Rapid Bispecific Antibody Characterization by Mass Photometry

Bispecific antibodies (bsAbs) are an emerging form of targeted immunotherapy that shows promise for treating a range of conditions. However, bsAb characterization presents significant challenges, as they are complex, artificially designed molecules that can be very variable in terms of structure, potency, immunogenicity, and the presence of aggregates or impurities. Among the main quality attributes that need to be measured are molecule size, fragmentation, aggregation and binding affinity. Characterizing bsAb binding affinities is especially challenging, as they have two different binding sites with their own binding affinities. These sites can cooperate or interfere with one another, and lead to the formation of higher-order assemblies. Conventionally used techniques for determining binding affinities include surface plasmon resonance (SPR) and biolayer interferometry (BLI). However, they are bulk techniques that do not accurately capture the contributions of all components in the sample. Also, because they tend to assume 1:1 binding, they struggle to fully characterize the complexity of bsAb interactions. Mass photometry is an emerging analytical technique that measures the mass of single particles in solution, in minutes and without labels. As it detects and quantifies all components in a sample and their masses, mass photometry can characterize the following key attributes of bsAb samples with a single, quick measurement: • Presence and quantity of fragmentation, impurities and aggregates • Molecular mass of the bsAb – to compare with the expected mass • Higher-order interactions with target antigens (and their stoichiometry) • Binding affinities for each of the target antigens • Cooperativity or interference between bsAb components To demonstrate the strengths and limitations of mass photometry for bsAb analysis, we used it to analyze a panel of different bsAbs presenting HER2 and CD3 binding sites (Table 1). We show that mass photometry readily informs on bsAb sample purity and binding functionality. APPLICATION NOTE Rapid analysis of bispecific antibody stability and target binding by mass photometry Analyzing bispecific antibodies can be challenging, as commonly used techniques struggle to provide information on their different binding sites. In this application note, we show how mass photometry efficiently characterizes the purity, stability, and binding of multiple bispecific antibody candidates. In addition, we show that it can easily characterize high-affinity binding, and can also measure low-affinity binding with a previous crosslinking step or by using Refeyn’s MassFluidixTM HC microfluidics add-on. Table 1. Theoretical mass, mass measured with mass photometry and binding sites of the bsAbs characterized in this application note. The agreement between the expected mass and the mass measured by mass photometry (see also Fig. 1) shows that the bsAbs are properly assembled and that mass photometry accurately measures their molecular masses. bsABTheoretical mass (kDa)Measured mass (kDa)Binding sitesHER275 – 10583n/aCD375 – 8585n/aOKT-31501542 CD3Trastuzumab1501532 HER2bsAb-I1251251 CD3, 1 HER2bsAb-H1731721 CD3, 2 HER2bsAb-F1721731 CD3, 2 HER2bsAb-R54591 CD3, 1 HER2bsAb-A1992012 CD3, 2 HER2 This application note was created in collaboration with Absolute Antibody Evaluating mass, purity and aggregation Using mass photometry, we measured a panel of bsAbs with different numbers of HER2 and CD3 binding sites (Fig. 1E-I). We also measured isolated HER2 and CD3 antigens to confirm their molecular masses (Figs. 1A and 1B) and two monoclonal antibodies as controls and (OKT-3, Fig. 1C; trastuzumab, Fig. 1D). Fig.1 Mass photometry accurately measures the mass of monoclonal and bispecific antibodies and antigens. A) HER2 antigen. B) CD3 antigen. C) OKT-3 mAb. D) Trastuzumab mAb. E) bsAb-I. F) bsAb-H. G) bsAb-F. H) bsAb-R. I) bsAb-A. Our