Analyzing mRNA Therapeutics With Mass Photometry

1 www.refeyn.com Accelerate mRNA analytics with mass photometry How mass photometry accurately assesses multiple critical quality attributes of mRNA – in the native state, in minutes, with nanograms of sample 2 www.refeyn.com Contents Introducing mRNA analysis with mass photometry Mass photometry in action Measuring mRNA length and integrity Accuracy and reproducibility Analyzing mRNA purity in non-denaturing conditions Analyzing mRNA stability Mass photometry vs. other methods Mass photometry vs. CDMS Summary References Mass photometry solutions for mRNA analysis Mass photometers Mass photometry software Mass photometry consumables 3 6 6 7 8 9 11 11 13 14 15 15 16 17 3 www.refeyn.com Introducing mRNA analysis with mass photometry Messenger RNA (mRNA) has become a central focus in modern therapeutics, driven by its success in vaccines as well as its growing potential in treatments for cancer and other conditions. Developing and producing these products requires thorough characterization by assessing a range of critical quality attributes (CQAs). mRNA CQAs relate to several sample qualities, including potency, safety, identity, content, purity and integrity (Fig. 1). Figure 1. Mass photometry offers a rapid, end-to-end solution for mRNA CQAs related to sample purity and integrity. For some CQAs, such as those related to sample purity and integrity, existing methods (Table 1) are slow, costly and complex to run, and they require denaturation, digestion or other modifications that can affect the results, while often using large amounts of sample. By contrast, mass photometry, a relatively new addition to the mRNA analytics toolbox, is fast, accurate, easy to use, and able to analyze samples in their native state using little sample. Native analysis in minutes Analysis of large mRNA molecules Multiple attributes with minimal sample 4 www.refeyn.com Table 1. Mass photometry can inform on a range of mRNA CQAs, which would otherwise require multiple methods and measurements to assess. In this handbook, we describe how mass photometry can be used to assess these CQAs and discuss its advantages. CQAs for mRNA therapeutics need to be monitored during product development and reported on in regulatory submissions. Guidelines on methods to test the quality attributes of mRNA therapeutics such as these are being developed by the US Pharmacopeia,1 but methods are still evolving. Mass photometry is an attractive tool for analyzing mRNA sample purity and integrity. CGE = Capillary Gel Electrophoresis; ELISA = Enzyme-Linked Immunosorbent Assay; HPLC = High-Performance Liquid Chromatography; IP = Ion Pairing; LC = Liquid Chromatography; MS/MS = Tandem Mass Spectrometry; RP = Reversed-Phase; SEC = Size-Exclusion Chromatography. Quality Attribute Methods Integrity mRNA intactness CGE, Agarose gel, Mass photometry Purity mRNA purity IP-RP-HPLC, Mass photometry Presence of impurities (aggregation) SEC-HPLC, Mass photometry Presence of impurities (dsRNA) ELISA, Mass photometry Box 1: Introducing mass photometry Mass photometry works by illuminating 10–20 microliters of sample on a glass surface with a laser, and then measuring the interference of light scattered by molecules in a sample with light reflected by the glass surface (Fig. 2). For the measurement of nucleic acids, the glass surface is functionalized with a cationic coating that enables the negatively charged molecules to interact with the glass, enabling the measurement. The resulting signal, known as the ‘contrast’, is proportional to the mass of each molecule.2 Mass photometry reports the mass distribution of all the particles it detects in a sample, providing an overview of all the components present, with single-molecule resolution. With over 1,200 peer-reviewed studies now citing mass photometry, it is becoming an established technique for applications that benefit from rapid, label-free, single-molecule mass distribution analysis, including the measurement of antibody aggregation, adeno-associated virus (AAV) empty/full ratios, and protein-protein interactions. Mass photometry is also now emerging as a powerful tool for multiple-attribute mRNA analysis, and has been cited in several recent publications.3–6 This handbook demonstrates how mass photometry is an ideal analytical tool for analyzing mRNA purity and integrity. Using rapid, single-molecule mass measurement, mass photometry enables the detection and quantification of distinct populations within samples. It analyzes samples in their native state, returning a mass profile that reveals multiple attributes – intactness, purity, aggregation and the presence of product-related impurities. Measurements take just a few minutes and require only nanograms of sample, and mass photometry can be used to measure mRNA across a broad size range (including large mRNAs). For a deeper understanding of how mass photometry works, Read Refeyn’s handbook Understanding Mass Photometry, Watch the webinar Mass Photometry 101: A Deep Dive into Principles and Applications. 