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Analytical approaches must keep pace to ensure the identity, safety, and efficacy of evolving mRNA candidates.
The success of the messenger RNA (mRNA) vaccines against the SARS-CoV-2 virus brought attention to a field that has steadily advanced for many years. Investment in this space has ramped up dramatically, and many applications of mRNA technology are rapidly advancing through pre-clinical development as well as clinical trials.
As with all drug or vaccine candidates, proposed mRNA products must meet regulatory requirements for analytical characterization as outlined by the International Conference on Harmonisation (ICH), the World Health Organization (WHO), FDA, the European Medicines Agency (EMA), and other health authorities around the globe.
Unlike most other drug substances, mRNA must be formulated within specialized nanoparticles, most commonly consisting of lipids, both to protect it from degradation and to facilitate its transit across cell membranes. Additional analytics are therefore required to ensure the quality and safety of the nanoparticle formulation as well as the mRNA molecule itself.
A key activity in the early development phase of mRNA therapeutics is extensive characterization of mRNA and nanoparticle quality attributes as these are known to strongly influence biological efficacy. Given the shrinking timelines for drug development, access to faster and higher-throughput analytical methods has become vital. Indeed, it is essential that analytical approaches for use during mRNA product development and quality-control (QC) testing must keep pace to ensure the identity, safety, and efficacy of these new prophylactic and therapeutic modalities.
Although analytical requirements for mRNA candidates are more complex than those for traditional biologics, some testing needs are similar while others are quite different. “There is a common need to adequately characterize both protein therapeutics and mRNA therapeutics to confidently identify and quantify any quality attributes that will impact their efficacy and safety,” says Todd Stawicki, global marketing manager, mass spectrometry, for the biopharmaceutical industry at SCIEX. He points to the need to identify process-related impurities—host-cell proteins (HCPs) in the case of protein therapeutics and a variety of genetic impurities like truncated transcripts or incorrect DNA template in the case of mRNA.
The differences are notable, however. Rather than focusing on post-translational modifications as is the case for protein therapeutics, there is a lot of emphasis on characterization of mRNA, the delivery systems, and cellular uptake at the discovery phase of drug development, because unlike engineered proteins and monoclonal antibodies (mAbs) that are ready to start functioning upon delivery, mRNA once delivered to cells provides instructions for producing the proteins that do the work, according to Brian Liau, a solution development scientist at Agilent Technologies, Singapore.
Manufacturing for mRNA products, furthermore, requires unique process steps such as in vitro transcription (IVT) and lipid nanoparticle formulation, which naturally creates different analytical needs, adds Ryan Hanko, manager of quality control for Catalent. IVT is a chemical rather than a cell-based process, expounds Joe Fredette, senior business development manager of biopharmaceuticals at Waters. It also involves the use of an enzyme called an RNA polymerase that transcribes DNA into mRNA.
As an example of different analytical needs, Hanko notes that mRNA products require QC assays to be developed that measure residual enzyme (such as polymerase or endonuclease), which may be present after the IVT step, to ensure that it is not present in the final product. Confirmation of the RNA sequence and determination of the efficiency of capping at the 5’ end and poly(A) tail distribution at the 3’ end are also unique to mRNA, notes Ashleigh Wake, business development director for Intertek Pharmaceutical Services. The potential impurity profile is also quite different to other molecules, and methods to quantify any residual plasmid or double-stranded RNA are also required.
In addition, lipid analysis workflows are necessary for characterization and chemistry, manufacturing, and controls (CMC) testing of mRNA products, including liquid nanoparticle (LNP) composition analysis to ensure the right mix ratio of lipids within the formulation, lipid ID confirmation, and impurities analysis and screening, according to Fredette.
In essence, summarizes Wake, there is a similarity in the overall requirements for product characterization in that the intent is to assess or confirm identity, purity/impurities, stability, activity, etc. “However,” she underscores, “to fully accomplish this, there is a need to include mRNA-specific analytics that allow assessment of these attributes.”
Another important difference between the development of protein- and mRNA-based products is the analytical burden during the early discovery phase. As traditional drug development typically involves mAbs, says Dr. Liau, bioanalytical requirements tend to be relatively modest because even sub-optimal molecules may be used to demonstrate target engagement and a therapeutic effect. In contrast, mRNA products require several biological processes, including cellular uptake, endosomal escape, and protein translation to take place efficiently before any effect may be observed. Hence, analytical characterization of mRNA and delivery vehicle quality attributes are important from the outset.