mass photometry measurements showed a single, narrow peak close to the expected molecular mass of each analyzed species (bsAbs, control mAbs and antigens, Table 1) – confirming the purity and stability of the samples. Quantifying high-affinity interactions Mass photometry operates at nanomolar concentrations, and it can readily detect and quantify high-affinity antigen binding. Here, we took the interaction with HER2 – with a dissociation constant (KD) of 0.5 nM for trastuzumab according to SPR1– as an example of a high-affinity interaction and measured its binding affinity to our antibody panel using mass photometry. First, we measured bsAb-A (5 nM) mixed with HER2 at different concentrations (0.0, 2.5, 5.0, 10 and 20 nM) after reaching equilibrium (Fig. 2). As mass photometry measures the mass of each molecule in a sample and counts the number of molecules with a given mass, it is possible to separately quantify the proportion of bsAbs bound to one or two HER2 antigens at each concentration and calculate the KD for each interaction (Table 2), as described before.2 We performed similar mass photometry analysis with the rest of our antibody panel (Fig. 3) and quantified their binding affinities to HER2 (Table 2). For these measurements, both the antibody and HER2 antigen were present at a concentration of 5 nM. A Welch’s t-test found significant differences in the affinity for HER2 between trastuzumab and bsAb-A, bsAb-I and bsAb-R. There were no significant differences in the affinity for HER2 between trastuzumab and bsAb-F or bsAb-H. These measurements show that mass photometry can readily determine site functionality and binding affinity for bsAbs. For bsAbs with one HER2 binding site, only 1:1 HER2-bsAb complexes were present, while in the case of bsAbs with two HER2 binding sites, only 1:1 and 2:1 complexes were present (Fig. 3). Fig. 2 Mass photometry resolves complex bsAb-antigen interactions. The concentration of bsAb-A was kept constant at 5 nM, while the HER2 concentration was varied (0.0, 2.5, 5.0, 10 and 20 nM). Mass photometry histograms show peaks and corresponding counts for each individual species as well as 1:1 HER2-bsAb complexes and 2:1 HER2-bsAb complexes. As the HER2 concentration increases, the peaks corresponding to free antigen and the 2:1 complex become more prominent. KD ± SD (nM) Antibody Ab:HER2 (1:1) Welch’s t- test Ab:HER2 (1:2) Trastuzumab 1.00 ± 0.90 n/a 1.21 ± 0.57 bsAb-A 2.06 ± 0.67 0.020* 3.66 ± 1.31 bsAb-F 0.92 ± 0.16 0.824 2.09 ± 0.91 bsAb-H 0.40 ± 0.08 0.126 0.85 ± 0.34 bsAb-I 0.25 ± 0.21 0.004* n/a bsAb-R 0.29 ± 0.13 0.040* n/a This suggests that there was no non-specific binding occurring at the CD3 binding sites of these antibodies. Finally, mass photometry quickly distinguished and quantified single and double HER2 binding, revealing small differences in the binding affinities that may be helpful for optimizing protein design. Table 2 Binding affinities of HER2 to the different bsAbs. Dissociation constant values were calculated from equilibrium measurements with mass photometry (Figs. 2-3). Because mass photometry can resolve both 1:1 and 2:1 binding, the KD for each interaction can be calculated separately. Both the antibody and HER2 antigen were present at a concentration of 5 nM. The bsAb-I and bsAb-R antibodies have a single HER2 binding site, so the KD for the 2xHER2 complex cannot be calculated. The middle row shows the p value of a Welch’s t-test for the binding affinity of bsAb:HER2 (1:1) against Trastuzumab:HER2 (1:1). An asterisk indicates a satistically significant difference. Evaluating low-affinity interactions Standard mass photometry measures samples at nanomolar concentration, which makes it challenging to study low-affinity interactions. This can be seen when we measure the interaction between the OKT-3 monoclonal antibody and its CD3 antigen. First, we incubated both species at μM concentrations to establish equilibrium, and