5 www.refeyn.com Figure 2. Mass photometry measures the molecular mass distribution of samples at the singleparticle level. It can be applied to a variety of biomolecule types, including nucleic acids. 4 How does mass photometry work? The particles which interferes with the light at the interface 2 Mass (kDa) Contrast The resulting interference contrast scales linearly with the particles’ mass 3 A mass histogram is generated from the single-particle measurements Particles, such as proteins, nucleic acids and viral vectors, on a glass surface are illuminated by a laser Incident light Particles in solution 4 1 Mass (kDa) Counts Contrast Scattered light Reected light Particles in solution 1 3 Particles in a sample on a glass surface are illuminated by a laser. The particles scatter light, which interferes with the light reected at the interface Mass (kDa) Contrast The resulting interference contrast scales linearly with the particles’ mass 3 A mass histogram is generated from the single-particle measurements Particles, such as proteins, nucleic acids and viral vectors, on a glass surface are illuminated by a laser Incident light Particles in solution 4 1 Mass (kDa) Counts Contrast Scattered light Reected light Particles in solution The resulting interference contrast scales linearly with the particles’ mass. 2 The particles scatter light, which interferes with the light reected at the interface 2 Mass (kDa) Contrast The resulting interference contrast scales linearly with the particles’ mass 3 A mass histogram is generated from the single-particle measurements Particles, such as proteins, nucleic acids and viral vectors, on a glass surface are illuminated by a laser Incident light Particles in solution 4 1 Mass (kDa) Counts Contrast Scattered light Reected light Particles in solution The particles scatter light, which interferes with the light reflected at the interface. 4 The particles scatter light, which interferes with the light reected at the interface 2 Mass (kDa) Contrast The resulting interference contrast scales linearly with the particles’ mass 3 A mass histogram is generated from the single-particle measurements Particles, such as proteins, nucleic acids and viral vectors, on glass surface are illuminated by a laser Incident light Particles in solution 4 1 Mass (kDa) Counts Contrast Scattered light Reected light Particles in solution MassFerence® P1 calibrant A mass histogram is generated from the single-particle measurements. TM 6 www.refeyn.com Mass photometry in action To demonstrate how mass photometry performs as a tool for measuring mRNA purity and integrity, we present a series of examples in this section. They show that mass photometry: • Accurately measures the length and integrity of mRNA, including long mRNAs • Analyzes purity and other CQAs in non-denaturing conditions • Has high accuracy and reproducibility • Compares favorably to other methods Measuring mRNA length and integrity Accurately measuring the length of mRNA – including long mRNAs, such as those encoding Cas9 (>4 kb) as well as saRNA (~10 kb) – is a persistent challenge in therapeutic development. Large transcripts, in particular, are prone to partial degradation, and even small deviations in length can affect translation efficiency, protein folding, or potency. Confirming that mRNA is full-length and intact is therefore essential. Mass photometry can quickly measure mRNA length and integrity because it reports the mass distribution of all the particles in a sample. If the distribution shows that there is just one population of molecules present, with a peak at the expected mass (or length) value, it indicates that the molecules in the sample are intact. To demonstrate this, we used mass photometry to analyze three samples of mRNA transcripts of different lengths (eGFP, 980 bases; FLuc, 1.9 kb; and eSpCas9, 4.5 kb) and one saRNA sample (eGFP, 10.1 kb). Figure. 3. Mass photometry accurately measures mRNA and saRNA length. (A) Samples of mRNA (EGFP, FLuc and eSpCas9) and saRNA (EGFP) varying in length from 980 bases to 10.2 kb were analyzed by mass photometry. In every case, the mass photometry measurement was highly accurate, returning lengths (determined from the mean of the main peak in the measured distribution) that were in close agreement with the expected values. Note that while mass photometry measures an optical interference signal known as the contrast, this can be readily converted to mass (or mRNA length) through initial calibration with an mRNA molecule (or ladder) of known length(s). (B) Plotting the measured vs. expected mRNA length values shows that the mass photometry measurements are highly accurate. Triplicate measurements for the same samples as in A. are shown; error bars (±standard deviation, SD) are smaller than the markers. Measured on the TwoMP mass photometer. 