Mass Photometry vs SEC: Antibody Aggregation Assessment

Antibody characterization is essential in biopharmaceutical development for ensuring the safety, efficacy and consistency of the therapeutic product. One important critical quality attribute (CQA) investigated is aggregation. Aggregation can affect the final product in three ways: 1. Increased immunogenicity risk, as aggregated antibodies are more likely to be seen as ‘foreign’ by the immune system. 2. Reduced efficacy, as aggregates may be inactive or interfere with the function of the monomeric antibody. 3. Impaired stability, as aggregation can indicate or lead to protein instability over time, affecting shelf life and formulation robustness. A powerful and widely accepted method for detecting and monitoring aggregation during development and release testing is size exclusion chromatography on a high-performance liquid chromatography setup (SEC-HPLC) coupled to UV detection (simply denoted as SEC in this article). SEC can resolve monomers, dimers and higher-order aggregates of antibodies in a single run. However, it frequently requires time-consuming column optimization, as some antibodies may stick to a column matrix or interact in unexpected ways, increasing the retention time and complicating the interpretation of traces. In addition, SEC is biased towards higher-mass species, as those with a greater mass generate a larger signal. Mass photometry is an emerging tool for characterizing multiple antibody CQAs, including aggregation. As a single-molecule technique, mass photometry can detect and quantify lowabundance populations. Its high resolution (25 kDa for a 66 kDa protein) enables population differentiation. It can detect antibody fragments, monomers, dimers and higher-order aggregates – and its mass readout makes it straightforward to identify the different populations detected. Mass photometry can also analyze antibody binding to targets. Measurements take just minutes and require only nanograms of sample. SEC and mass photometry (compared in detail in Box 1) both offer powerful ways to assess antibody aggregation. But, as APPLICATION NOTE Comparing mass photometry and SEC for antibody aggregation assessment Mass photometry is an emerging tool for assessing multiple antibody attributes, including aggregation. Here, a comparison to size-exclusion chromatography (SEC) shows the two techniques agree, but mass photometry has superior resolution, particularly for larger modalities. Mass photometry requires no optimization beyond sample dilution and readily characterized samples that could not initially be measured with SEC. In addition, mass photometry requires >100x less sample and is 20x faster than SEC, and provides an informative mass readout. they work very differently, it is crucial to understand the relative strengths and limitations of each – and when they are most suitable. Here, we provide a detailed comparison of the techniques in the context of antibody aggregation analysis. We characterize a series of antibodies with SEC and mass photometry. Samples were only selected for inclusion in this study if SDS-PAGE analysis showed the intact antibody under non-reducing conditions, with disassembly into heavy and light chains under reducing conditions. SEC and mass photometry comparison experiments were carried out side-by-side using the same samples and illustrate different cases for how SEC and mass photometry compare. We show that: 1. Mass photometry and SEC results generally agree, although they are not directly comparable due to differences in what they measure. Mass photometry provides a mass readout. 2. Mass photometry, unlike SEC, does not require prior optimization and cannot be affected by column interactions. 3. For larger modalities, mass photometry has superior resolution to SEC and provides much more detailed insights about sample composition. 4. Standard mass photometry is run at a lower concentration than SEC, which could affect concentration-dependent multimers. This can be overcome by combining mass photometry with the rapid-dilution MassFluidix HC microfluidics system. This application note was created in collaboration with Absolute Antibody Case 1: SEC and mass photometry agree We first measured a monoclonal IgG antibody, IgG1 Human 1. The results from SEC generally aligned with mass photometry, but the superior resolution of mass photometry enabled detection of a fragment population. The SEC results showed a main peak at around 7.5 min, as expected, as well as higher-order structures that eluted prior to the main peak (Fig. 1, Table 1). Smaller populations also eluted around 12–18 minutes, but the peaks were too small to be fitted. This result agreed with SDS-PAGE, which showed