then diluted the sample to the nM concentration required by mass photometry. In these conditions, OKT-3 and CD3 were dissociated (Fig. 4A). To measure low-affinity interactions with mass photometry, Refeyn offers MassFluidix HC – a microfluidics add-on to the TwoMP mass photometer. This system can perform a rapid dilution in only 37 milliseconds, so the mass photometry measurement is performed before the reaction has shifted significantly from its higher-concentration equilibrium state. We tested this approach to measure CD3:OKT-3 interactions by incubating the sample at μM concentration until equilibrium was reached, then measuring it after rapid dilution with MassFluidix HC (Fig. 4B). Under these conditions, CD3:OKT-3 complexes were visible, with 1:1 complexes representing 36.3% of counts and 2:1 complexes representing 11.7%. Next, we compared the MassFluidix HC measurement results against a more traditional crosslinking approach by using a rapid crosslinking protocol.3 OKT-3 and CD3 were incubated at micromolar (μM) concentration for 10 minutes, at a 1:4 ratio (CD3:OKT-3). We then crosslinked the sample with disuccinimidyl dibutyric urea (DBSU), incubated the reaction for 45 minutes Fig. 3 Mass photometry characterizes HER2 binding for bsAbs with different numbers of HER2 binding sites. A) Trastuzumab mAb + HER2. B) bsAb-I + HER2. C) bsAb-H + HER2. D) bsAb-F + HER2. E) bsAb-R + HER2. F) bsAb-A + HER2. For all Abs and HER2, concentration was 5 nM. Fig. 4 Mass photometry can be used to characterize low-affinity interactions. A) Measurement of a mix of OKT-3 (1 μM) and CD3 (5 μM) diluted to nM concentration. B) Mix of OKT-3 (1 μM) and CD3 (4 μM) measured after a fast 2000x dilution with MassFluidix HC. C) Measurement of the same OKT-3 and CD3 sample diluted to nM concentration after a crosslinking step. and proceeded with dilution to nanomolar concentration. The mass photometry measurement showed that 50.8% of the counts corresponded to 1:1 CD3:OKT-3 complexes and 23.6% to 2:1 complexes (Fig. 4C), indicating that crosslinking preserved the weak interactions between OKT-3 and CD3 after dilution. A small mass increase could be seen on all components of the crosslinked sample when compared to the MassFluidix HC measurement due to the presence of the crosslinker. Fig. 5 Mass photometry captures low-affinity Ab-CD3 interactions with MassFluidix HC. For each antibody, the top histogram shows a standard mass photometry measurement, the middle histogram shows a measurement taken with MassFluidix HC and the bottom one shows a measurement of a crosslinked sample. A) bsAb-I + CD3. B) bsAb-H + CD3. C) bsAb-F + CD3. D) bsAb-R + CD3. E) bsAb-A + CD3. Inset: Zooming in on the measurement of the bsAb-A + CD3 crosslinked sample shows that both 1:1 and 2:1 complexes are visible. To test CD3 binding in our panel of antibodies, we applied the MassFluidix HC approach to measure mixtures of each with CD3 (Fig. 5). No CD3:Ab complexes were visible with standard mass photometry alone, while the MassFluidix HC mass photometry measurements showed the expected CD3:Ab complexes. As expected, measurements of antibodies with two binding sites (OTK3 and bsAb-A) showed 1:1 and 2:1 antigen:antibody complexes. For antibodies with only a single binding site, only 1:1 complexes were detected. These results show that mass photometry – in combination with either MassFluidix HC or crosslinking – can characterize low-affinity interactions between antibodies and their antigens. With this type of analysis, binding site functionality can be confirmed. However, with neither approach can the sample be assumed to be at equilibrium, so they should not be used to calculate KD values. Observing double binding in bsAbs Next, we explore whether mass photometry and crosslinking can be used to characterize bispecific antibodies with two targets of different affinities. One example is bsAb-I, which has one HER2 binding site (with higher