7 www.refeyn.com In each case, the sample was simply diluted (to 1-10 nM concentration) and measured, and the analysis revealed one main population with a length in close agreement with the expected value for that sample (Fig. 3A). Plotting the measured values against the expected values yielded an R² value of 1.00, indicating excellent linearity and agreement (Fig. 3B). Overall, this example shows that mass photometry accurately measures mRNA length, helping reliably assess the integrity of the molecules in a sample. It also shows that mass photometry’s broad dynamic range readily accomodates the different lengths of mRNA molecules that require analysis. It can detect and measure single stranded RNA (ssRNA) molecules of 200 to ~10,000 bases in length, with the exact upper limit dependent on characteristics of each sample and buffer. Accuracy and reproducibility Comprehensive evaluation has demonstrated that mass photometry consistently measures mRNA length with a relative error below 5% and precision within 2%. This error bound has been repeatedly shown in data produced internally by mass photometer manufacturer Refeyn (Fig. 4), and it has been verified independently in publications (see, e.g. Fig. 8).3,4,6 In contrast, caplillary gel electrophoresis (CGE) typically only achieves 15-30% relative error for large mRNAs. Even charge-detection mass spectrometry (CDMS), another single-particle mass measurement technique, had slightly greater mRNA mass measurement error than mass photometry when the two methods were compared (Fig. 8).4 Another study, by authors from Sanofi, assessed the accuracy and reproducibility of mass photometry under different buffer conditions. During process optimization, buffer conditions may change, and it is important to check whether an analytical method is sensitive to these changes. In this study, when the authors used mass photometry to analyze the same RNA molecule while varying salt, surfactant and other buffer conditions, they found that mass photometry consistently returned highly accurate results (<3% error) (not shown).3 Figure 4. Mass photometry measurements of RNA length are accurate and reproducible. Length measurements of four different mRNA samples (eGFP saRNA; and eGFP, FLuc and SpCas9 mRNAs) were consistently within 4% error of the expected length for each mRNA. The coefficients of variation for each set of measurements were within the range 1.5–2.0%. The boxes represent the middle 50% of the data, with the horizontal lines inside each box showing the median, the vertical whiskers the range, and the X marks the mean of the data for each sample (n=17, 4, 4, and 4). Experiments were performed on the TwoMP mass photometer in 1x PBS at 5 nM mRNA. The authors observed that, with mass photometry, the “matrix robustness and overall simplicity of sampling is a large boon for in-process sampling.”3 These results confirm that mass photometry is a reliable tool for measuring samples in different matrices, making it an ideal analytical method to use in process monitoring for mRNA production. “Consistent measurements of mRNA lengths were observed with calculated errors of less than 2.3%... demonstrating the robustness and accuracy of MP as a reliable tool for accurately determining mRNA length across a wide range of sizes.” – Scientists at Genentech, who used mass photometry and other methods to characterize mRNA and associated impurities6 8 www.refeyn.com Analyzing mRNA purity in non-denaturing conditions Commonly used mRNA analysis techniques, including CGE and HPLC, are performed in reducing or denaturing conditions, which means they do not report on the mRNA in its native state. This is important when measuring mRNA integrity (size) as well as purity – and the presence of aggregation or dsRNA. Forced degradation studies, for example, are routinely done to assess stability and aggregation after exposure to stress conditions, such as elevated temperatures. It is important that the techniques used to analyze samples from degradation assays reliably represent the state of the sample after exposure to stress, but denaturation can compromise this accuracy. Mass photometry enables direct, label-free analysis of intact, degraded or aggregated mRNA in native conditions. As an example, we looked at analyses of eGFP mRNA using mass photometry – under both denaturing and native conditions – and CGE, which requires denaturing conditions. The results for both techniques under denaturing conditions were similar (Fig. 5); both indicated that monomers (with