one major band and a very faint second band at higher molecular weight under non-reducing conditions (Fig. 1). Mass photometry confirmed that the main species was a ~150 kDa molecule, as expected for a monoclonal antibody. Mass photometry further identified populations of probable dimers, with mass ~300 kDa, and a very small population of trimers (Table 1). Though the dimers and trimers had very low abundance relative to the monomer, they were detectable. The dimer and trimer peaks were more visible with SEC, in line with the mass weighting that occurs with this technique (see Box 1). Overall, quantification showed the two techniques were in general agreement about the relative sizes of the monomer, dimer and trimer populations (Table 1). However, mass photometry also clearly resolved an additional population around 40 kDa that SEC did not detect. This population is likely fragmentation, based on the mass. It is a striking difference between the two techniques that this population, which may have considerable importance for understanding the sample’s stability, was only detected by mass photometry and not SEC. Fig. 1 SEC and mass photometry results agree for analysis of two monoclonal IgG antibodies. For antibody IgG1 Human 1, results are shown for SDS-PAGE under non-reducing (NR) and reducing (R) conditions (left), SEC analysis (middle) and mass photometry (right). For SEC and mass photometry, the colored peak regions correspond to antibody fragments (purple), monomers (blue), dimers (yellow) and trimers (green). Oligomeric state SEC measurement (%) Mass photometry measurement (%) IgG Human 1 Fragment 0.0 5.4 Monomer 86.4 89.8 Dimer 10.8 4.1 Trimer 2.8 0.7 Table 1. Proportions of species, as determined by SEC and mass photometry for the monoclonal IgG antibody IgG1 Human 1 in Fig. 1. Case 2: SEC ambiguous, mass photometry clear and consistent Next, a different IgG antibody, IgG Rabbit 1, was analyzed by SEC and mass photometry. In this case, in the SEC analysis, the retention time for this antibody was ~9 min (Fig. 2), whereas the expected retention time was ~7.5 min under the column conditions used. The slower elution time would suggest that the proteins in the sample were in fact smaller than full IgGs (e.g. fragments). The mass photometry result, on the other hand, produced a main peak around 150 kDa, consistent with the sample containing mainly full, monomeric IgG antibodies, as expected. Mass photometry further identified a population of dimers, with mass ~300 kDa and even a very small trimer population (Table 2). The unexpected SEC result could have been caused by temporary retention of the molecules in the column. WIthout additional information from an orthogonal technique, it would be unclear from the SEC data whether the protein was the expected one (but slightly delayed due to column interactions), or in fact had fragmented into a lower-mass species. This example illustrates the advantage that mass photometry offers by eliminating the need for a column. It also demonstrates the value of analysis by orthogonal techniques. Fig. 2 SEC suggested possible fragmentation or column interactions, while mass photometry confirmed the presence of a monomeric full IgG population. For an IgG rabbit antibody, results are shown for SDS-PAGE under non-reducing (NR) and reducing (R) conditions (left), SEC analysis (middle) and mass photometry (right). For SEC and mass photometry, the colored peak regions correspond to monomers (blue) and dimers (light orange). Table 2. Proportions of species, as determined by SEC and mass photometry for the monoclonal IgG antibody IgG Rabbit 1 in Fig. 2. Oligomeric state SEC measurement (%) Mass photometry measurement (%) IgG Rabbit 1 Monomer 99.1 97.4 Dimer 0.9 2.2 Trimer 0.0 0.2 Case 3: SEC fails, while mass photometry works We next analyzed another human antibody, IgG1 Human 2 (Fig. 3A). In this case, the SDS-PAGE results clearly indicated the presence of an antibody, but the SEC trace provided negligible information – despite being run under identical conditions to the antibodies shown in Cases 1 and 2. This result is likely due to the antibody having been retained within the column matrix – a common issue in SEC that can take considerable time to resolve. Conversely, mass photometry provided a clear readout of the sample’s aggregation state, and with no optimization required. A mouse fab fragment (Fab Mouse 2) also produced inconsluive results in the SEC analysis, but again with a clear mass photometry readout (Fig. 3B). The mass photometry data showed a main peak at ~80 kDa as well as a multitude of higher-order species present with low abundance, in agreement with SDS-PAGE, which showed a smear in non-reducing conditions and