“For process scale-up and GMP [good manufacturing practice] manufacturing/product release, chemical characterization of mRNA remain important. However, the focus tends to shift towards ensuring consistent size polydispersity and mRNA encapsulation in the formulated product, as well as identifying process-related impurities,” Dr. Liau comments.
With mRNA candidates, Stawicki adds that scale up of the enzymatic process requires significant and difficult scale up for all upstream raw materials—nucleoside triphosphate (NTPs), enzymes, capping reagents, cofactors, and multiple classes of lipids. “The majority of these components are large, complicated biomolecules with their own associated scale up, purification, and analytical challenges,” he explains.
There are also some differences in the attributes that are monitored during production, according to Hanko. A260 concentration checks and osmolality testing are the primary monitoring assays in early development and process scaleup for in-process observations, while during GMP manufacture RNase testing is important to ensure the final product will not be compromised by incidental contamination from non-product materials.
Core release tests include identity determinations, final concentration, impurity content (e.g., residual protein, dsRNA, rDNA), capping efficiency, and poly-A tail, in addition to typical compendial evaluations for safety and quality attributes.
There is an extensive list of critical quality attributes (CQAs) that must be monitored to meet regulatory requirements regarding the assessment of the identity, functionality, and safety of mRNA therapeutic/vaccine products (see Table I). “Confirmation that the specific mRNA sequence has been generated, that the mRNA solution is at target pH, and that any potential process impurities (solvent, protein, DNA-related) have been controlled will be required,” says Matthew Howard, a senior specialist in quality assurance at Catalent.
Assurance will also be needed that the translation-promoting and
degradation-preventing characteristics of the 5’ cap and poly-A tail are sufficient, says Howard. The efficiency of capping at the 5’ end can influence both target protein production and overall immunogenicity, while the length/distribution of the poly(A) tail is critical for translation and overall protection of the mRNA, Wake explains.
Adds Maxwell Meller, senior scientist, quality control, with Catalent: “The 5’-capping of the mRNA molecule is critical to its integrity upon delivery to the target site and is a unique requirement for mRNA production compared to other kinds of drugs. Characterization and monitoring of capped material, however, have proven to be time- and resource-intensive.”
For the lipid components, confirmation of the identity of each lipid in the LNP mix and that they are present in the correct ratio is essential as these CQAs can directly impact LNP formation and efficacy, according to Fredette. It is also important, he emphasizes, to ensure that lipid impurities are well controlled for and below critical thresholds, as both lipid and mRNA impurities can adversely impact process outcomes and in some cases lower the potency of the mRNA.
The sensitivity of mRNA to degradation by enzymes, particularly RNase, creates additional unique requirements when working with mRNA-based therapeutics and vaccines. For instance, Hanko observes that the use of RNase-eliminating preparations is vital and laboratories should consider physical space segregation in addition to personnel training and awareness regarding the maintenance of RNAse-free zones, equipment, and personal protective equipment (PPE).
These actions are important, Hanko says, because a compromised mRNA sample will show lower-than-expected purity in those assays. Some analyses, as those for residual impurities, are arguably not impacted by compromised mRNA and others may only require heightened RNase sensitivity depending on solution preparations and material manipulations. “Even so,” Hanko states, “the core method performance expectations must be understood for all methods regardless of the level of mitigation required.”
One of the biggest issues facing mRNA analysis is experienced across the board in the biopharmaceutical industry. “The great enemy here is time,” Stawicki asserts. “The unfortunate reality is that many of the current functional and biophysical tests are still too slow to generate actionable answers in a timeframe that enables intervention in the process,” he explains.
For instance, Stawicki highlights cell-based functional assays, which are the gold standard today. “This test is the best early indication that you’ve got everything right,” he says. “You dose your formulated mRNA LNP to cells and see if you get sufficient yield of the right protein or antigen. The problem is that it can take days to get a read out. Therefore, often times developers have to proceed at risk in their processes without having the analytical data to back up their process parameters.”
Bioanalysis of mRNA products requires a combination of familiar and more novel analytical techniques and technologies. It also necessitates, Hanko reiterates, the control and elimination of RNase enzymes, which may compromise product integrity.
A wide variety of analytical techniques are employed to evaluate these properties. What is interesting to Stawicki is that they have come from a variety of different backgrounds. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) have migrated from protein-therapeutic characterization. Electrophoretic techniques (most notably capillary electrophoresis) have come from classic molecular biology use. Particle sizing techniques that have come from polymer analysis or materials lab use.