a peak around 1000 b) accounted for about 80% of the sample and no higher-order species were detected. However, the mass photometry analysis of the sample under non-denaturing conditions showed that higherorder species were present. In this sample, monomers accounted for only around 61% of the particles detected, with dimers accounting for 12% and trimers 4%; low-mass species were also present (Fig. 5). This example shows that relying on the analysis of the denatured sample could yield a misleading assessment of a sample’s purity. Avoiding this problem, mass photometry analyzes samples in the native state. It can detect impurities such as aggregation across time points or stress cycles, without the artifacts that can result from denaturation. Figure 5. Mass photometry does not require denaturation, so it can detect species that are missed by other techniques. Mass photometry was used to measure eGFP mRNA under native (non-denaturing, left) and denaturing (middle) conditions. Mass photometry analysis of the native sample revealed distinct molecular species (monomers, dimers, and trimers), but only a single mRNA species (monomers) was apparent in the denatured sample. For each peak, the percentage of detected particles corresponding to that peak is also indicated (as the mean ± SD from a Gaussian fit to the peak). CGE analysis under denaturing conditions, as reported in the manufacturer’s certificate of analysis (right), also detected only monomers. Measured on the TwoMP mass photometer. 9 www.refeyn.com Analyzing mRNA stability Degradation studies are routinely used to assess the stability of mRNA samples – to assess therapeutic products and define shelf life and storage conditions, as well as to validate analytical methods. Mass photometry is a valuable tool for stability analysis due to its broad dynamic range and ability to detect low-abundance populations with low or high mass. The values of mass photometry as a tool for monitoring stability has been demonstrated in work by Refeyn as well as in a separate study by authors at Genentech.6 In Refeyn’s study, mRNA eGFP samples were exposed to thermal stress – either 5 free/thaw cycles (Fig. 6A) or 2 min at 60°C, 70°C or 80°C (Fig. 6B) – and analyzed by mass photometry. High-mass species, suggesting aggregation, were visible in the sample exposed to freeze/thaw cycles (Fig. 6A), while low-mass species appeared in the samples exposed to heat stress, indicative of degradation. Mass photometry also made it straightforward to quantify changes in monomer abundance (Fig. 6C) and monomer length (Fig. 6D) as a result of heat stress. In the Genentech study, mRNA stability was assessed after samples were heated to 37°C for 5 days. Mass photometry revealed that one sample contained 65% monomers (771 nt) and 16% dimers (1556 nt) before heat stress, and heat disrupted the dimers (not shown). After heat stress, there was a 34% increase in a prepeak region, in agreement with CGE data.6 In addition, in that study, mass photometry revealed non-covalent dimer populations that were not visible in techniques using reducing conditions (i.e. IP-RP-HPLC and CGE). The authors concluded that mass photometry is “a fast and simple orthogonal method that provides insights into the homogeneity and stability of mRNA samples.”6 “...a fast and simple orthogonal method that provides insights into the homogeneity and stability of mRNA samples.” – Scientists at Genentech, who used mass photometry and other methods to characterize mRNA and associated impurities6 The “matrix robustness and overall simplicity of sampling is a large boon for inprocess sampling.” – Scientists at Sanofi, who assessed how mass photometry performs under different buffer conditions3 10 www.refeyn.com Figure 6. Mass photometry reveals changes in composition of mRNA samples after freeze/thaw cycles or exposure to heat stress (2 min). Mass photometry was used to measure eGFP mRNA under control conditions as well as a range of stress conditions. Plotted are histograms of the eGFP mRNA samples exposed to control conditions or (A) 5 freeze/thaw cycles or (B) 2 minutes of exposure to 60, 70 and 80ºC. (C) Percentage of mRNA monomers detected in samples after exposure to 60, 70, and 80ºC for 1 and 2 minutes. (D) Length of mRNA monomers measured after exposure to 60, 70, and 80ºC for 1 and 2 minutes. The TwoMP mass photometer was used. 11 www.refeyn.com Other techniques that are used to assess the mRNA CQAs that can be assessed using mass photometry include CGE (for integrity), HPLC (for aggregation) and ELISA (to detect dsRNA). Compared to mass photometry, CGE and HPLC have the drawbacks that they require denaturation. Furthermore, method development tends to be more complex for liquid chromatography (LC) methods, and to a lesser extent for CGE, than for mass