many different sized bands under reducing conditions. This could explain the poor SEC results, as the higher-order species could have clogged the column, resulting in the antibody’s retention. Fig. 3 Mass photometry, but not SEC, returned clear aggregation readouts for an IgG antibody and a mouse fragment. For antibodies (A) IgG1 Human 2 and (B) Fab Mouse 2, results are shown for SDS-PAGE run under non-reducing (NR) and reducing (R) conditions (left), SEC analysis (middle) and mass photometry (right). For mass photometry, the colored peak regions correspond to monomers (blue) and higher-order species (light orange). Case 4: Only mass photometry could resolve higherorder IgM oligomers Finally, we analyzed samples of larger antibodies, IgMs, which feature a more complex structure than IgG antibodies, forming pentamers or hexamers. Hexameric IgM samples, IgM Human 1 and IgM Human 2, were obtained by expressing IgM without the J-chain. In both cases, SDS-PAGE under reducing conditions showed the expected heavy and light chains (Fig. 4). In both cases, in the SEC trace, three peaks were distinguishable in the expected range (Fig. 4AB, mid left). More peaks were visible from the mass photometry analysis, and the mass information from mass photometry makes it possible to identify the species. In addition to the fully assembled hexameric IgM, mass photometry revealed significant populations of pentamers, tetramers, dimers and monomers (Fig. 4AB, mid right). The mass photometry results suggest that the major eluting peaks from the SEC traces are associated with at least three species: Tetramers, pentamers and hexamers. The observation that SEC does not always resolve all oligomeric states in IgM aggregation has been made before, such as in comparison to data from analytical ultracentrifugation.1 Overall, it was only possible with mass photometry to identify and quantify the range of oligomeric species in each sample (Fig. 4). Mass photometry requires that samples be at low (nanomolar) concentrations for measurements. The low concentrations can affect the formation of multimers, such as the IgM assemblies here, which form reversibly. Because SEC and mass photometry require different concentrations for measurement, they may report different oligomeric distributions of the IgM species. We can overcome mass photometry’s low-concentration requirement by using a rapid-dilution microfluidic system (Refeyn’s MassFluidix HC) coupled to the mass photometer. The MassFluidix approach allows samples to be diluted up to 10,000x immediately prior to measurement, before significant dissociation can occur, so the observed distribution of species reflects what was present in the sample at the higher concentration. To assess the effect of concentration on the oligomeric distribution, we tested the two IgM samples using the MassFluidix mass photometry approach (Fig. 4AB, far right). The MassFluidix measurements verified that the additional species observed (monomers-pentamers) were also present at higher concentrations, so they were not due to the low concentration used for standard mass photometry. However, the higher-order oligomers were present in higher proportions than in the standard mass photometry measurement, indicating that some IgM hexamer disassembly occurs when the sample is diluted. Fig. 4 IgM pentamers and hexamers were only resolved by mass photometry. For IgM antibodies (A) IgM Human 1 and (B) IgM Human 2, results are shown for SDS-PAGE run under reducing (R) conditions (far left), SEC analysis (mid left), standard mass photometry (mid right) and mass photometry with MassFluidix HC (far right). The colored peak regions correspond to monomers (blue), dimers (light orange) and higherorder species (green). In (B), an additional population eluted around 14 min, likely due to buffer contaminants. It was not detected by mass photometry, suggesting the species had mass below the 30 kDa minimum for mass photometry analysis. Discussion Here, we have shown that mass photometry is an effective tool for antibody aggregation analysis that outperforms SEC due to having superior resolution, providing an informative mass readout, and eliminating both the need for optimization and the risk of column interactions. Mass photometry also has the advantages of being fast (with a measurement taking only 1 min, vs. 20–25 min for SEC) and using >100x less sample. A dedicated Antibody Stability software module2 further facilitates mass photometry analysis of antibody aggregation. Differences between the methods are detailed in Box 1 and summarized in Table 3. Another important difference between SEC and mass photometry is the concentration at which samples are measured. Mass photometry requires a low (nanomolar) concentration and small volume (10–20 μL). While mass photometry’s low sample consumption is beneficial, the low concentration can cause the dissociation of higher-order species. Here, we have shown that this issue can be overcome by using the MassFluidix HC rapid dilution approach with mass photometry. This rapid dilution approach could also be used to assess whether measured aggregates are dissociable or not. In practice, both SEC and mass photometry are valuable tools for antibody aggregation analysis and can be used in complementary ways (Table 3). SEC is ideal for high-throughput screening and quality control (QC) in GMP-regulated environments (as there is not yet a 21CFR11-compliant mass photometry solution for antibody aggregation analysis). Mass photometry, meanwhile, is ideal for rapid screening and situations where sample is limited, and its superior resolution is valuable for analyzing larger modalities. The availability of the MassFluidix HC rapid dilution system means that mass photometry is not limited to low-concentration samples. Materials and methods Samples All samples were provided by Absolute Antibody. Any requests for samples can be directed to support@absoluteantibody.com. Mass photometry All mass photometry measurements were carried out on a Refeyn TwoMP mass photometer. All measurements were acquired using AcquireMP 2024.1.1 and analyzed with DiscoverMP 2024.1.0. All measurements were carried out using the large field of view setting and each movie was recorded for 60 seconds. Calibrations, to enable conversion from the measured contrast to mass, were carried out using Refeyn’s MassFerenceTM P1 calibrant. Prior to measurement, samples were diluted from 1 mg/mL (approximately 6.6 μM for a 150 kDa protein) to 10 nM using PBS. Mass photometry with MassFluidix HC A Refeyn MassFluidix HC system was used with a Refeyn TwoMP mass photometer. IgM samples were at 1 mg/mL (~0.92 μM, for hexameric IgM). Prior to loading, samples were diluted 2x to 0.55 μM, followed by rapid 1,000x dilution in the microfluidic chip. SEC-HPLC with UV detection Size-exclusion chromatography HPLC with UV detection was carried out using an Agilent 1100 series HPLC with a Superdex 200 Increase 5/150 GL column equilibrated in PBS, pH 7.2, at a flow rate of 0.2 mL/min. Proteins were analyzed following concentration to 1 mg/mL by injecting 5 μL of sample. Absorbance was measured at 280 nm and proteins assessed for percentage purity (peak area) and size (peak retention time). SDS-PAGE Qualitative protein analysis by SDS-PAGE was performed on samples concentrated to 1 mg/mL. Samples were loaded to pre-cast gels (10% Bis-Tris) from Invitrogen™. Gels were run in MES at 180 V for 45 minutes before staining with Invitrogen SimplyBlue™ SafeStain and destained for 16 hours in deionized water. Gel images were captured using a Bio-Rad GS-900 densitometer. References 1 Chouquet et al., Front. Bioeng. Biotechnol. 2022 https://doi.org/10.3389/fbioe.2022.816275 2 Refeyn product datasheet: Antibody Stability Module https://info.refeyn.com/antibody-stability-module 3 Refeyn tech note: Assessing proteins samples by MP and SEC https://refeyn.com/mass-photometry-vs-sec-in-protein-analysis Box 1: How do SEC and mass photometry compare? SEC and mass photometry operate in very different ways and these differences must be taken into consideration when comparing data from the two methods. We explain differences in key areas below and present an overall comparison (Table 3), highlighting differences related to sample requirements, resolution and other aspects. of species represented in the histogram counts are the same as their concentration in solution, so relative concentrations can be assessed directly from peak sizes. In SEC, by contrast, the amount of UV light absorbed by a protein depends on its extinction coefficient – which is a function of the number of aromatic (particularly tryptophan) residues in its sequence. A dimeric molecule, for example, would contain twice as many aromatic residues and so absorb roughly double the UV light, giving twice the signal of a monomer. Therefore, in SEC, the signal from a given species depends on its mass as well as its amino acid composition. The need to factor in extinction coefficients when interpreting SEC data is particularly important in the analysis of samples such as antibody-drug conjugates (ADCs), where the variability in the extinction coefficients of different species may be significant. SEC and MP data are not directly comparable Due to the differences between the techniques, mass photometry and SEC data are not directly comparable. To compare them quantitatively, it is necessary to use the extinction coefficients of the species