“Since mRNA therapeutics are such large, heterogenous complexes, it really comes down to which part or parts of the candidate require characterization,” Stawicki remarks. For the mRNA component, he notes that capillary electrophoresis or even slab-gel electrophoresis methods are preferred. For characterization of lipids and LNPs, HPLC with charged aerosol detection (CAD) or LC-MS are generally the methods of choice. For complete mRNA-LNP complexes, techniques such as particle sizing or cryo-electron microscopy are preferred.
Some common activities during early development are mRNA codon optimization and selection of optimal 5’ and 3’ untranslated regions (UTRs), says Dr. Liau. As this entails cloning coding sequences out of libraries and combining them in multiple plasmids with different UTRs, Sanger sequencing, capillary or slab-gel electrophoresis, and polymerase chain reaction (PCR) remain mainstay analytical methods during this phase of development.
Characterization and CMC testing for the mRNA component of the drug, according to Fredette, includes a range of nucleic acid (DNA/RNA) analytical workflows to confirm the identity and purity of the mRNA, including 5’ Cap analysis, PolyA tail analysis, oligo mapping (similar to peptide mapping, but for nucleic acids), and MS-based sequencing.
MS, meanwhile, is also used to establish key quality attributes such as capping efficiency and poly(A) tail distribution, notes Wake. She adds that all of these analyses are performed in addition to more traditional QC requirements, such as assay [ultraviolet (UV)/HPLC)] analytics, residual solvents, metals, etc.
To confirm the biological activity of mRNA candidates, well-studied lipid delivery systems using cationic or ionizable lipids may be used to transfect standard cell lines in vitro. “For such early-stage experiments,” Liau observes, “it is often unnecessary to use highly purified mRNA or monodisperse preparations of lipid nanoparticles. Instead, bulk dynamic light-scattering and zeta potential measurements are often used to ensure the nanoparticles have approximately the correct size and surface-charge properties to permit efficient cell transfection. Biological activity may then be determined by fluorescence microscopy, enzymatic assays, or immunostaining as appropriate.”
Similar analytical techniques are generally used from early in development through the GMP environment. For instance, LC and LC-MS workflows bring value across all stages of development and for process monitoring in manufacturing, Fredette observes.
Because the goals of process optimization and scaleup are often quite different from those of early-stage development studies, however, Liau comments that different analytical techniques are required in some cases. “During process optimization and scaleup, the mRNA sequence is fixed, and its biological activity known, and the focus is on monitoring and/or improving the process yield and mRNA quality attributes,” he says.
LC-MS is very useful both for monitoring the progress of in vitro transcription reactions and determining the status of critical quality attributes such as 5’ capping, Liau adds. Separation-based techniques such as size-exclusion chromatography-multiangle light scattering (SEC-MALS) and field-flow fractionation (FFF)-MALS may, meanwhile, be useful for monitoring the properties of LNPs and optimizing the complexation process.
One important trend noted by Stawicki that is also seen with protein therapeutics is the preference for increasingly robust and simpler techniques the further down the pipeline programs get, Meller agrees. For instance, he notes that while LC-MS is a promising tool for characterizing and quantitating capped species, a more simplistic approach lends itself more appropriately to routine use. “In the QC environment,” says Meller, “the chromatography analysis must be condensed to an HPLC assay to prioritize speed and efficient data analysis.”
In general, Wake believes that many of the methods or approaches used to fully understand mRNA are novel in the QC area, when compared to those used for other modalities. “To fully support all regulatory requirements, greater use of technologies such as next-generation sequencing, which have not been typical technologies operated in a GMP environment, has been necessary,” she explains.
For other considerations such as capping, however, Wake says that the technologies used are more familiar and tend to be heavily MS-based. Even in these cases, though, she remarks that the overall sample preparation approach is often novel and challenging. “A lot of work is being done in our and other laboratories to develop platform approaches to overcome these challenges considering best science, quality data, and regulatory compliance,” she concludes.
There is, in fact, tremendous effort being made to advance analytics for mRNA vaccines and therapeutics. “Scientists have been working with mRNA for decades, but a lot of that effort has focused optimizing analytical methods to enable production-grade techniques. With the explosion of genetic therapies and the now intense interest in mRNA, you are seeing a tremendous amount of investment and innovation by scientists and instrument companies to develop the best analytical solutions to support that work,” Stawicki contends.