photometry. In terms of the speed of analysis and sample consumption, mass photometry is again the more favorable technique. Mass photometry uses 10 – 20 μL of sample in the nanomolar concentration range; this is 10x less sample than CGE or HPLC, and about 50x less than ELISA. Meanwhile, with each measurement taking one minute and the entire measurement process (from sample prep to analysis) taking less than five minutes, mass photometry is ~20x faster than other methods. While the body of comparative data is still growing, initial findings suggest that mass photometry yields results that align well with those from other analytical techniques (see, e.g., comparison to CGE in Fig. 2). Mass photometry vs. CDMS One recent publication, from authors at the University of Utrecht and Pfizer, evaluated mass photometry and single-particle Orbitrap-based charge-detection mass spectrometry (CDMS) as methods to analyze large (1,000 – 10,000 nt), intact mRNA molecules (Fig. 7).4 Mass photometry vs. other methods Both methods perform analysis based on single-particle mass measurement. The authors used mass photometry and denaturing CDMS to measure a series of nine mRNAs, with lengths ranging from 859 to 9,472 bases (Table 2). While the CDMS analyses generally returned results with slightly lower standard deviation (sharper resolution), mass photometry had better accuracy, with excellent linearity and mass measurement error consistently <2% (Table 2, Fig. 8). In addition to high molecular weight species (HWMS) including dimers, they also found that, in at least one sample, mass photometry also detected low molecular weight species (LWMS). Furthermore, there was no need for denaturing in the mass photometry analysis. Figure 7. Deslignière et. al (2025) compared CDMS and mass photometry for the analysis of large mRNAs.4 For CDMS, methanol was added to induce partial denaturation to improve the signal-to-noise ratio and thus the mass resolution. Reproduced from Deslignière et. al (2025).4 “MP enabled the measurement of mRNAs with high accuracy, while revealing low amounts of mRNA fragments and dimers that are sometimes overlooked in CDMS.” – Scientists at the University of Utrecht and Pfizer, who evaluated mass photometry and CDMS as methods to analyze large, intact mRNAs4 12 www.refeyn.com Sample Length (bases) Theoretical mass (kDa) CDMS (denatured) Mass photometry Measured mass (kDa) Deviation vs. theory (%) Measured mass (kDa) Deviation vs. theory (%) EPO 5moU 859 282.9 288 ± 38 1.8 281 ± 56 -0.7 EGFP 997 323.5 327 ± 29 1.1 329 ± 54 1.7 Cre 5moU 1351 444.4 451 ± 30 1.5 439 ± 55 -1.2 OVA 1468 467 473 ± 44 1.3 459 ± 56 -1.7 Fluc 1922 622.3 633 ± 35 1.7 624 ± 67 0.3 DS3 m1Ψ 2162 702.2 743 ± 30 5.8 696 ± 61 -0.9 β-Gal 3421 1106.90 1172 ± 44 5.9 1,092 ± 70 -1.3 DS1 m1Ψ 9432 3041.70 3111 ± 65 2.3 3,050 ± 80 0.3 DS2 m1Ψ 9472 3054.90 3174 ± 75 3.9 3,097 ± 80 1.4 Table 2. Comparison of theoretical vs. measured masses for all mRNA products (monomers only) measured by mass photometry and denaturing CDMS. This table was reproduced from Deslignière et. al (2025).4 Figure 8. Mass error for denaturing CDMS vs. mass photometry for mRNAs of varying lengths. Data are from Table 2 (Deviation vs. theory (%) columns for CDMS and mass photometry). “Compared to [SEC–MALS], MP does not require a large amount of mRNA sample (only 50 ng versus a few μgs) and does not suffer from poor separation resolution due to the limited pore size of SEC columns, enabling efficient separation of different aggregate forms.” – Scientists at Genentech, who used mass photometry and other methods to characterize mRNA and associated impurities6 Based on their evaluation, the authors of the study assessing mass photometry and CDMS concluded: “MP enabled the measurement of mRNAs with high accuracy, while revealing low amounts of mRNA fragments and dimers that are sometimes overlooked in CDMS.”4 13 www.refeyn.com Summary As emerging therapeutic modalities push the limits of traditional analytical tools, there is a growing need for new methods that can meet the demands of these advanced modalities while also delivering efficiency and accessibility. For mRNA therapeutics, there is a need for methods to assess mRNA integrity and purity that do not require denaturation or struggle with large molecules. An ideal method, in mRNA as in all other applications, would also be fast and use little sample. In this handbook, we have described how mass photometry, an emerging technology in mRNA analytics, fits the profile of an ideal mRNA analysis tool. Other methods require more sample (10–50x more) and more time (20x more), but provide less information. In contrast, mass photometry represents a single, multipleattribute tool. In just one measurement, it gathers data to report on multiple CQAs related to integrity and purity (Fig. 9). Because it directly detects individual molecules in solution, it has the necessary resolution to detect subtle truncations or degradation products, which are often missed by conventional sizing techniques. Mass photometry also offers the advantages that it can be used in a wide range of buffer conditions and can measure samples in the native state – unlike CGE and LC methods. Figure 9. As a multiple-attribute tool, mass photometry dramatically simplifies mRNA analytics. As mass photometry measures the mass of individual molecules in solution across a broad size range, it can reliably report on mRNA inactness and length, as well as the presence of degradation products and other impurities. It replaces multiple tools, which require denaturation and use more sample, with a single measurement. “… mass photometry affords native mass measurement which includes details such as higher order structure. The technique worked with both in-process crude samples and purified nucleic acids.” – Scientists at Sanofi, who assessed how mass photometry performs under different buffer conditions3 14 www.refeyn.com While mass photometry is a relatively new technique for mRNA analysis, it is already being used successfully by groups in industry as well as academia and its adoption continues to expand. It has immense potential to strengthen and streamline mRNA analysis of integrity and purity in the near future. Overall, as the examples presented in this handbook have shown, mass photometry offers: • Analysis in native conditions • A broad dynamic range – covering mRNA lengths of 200 to 10,000 bases • High accuracy (<5% error) across the size range • Speed (< 5 min incl. sample prep to analysis) • Ease of use with minimal sample prep or method development • An end-to-end solution comprising hardware, software and consumables References 1. US Pharmacopeia. Analytical Procedures for Quality of mRNA Vaccines and Therapeutics (Draft Guidelines: 3rd Edition) | USP-NF. (2024). https://www.uspnf.com/notices/analytical-procedures-mrna-vaccines-20240802 2. Young, G. et al. Quantitative mass imaging of single biological macromolecules. Science 360, 423–427 (2018). https://doi.org/10.1126/science.aar5839 3. Schmudlach, A., Spear, S., Hua, Y., Fertier-Prizzon, S. & Kochling, J. Mass photometry as a fast, facile characterization tool for direct measurement of mRNA length. Biol. Methods Protoc. 10, bpaf021 (2025). https://doi.org/10.1093/biomethods/bpaf021 4. Deslignière, E., Barnes, L. F., Powers, T. W., Friese, O. V. & Heck, A. J. R. Characterization of intact mRNA-based therapeutics by charge detection mass spectrometry and mass photometry. Mol. Ther. Methods Clin. Dev. 33, 101454 (2025). https://doi.org/10.1016/j.omtm.2025.101454 5. De Vos, J. et al. Evaluation of size-exclusion chromatography, multi-angle light scattering detection and mass photometry for the characterization of mRNA. J. Chromatogr. A 1719, 464756 (2024). https://doi.org/10.1016/j.chroma.2024.464756 6. Camperi, J. et al. Comprehensive Impurity Profiling of mRNA: Evaluating Current Technologies and Advanced Analytical Techniques. Anal. Chem. 96, 3886–3897 (2024). https://doi.org/10.1021/acs.analchem.3c05539 Visit Refeyn online to learn more! “Mass photometry provides a fast screening tool to investigate mRNA integrity and size.” – RIC group scientists, whose study evaluated SEC, MALS and mass photometry for the characterization of mRNA 15 www.refeyn.com The TwoMP and TwoMP Auto mass photometers are easy-to-use instruments that quickly deliver multiple-attribute measurements of mRNA therapeutics. To facilitate fast, informative analysis of mRNA samples across mRNA therapeutic development, Refeyn offers end-to-end mass photometry solutions. These include: • Mass photometers: TwoMP and TwoMP Auto • Software: AcquireMP for data acquisition and DiscoverMP for data analysis and visualization • Consumables: MassGlass™ NA sample preparation kits The TwoMP product family is a core part of Refeyn’s broader product portfolio (Table 3). Mass photometers The TwoMP mass photometer is a benchtop instrument that delivers multiple-attribute measurements of mRNA therapeutics. It uses mass photometry to accurately characterize individual mRNA molecules in their native buffer – completely label-free and with minimal sample preparation. It is easy to use, with new user training usually taking less than one day. It is also fast. A typical measurement takes 60 seconds, and the complete process – from sample preparation to analysis – takes under 5 minutes. The TwoMP Auto enables up to 24 automated measurements, ensuring repeatability and providing walkaway time. It offers the same measurement capabilities as the TwoMP Auto, but with the added benefit of automation. A complete, 24-measurement run takes about 90 minutes and mass