present in a SEC profile to convert the data so that the signal is purely based on concentration, as it is in mass photometry. For a demonstration of the conversion, see Refeyn’s recent technical note, Assessing protein samples by mass photometry and SEC.3 However, when SEC peaks contain multiple different species (which may occur due to SEC’s limited resolution for larger species), normalization by extinction coefficients may not be possible. Additional considerations Unlike SEC, which separates based on size and may dilute or disturb equilibrium, mass photometry measures the native distribution of species in near-physiological conditions. Other advantages of mass photometry are its versatility with respect to sample type, conditions and buffer, low sample consumption and rapid measurement time. For antibody aggregation studies using mass photometry, samples only need to be diluted to appropriate concentrations (nanomolar) and then can be measured in just one minute, giving a clear read-out on aggregation state. This makes mass photometry particularly useful for rapid aggregation profiling, and for stability studies in early and late-stage biopharma development. The measurement process SEC separates molecules based on their size by passing them through a column packed with porous beads. Large molecules are excluded from entering the pores and elute first, while smaller molecules enter the pores and take longer to pass through, eluting later. UV absorbance (at 280 nm) is then measured by a detector as the sample elutes. Mass photometry, meanwhile, measures the mass of single proteins in solution by measuring the interference between light scattered by individual proteins and light reflected at the measurement surface. The resulting contrast scales linear with the proteins’ mass. Data presentation SEC data are typically represented in a graph showing UV absorbance vs. elution time (or elution volume). As larger molecules elute first, the first peak in the graph represents the largest molecule and the last peak the smallest. This is opposite to mass photometry, where data are represented in a mass histogram (counts vs. mass) with the first peak corresponding to the lowest-mass species. An advantage of mass photometry is that, provided a masscontrast calibration step has been done, the molecular mass of the species in each peak is immediately apparent. This makes it possible to identify which species is most likely present in each peak (e.g. the dimer peak would be the one with a mean around the expected mass value of a dimer). Data interpretation Another important difference relates to what peak area and height represent. In mass photometry, the area under the peak corresponds to the relative number of proteins in that population (with that mass value). For example, a population with a peak around twice as large as another would have about twice the concentration. Generally, in mass photometry, the proportions Table 3. A comparison of SEC vs. mass photometry. Mass photometry Size-exclusion chromatography Separation mode None Size-based separation by a column Readout Molecule counts over molecular mass UV protein absorbance over retention time Sample requirement ~38 ng (10 μL of 10–55 nM) 10 μg for measurements with MassFluidix HC ~5 μg (5 μL of 1 mg/mL) Labeling Label-free Label-free Resolution Detects fragments, monomers and all higherorder multimers with baseline resolution Depends on column, with molecular weight range and operational limitations Run time 1 min 20–25 min Operational costs <$5 per sample <$5 per sample Ease of data interpretation Straightforward, direct mass readout with particle counts Cumbersome, requiring conversion based on extinction coefficients Limitations Non-preparative, Mass detection range ~30 kDa to ~5 MDa, Sample concentration limited to <50 μM Non-specific column interactions, Column exclusion limits for larger aggregates (typically ~0.6 MDa), Sensitive to extinction coefficients Ideal use cases Rapid, native-state profiling, Measurement in physiological conditions (i.e. native buffer, nM concentration) Fractionation with quantitation, High-throughput analysis, GMP-regulated environments V1-Jul 25 ©2025 Refeyn Ltd Unit 9, Trade City, Sandy Lane West, Oxford OX4 6FF, United Kingdom For information on products, demos and ordering, write to orders@refeyn.com MassFluidix, MassFerence and Refeyn are trademarks of Refeyn Ltd. orders@refeyn.com 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 information on products, demos and ordering, write to info@refeyn.com 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 information on products, demos and ordering, write to info@refeyn.com 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.