibility as their candidates advance to the clinic and the market, accelerating development while significantly reducing risk.
SD: User experience is always a primary consideration, because we hear from our customers when it’s not what they want. If you introduce a tool or method into a QC lab, it has to be straightforward. It has to be user friendly, because the analysts in QC tend to be entry level and right out of school. In addition, there are typically eight to ten different techniques to run in the lab, whether in QC or development. The easier you can make sample preparation and system operation, the better the whole experience. This also helps researchers accelerate their projects, because they’re not futzing around trying to learn complex software or hardware or doing a whole bunch of sample prep.
With this kit, we wanted every single thing that technicians need in the kit, beyond the pipettes and the tubes and other basic laboratory consumables. Instead of having to get reagents from different vendors, everything required for the analysis is in that one box, so the user experience is simple and easy.
QM: With each new product that we develop, SCIEX is always focused on providing cutting-edge analytical tools to accelerate pharmaceutical research and development and to reduce the time to commercialization of new drugs and vaccines. We want to help drug developers bring novel medicines to the market that can change patient lives.
For next-generation molecules like RNA, we know that the existing, traditional research-grade molecular biology tools and protein analysis tools that have been adapted to support new drug development were not fit-for-purpose and often lacked the robustness and performance required for drug manufacturing. With products like the new RNA kit, we are working to fill in that gap while also making them as easy to implement as possible.
QM: We are always looking to the future, because everyone in the biopharma industry is always trying to push the envelope to find new treatments for patients in need. For instance, drugs moved from simple small molecules like acetaminophen to more complex small molecules and then the first biologics, including recombinant proteins and monoclonal antibodies. The next wave of evolution is upon us now with next-generation biologics, including cell and gene therapies, nucleic acid–based medicines, and treatments delivered using viral vector systems. Going forward, we will not only see advances in how these therapeutics are manufactured, but improvements in efficacy and further developments in technologies with even greater complexity. Analytics will need to continue to evolve in order to support these advances.
SD: In fact, at SCIEX we are already being asked what else we can do in the RNA space. We are already back at the drawing board looking for the next innovations. We have applications that support gene therapy and cell therapy development and enable genomic integrity analysis of viral vectors. We of course also offer solutions for monoclonal antibodies and next-generation multispecifics, antibody fragments, and so on.
What is great about these solutions is that all of these kits and methods are run on just one system — the PA800 Plus and more recently the BioPhase 8800. Researchers come to us because they already know how to use the system for many molecules in their candidate and approved product portfolios and they want to do even more using what is familiar to them. SCIEX is committed to helping them by developing other analytics they can use on the same system.
SD: One of the key areas of our SCIEX strategy is what we call “closing the developability gap.” What that means is providing researchers with solutions that allow them to better and more quickly characterize potential drug candidates upfront to weed out the ones that will fail and avoid discovering problems during manufacturing or formulation or even in the clinic.
Closing the developability gap is difficult enough with recombinant proteins and antibodies for which there is a large, established knowledge base. It is an even bigger challenge for the newer modalities in the pharma pipeline, because not only are the drug molecules new and highly complex, there is even greater pressure to get them to market as soon as possible, because the first to market wins. For most next-generation molecules, ideal analytical tools are not available, and therefore candidates are being moved through development with more risk.
At SCIEX, our strategy is to look for opportunities where we can help customers close that gap and link structure to function; not just provide a really rapid assay but make it possible for them to do the characterization they need upfront in order to make informed decisions and get drugs to market faster.
Getting drugs to market faster is a big focus in the industry right now, driven further by the successful development of the COVID-19 vaccines. We’ve had customers say that they want everything to move at pandemic speed — that they want to go from IND filing to BLA in a thousand days. That’s just a little under three years. It seems mindboggling, but that is what our customers are telling us, so that is what our strategy is all about — developing solutions that help them achieve that goal.
QM: I would like to underline that the multi-capillary BioPhase 8800, which we launched late last year, is an important tool that is already helping customers on that path. It supports rapid movement through early- to late-stage development and commercialization, because the highly reproducible and robust methods developed on the BioPhase 8800 can be seamlessly transferred to the single capillary PA 800-Plus.
The RNA 9000 Purity and Integrity kit is another important example, because it has been qualified and validated on both of those systems. Developers of RNA-based therapies and vaccines now have a method and platform that can be used from early-phase development all the way to QC release, which definitely helps accelerate the entire process.
Susan Darling is the Senior Director of capillary electrophoresis and capillary electrophoresis (CE)–mass spectrometry (MS) product lines at SCIEX. She is responsible for strategic direction of CE and CE-MS at SCIEX as well as new product and workflow development.