photometry analysis has confirmed that mRNA samples remain stable for the duration of a full TwoMP Auto run (Fig. 10). Mass photometry solutions for mRNA analysis TwoMP Auto Mass Photometer TwoMP Mass Photometer TwoMP TwoMP Auto Click below to learn more: i 16 www.refeyn.com Figure 10. mRNA remains stable for the duration of an automated mass photometry run. Samples of eGFP, FLuc and SpCas9 were measured in triplicate by automated mass photometry immediately after dilution in 1x PBS at the start of a full run of automated measurements (solid bars) and then again at the end (dotted bars). (A) The relative error for both sets of measurements (based on the measured length vs. the expected length) was very low (<3%) at both time points. (B) The percentages of full-length (monomeric) RNA in the samples also remained consistent. Both results indicate that mRNA is stable enough for analysis by automated mass photometry. The TwoMP Auto, used here, enables up to 24 automated measurements in a single run, which can take as little as 90 min, and operates at ambient temperature. MP = Mass photometry. Mass photometry software Refeyn’s AcquireMP acquisition software and DiscoverMP analysis software are intuitive tools for mass photometry data capture and analysis. Their ease of use complements the intuitive nature of mass photometry’s single-molecule mass measurement approach. The AcquireMP software supports highquality data capture, providing real-time data during acquisition and clear user guidance, with few parameters to optimize. The resulting data can then be analyzed in the DiscoverMP software, which allows the user to understand the different populations in their sample. DiscoverMP also contains tools to quickly produce report-ready graphs and tables – helping teams make data-driven decisions, fast. A. B. Software 17 www.refeyn.com Mass photometry consumables An important factor in the quality of mass photometry measurements is the sample carrier slide. For mRNA samples, it is important to use a high-quality surface that promotes efficient mRNA binding (Fig. 11). MassGlass NA are ready-to-use slides that are optimized for reliable mRNA quantification using the TwoMP or TwoMP Auto mass photometers. They use advanced surface technology to ensure strong mRNA interaction with the glass and consistently enable accurate quantification. They are available within the MassGlass NA Sample Preparation Kits, which are available for manual and automated systems and contain all the consumables needed to run mass photometry measurements of mRNA samples. Figure 11. MassGlass NA slides enable mass photometry analysis of mRNA. (A) Commercially available mRNA samples were prepared and measured using both uncoated (MassGIass UC, blue) and cationic-coated (MassGIass NA, orange) slides. The mRNA’s strong negative charge prevented its interaction with the uncoated glass, but the positive charge on the MassGlass NA surface made it possible to measure the molecular mass distribution. (B) The difference can also be seen in the ratiometric images generated by mass photometry. A representative image from MassGlass UC analysis shows very few landing events (which appear as dark dots), while the MassGlass NA image clearly shows many landing events. MassGlass NA Sample Preparation Kits The MassGlass NA Sample Preparation Kits contain everything needed for mass photometry measurements of mRNA samples, including the sample well cassettes (left) and MassGlass NA slides (middle and right) shown here. Pictured here is the kit for use with the TwoMP mass photometer. The MassGlass NA Auto Sample Preparation Kit, for use with the TwoMP Auto, is also available. Click here to learn more about Refeyn consumables i 18 www.refeyn.com Table 3. Refeyn’s end-to-end mass photometry solutions enable rapid, user-friendly analysis of mRNA as well as other sample types. 18 www.refeyn.com TwoMP SamuxMP KaritroMP Technology Mass photometry Macro mass photometry Parameters measured Mass Diameter (size) Contrast (mass proxy) Concentration range 100 pM – 100 nM** 1011 particles/mL 108 – 109 particles/mL Particle type measured (Mass or size range) Proteins (30* kDa – 5 MDa) Nucleic acids (200 – 10k b; 100 – 5k bp) AAVs (500 kDa – 6 MDa) Large viruses (e.g. AdV) (40 – 150 nm) Consumables Sample carrier slides MassGlass™ UC for protein analysis, MassGlass NA for nucleic acid analysis, MassGlass UC MassGlass KV Calibrant MassFerence™ P1 (For protein measurements within 90 – 1000 kDa) MassFerence P2 SizeFerence™ Add-ons High-concentration samples Automation TwoMP Auto*** SamuxMP Auto*** Software Data acquisition AcquireMP Analysis Standard DiscoverMP DiscoverMPK StreamlineMP Antibody stability module GMP environments GMP software package * Using slides hand cleaned according to Refeyn protocol. The lower limit