As mRNA therapeutics continue to evolve, analytical scientists face increasing pressure to characterize complex, fragile molecules that resist conventional approaches. According to Matt Ranaghan, Senior Applications Scientist at Refeyn, the structural complexity of mRNA, including the formation of higher-order structures and aggregates, creates significant analytical bottlenecks. Mass photometry (MP) offers a fast, accurate, and native-state solution to many of these challenges.
“Many of the challenges with mRNA characterization are attributed to the ability of mRNA to adopt various complex secondary and tertiary structures,” Ranaghan explains. “These higher-order structures can lead to the formation of larger aggregates, meaning a single sample may contain a mix of full-length transcripts, degraded fragments, and aggregates—all of which interfere with analytical techniques and complicate quality control.”
Traditional analytical tools, such as capillary gel electrophoresis (CGE) and high-performance liquid chromatography (HPLC), often require the use of harsh denaturants or elevated temperatures to analyze these molecules. These steps not only lengthen the process but also alter or damage the sample.
Ranaghan notes that these methods often lack the comprehensive resolution needed to accurately assess impurities in their native state. Mass photometry circumvents these issues by enabling direct, label-free measurement of intact mRNA molecules in simple biological buffers. “This allows researchers to characterize mRNA integrity and purity quickly and accurately—without compromising the molecule itself,” he adds.
Overcoming Size and Stability Barriers
As mRNA sequences scale up to 10 kilobases or more, their increased size and heterogeneity make accurate characterization even more difficult. Ranaghan says that MP excels with these large molecules: “We have shown that it can accurately measure the intact mass and length of large mRNAs (up to 10 kb) with less than 5% error,” he notes. "This accuracy is significantly better than the error often seen with conventional techniques like CGE."
“Unlike chromatographic techniques, which can require extensive sample preparation, mass photometry typically requires only a simple dilution—enabling high-quality data collection in minutes.” He adds that MP can simultaneously characterize multiple attributes of an mRNA sample—including length, purity, and aggregation state—which otherwise would require multiple complex methods. This capability makes it an efficient tool across discovery, process development, and quality control.
A Complementary Tool to LC-MS and CDMS
While LC-MS and charge detection mass spectrometry (CDMS) remain key analytical tools for nucleic acid characterization, Ranaghan emphasizes that MP fills an important gap, a finding also highlighted in recent research. LC-MS, for instance, often requires enzymatic digestion of the mRNA into short fragments, resulting in only 80– 85% sequence coverage.
“Mass photometry requires minimal sample preparation and measures the intact molecule in its native state,” Ranaghan observes. “That avoids misrepresenting the natural distribution of mRNA species and related impurities that can result from fragmenting or ionizing fragile molecules.”
Rather than replacing established methods, Refeyn envisions MP as a rapid first-line analysis tool. Quickly screening for sample integrity, fragmentation, and aggregation, it allows scientists to determine when a deeper, more complex analysis with LC-MS or CDMS is warranted. “MP provides a fast, accurate, and label-free measurement that preserves the native structure—a critical advantage for assessing fragile mRNA molecules,” advises Ranaghan.
From Research to Manufacturing
Refeyn’s technology is gaining traction among biotechnology and pharmaceutical developers who wish to streamline impurity profiling and in-process monitoring of mRNA production. Mass photometry's ability to analyze dilutions of crude samples provides two key advantages in the analytical workflow, as demonstrated in recent papers by Camperi et al. and Schmudlach et al.:
- Comprehensive impurity profiling: MP provides an accurate determination of nucleotide numbers and detects size variants, such as aggregates, degraded fragments, and poly(A) tail differences, that can affect product quality.
- Rapid in-process monitoring (IPM): MP can rapidly analyze dilutions of crude samples directly from production stages because it is insensitive to modest variations in salt concentration, buffer, or pH. This allows for quick, real-time quality checks that inform process decisions.
While GMP-compliant software for MP in mRNA workflows is not yet available, Refeyn has already implemented such solutions for its AAV (adeno-associated virus) analysis platform—a model the company expects to extend to RNA-based therapeutics. “We see MP becoming a valuable addition to manufacturing and quality control workflows, once GMP compliance for RNA analysis is achieved,” says Ranaghan.
Looking Ahead: Beyond mRNA
Refeyn continues to expand the capabilities of mass photometry to address emerging therapeutic modalities. “We are exploring how MP can address other mRNA critical quality attributes, like double-stranded RNA detection and 5' capping efficiency,” Ranaghan says. He also notes that the company is developing methods for analyzing CRISPR guide RNAs, which are prone to aggregation and difficult to assess with conventional techniques.
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Beyond RNA, Refeyn expands the mass photometry platform with specialized instruments designed to meet the analytical challenges of viral and non-viral vectors. The technology for characterizing adeno-associated virus (AAV) capsid integrity is already mature, enabling the rapid measurement of empty versus full capsids and their incorporation into regulatory guidance. Recent advancements also extend MP capabilities to larger viral systems, such as lentivirus and adenovirus, for assessing sample purity, heterogeneity, and titer.
A New Standard for Rapid, Native-State Analysis
As the field advances toward larger and more complex RNA therapeutics, Ranaghan believes mass photometry will play an increasingly central role in accelerating development and improving product quality. “MP offers a fast, easy, and accurate way to characterize critical quality attributes of intact mRNA with minimal sample requirements,” he concludes. “By enabling native-state measurements that are both rapid and quantitative, MP helps overcome long-standing analytical bottlenecks and supports innovation across the RNA therapeutics pipeline.”