SCIEX, for instance, has introduced electron activated dissociation (EAD) as a new MS fragmentation technology in the ZenoTOF 7600. Initially launched with small-molecule and protein therapeutics in mind, the company has since learned, according to Stawicki, that EAD technology is also powerful for analyzing mRNA products. “EAD has amazing utility for both oligonucleotide and LNP characterization, allowing, for example, the sensitive and complete structural characterization of lipids including double bond positions and isomer configurations in a single experiment.”
Stawicki also points to SCIEX’s new BioPhase 8800 multi-capillary CE instrument, which allows up to eight analyses to be performed simultaneously on the same or different samples. “We are exploring how to leverage the high throughput capabilities of the BioPhase for a whole range of different mRNA applications,” he says.
Agilent recently developed an improved method for rapid analysis of mRNA 5’ capping by LC-MS, according to Liau. Enzymatic cleavage is required for this analysis because mRNA is such a large molecule. The established method required two challenging sample preparation steps: affinity capture using biotinylated oligonucleotide probes, and site-directed cleavage of affinity-captured mRNA using RNase-H.
This method failed to work reliably with some mRNA sequences due to inefficient affinity purification, so Agilent developed an improved sample preparation method comprising site-directed cleavage in solution without affinity capture followed by purification of the cleaved oligos and mRNA sample matrix using silica-based columns. Using a thermostable version of RNase-H enzyme allowed the sample preparation time to be reduced as well.
The total process—sample prep, analysis, and data processing—requires just 75 minutes and allows separation of the oligos of interest from the mRNA sample matrix without further sample cleanup.“When used in conjunction with a specially-designed flushing protocol to mitigate sample matrix buildup on the column, this method yielded superior linearity, sensitivity, and reproducibility,” Liau says.
Howard highlights several promising techniques currently being refined for the evaluation of protein expression in cells, such as flow-cytometry for the detection of intracellular dsRNA levels and antigen-specific fluorescent detection of expressed proteins, both of which provide rapid results for cell uptake, RNA amplification, and protein production. “Such direct measurements could reduce the turn-around time for in vivo antigen studies. The new techniques certainly look promising and could be practical in the next few years,” he believes.
Wake returns her attention to NGS technology; even though it isn’t new in general, its use in the GMP environment is, and for mRNA products the introduction of NGS is vital to achieving the required quality of control. Similarly, she says that electron microscopy is an essential technique, along with approaches for establishing in vitro release performance, for evaluating encapsulated products. Doing so, however, has required the adaptation of a kit that has mainly been associated with US Pharmacopeia type 4 dissolution testing.
The importance of developing platform analytical technologies is also reiterated by Wake. The continual improvement of platform approaches suited for establishing if mRNA is truly encapsulated within the nanoparticle system at the right internal liposome pH to support early-phase products without the need for extensive development is a promising example, she says. As the product moves into later phases, these same methods are optimized and eventually validated for each specific product. “However,” Wake argues, “understanding these critical aspects as early as possible in the development cycle necessitates the need for a general approach.”
It is important to remember that mRNA technology itself is advancing rapidly and evolving on a continual basis. Developers of analytical methods for mRNA therapeutics and vaccines are therefore challenged not only by the complexity of these biomolecules and their delivery vehicles, but also by the need to develop flexible methods that can accommodate the ongoing changes in mRNA structures and final formulations.
As an example, Wake notes that while to date mRNA products have been developed as injectables, inhalation and nasal delivery present viable alternatives. “These routes of delivery can not only aid in achieving improved bioavailability, but also lead to products that can be delivered in a less evasive manner, even in the absence of a healthcare lead administration system,” she explains.
Scientists, Fredette recommends, should think about the analytical platforms and workflows they need to deploy across their organizations as they grow and how best to harmonize everything, from method development and method/data transfer; to ensuring data integrity, traceability, and compliance; to the level of service and support they want and need.
“I don’t even think we have started to approach the steady state for what analytics look like for mRNA therapeutics,” states Stawicki.“Just the space of what we consider mRNA therapeutics continues to grow rapidly since elasomeran (Spikevax) from Moderna and tozinameran (Comirnaty) from Pfizer-BioNTech became world-changing vaccines. Many companies are now developing variations on mRNA such as self-amplifying mRNA and circular mRNA.In addition, scientists are developing new types of vehicles and targeting strategies for mRNA therapies.So yes, we are still at the beginning stages of this technology revolution,” he concludes.
Cynthia A. Challener, PhD, is contributing editor to BioPharm International.
Vol. 35, No. 2
When referring to this article, please cite it as C. Challener, “Analysis of mRNA Therapeutics and Vaccines,” BioPharm International 35 (2) (2022).