for Refeyn’s pre-cleaned MassGlass UC slides is 50 kDa without further cleaning. ** The MassFluidix HC add-on expands the TwoMP sample concentration range up to the tens of micromolar. *** Autonomous measurement of 24 samples in as little as 90 min. Refeyn mass photometers are Class 1 laser products. Table 3. Refeyn’s end-to-end mass photometry solutions enable rapid, user-friendly analysis of mRNA as well as other sample types. i Please note that in practice, exact specifications are sample and buffer dependent. * Using slides hand cleaned according to Refeyn protocol. The lower limit for Refeyn’s pre-cleaned MassGlass UC slides is 50 kDa without further cleaning. ** The MassFluidix HC add-on expands the TwoMP sample concentration range up to the tens of micromolar. *** Autonomous measurement of 24 samples in as little as 90 min. Oces Boston (Waltham) (US HQ, Service Depot and Customer Interaction Center) Berlin San Francisco San Diego Baltimore Frankfurt Munich Shanghai Kobe Singapore Oxford (Global HQ and Service Depot) Service Team Tokyo Our vision is to accelerate discovery through innovation, empowering the latest scientific breakthroughs in basic research and transforming biotherapeutic development and manufacturing. Get in touch to speak with one of our mass photometry experts. Refeyn Headquarters (UK) Unit 9, Trade City Sandy Lane West Oxford OX4 6FF United Kingdom Phone: +44 1865 800175 Refeyn Headquarters (USA) 21 Hickory Drive Suite 200A Waltham MA 02451 USA Phone: + 1 (971) 200 1370 Sales Directors: EMEA – Rebecca Elston Phone: +44 7804 629158 E-mail: [email protected] USA – Candi Mach Phone: + 1 (971) 200 1370 E-mail: [email protected] APAC and Global – Guillaume Kohen Phone: +44 7511 183320 E-mail: [email protected] Mass photometry technologies ® © 2025 Refeyn Ltd Unit 9, Trade City, Sandy Lane West, Oxford OX4 6FF, United Kingdom For information on products, demos and ordering, write to [email protected] MassFerence, MassFluidix, MassGlass, Karitro, Samux, SizeFerence and Refeyn are registered trademarks of Refeyn Ltd. [email protected] refeyn.com Refeyn @Refeyn 20 Testimonials “Mass photometry provides a fast screening tool to investigate mRNA integrity and size.” De Vos et al. (2024), J Chromatogr A “The data confirm the great potential of [mass photometry] technology... as a fast and simple orthogonal method that provides insights into the homogeneity and stability of mRNA samples.” Camperi et al. (2024), Anal Chem Unit 9, Trade City, Sandy Lane West, Oxford OX4 6FF, United Kingdom ©2024 Refeyn Ltd For information on products, demos and ordering, write to [email protected] Samux and Refeyn are registered trademarks of Refeyn Ltd. refeyn.com @refeynit Refeyn Refeyn About Refeyn Refeyn pioneers analytical instruments that put molecular mass measurement capabilities within easy reach for scientists. Refeyn’s unique products measure the mass of individual proteins, nucleic acids, complexes and viruses directly in solution – providing vital insights for scientific discovery, R&D and therapeutics production. Our instruments feature mass photometry technology, which uses light to quantify the mass of single particles in solution without labels, and macro mass photometry technology, which uses light to characterize large viral vectors. Providing intuitive data in minutes, mass photometry technologies help scientists solve their research questions, optimize R&D processes and focus on innovation. 20 Testimonials “Mass photometry provides a fast screening tool to investigate mRNA integrity and size.” De Vos et al. (2024), J Chromatogr A “The data confirm the great potential of [mass photometry] technology... as a fast and simple orthogonal method that provides insights into the homogeneity and stability of mRNA samples.” Camperi et al. (2024), Anal Chem Unit 9, Trade City, Sandy Lane West, Oxford OX4 6FF, United Kingdom ©2024 Refeyn Ltd For information on products, demos and ordering, write to [email protected] Samux and Refeyn are registered trademarks of Refeyn Ltd. refeyn.com @refeynit Refeyn Refeyn About Refeyn Refeyn pioneers analytical instruments that put molecular mass measurement capabilities within easy reach for scientists. Refeyn’s unique products measure the mass of individual proteins, nucleic acids, complexes and viruses directly in solution – providing vital insights for scientific discovery, R&D and therapeutics production. Our instruments feature mass photometry technology, which uses light to quantify the mass of single particles in solution without labels, and macro mass photometry technology, which uses light to characterize large viral vectors. Providing intuitive data in minutes, mass photometry technologies help scientists solve their research questions, optimize R&D processes and focus on innovation. V1 Aug-2025