affinity) and one CD3 binding site (lower affinity). We used mass photometry to analyze a sample of bsAb-I (1 μM), HER2 (2 μM) and CD3 (4 μM), with and without crosslinking – following the procedure described in the previous section. Without crosslinking, only HER2:bsAb-I complexes were visible after diluting the sample to the nM concentrations required for mass photometry. However, when a crosslinking step was performed before dilution, the bsAb-I:HER2:CD3 complex was also detected (Fig. 6). Conclusions Mass photometry is an ideal, all-in-one technique for bispecific antibody characterization. In a matter of minutes and consuming very little sample, it can detect fragmentation, assess purity and determine if the measured antibody has the expected mass – providing straightforward information into the success of protein design and purification processes. Furthermore, mass photometry can be used for binding studies, quickly determining binding functionality, complex stoichiometry and the KD for high-affinity bsAb-antigen interactions. Lower-affinity interactions can be detected by using the MassFluidix HC rapid dilution add-on or a crosslinking step. As an additional benefit, the single-molecule nature and high mass resolution of mass photometry make it possible to observe and measure the binding affinities and stoichiometries of antibodies with multiple targets. Widely used techniques like biolayer interferometry (BLI) or surface plasmon resonance (SPR) are powerful when studying 1:1 antibody-antigen interactions but, as they measure interactions in terms of the sample average, do not give the full picture when References 1 Bostrom et al., PLoS one. 2011 https://doi.org/10.1371/journal.pone.0017887 2 Soltermann et al., Angew. Chem. Int. Ed. 2020 https://doi.org/10.1002/anie.202001578 3 Gizardin-Fredon et al., Nat. Comm. 2024 https://doi.org/10.1038/s41467-024-47732-4 4 Wu and Piszczek, Anal. Biochem. 2020 https://doi.org/10.1016/j.ab.2020.113575 5 Rapid, reliable antibody aggregation analysis with mass photometry https://refeyn.com/mass-photometry-antibody-aggregation Fig. 6 Complex formation of a bispecific antibody with two targets of varying affinities can be detected through mass photometry and crosslinking. Top: Control mass photometry measurement without crosslinking. Here, only the high-affinity HER2:bsAb-I complex is observed. Bottom: Mass photometry measurement of the same sample after crosslinking, showing the HER2:CD3:bsAb-I ternary complex. dealing with the more complex binding behavior of bsAbs. Mass photometry, on the other hand, gives an overview of all the different species and complexes in a sample and their relative abundances, characterizing the binding affinities of multiple binding sites with a single measurement.4 In addition, mass photometry measures samples in minutes, consumes only nanograms of sample and is very user-friendly – needing only a simple dilution step as sample preparation. These practical advantages make it an ideal technique for repeated, in-house analysis of bsAb binding as well as other critical quality attributes like aggregation.5 The fast, frequent analyses enabled by mass photometry derisk bsAb development and manufacturing by enabling early decision-making and supporting day-to-day quality assurance. Materials and methods Reagents - DMSO buffer, cat. No: 855190 ThermoFisher Scientific, LC-MS grade - DSBU (disuccinimidyl dibutyric urea, BuUrBu), ThermoFisher Scientific, cat. no. A33549 - 1M Tris HCl pH8.0, ThermoFisher Scientific, J22638.AE - MassGlassTM UC slides, Refeyn Ltd. Antibodies - Anti-CD3e, OKT-3, 1 mg/mL, mouse IgG2a, Ab00122-2.0, T1528802 - Anti-erbB2, Trastuzumab, 1 mg/mL, human IgG1, Ab00103-10.0, T1818A54 - HER2 from AcroBiosystems, Human Hert2/ErbB2 protein, His-Tag, cat. no. HE2-H5225 - Human CD3 epsilon&CD3 delta Heterodimer Protein, Fc Tag&Fc Tag, cat. no. CDD-H5225 - HER2-hOKT3 bsAb-A, 25 - IgG-scFv (HC C-term), cat. no. bAb0515 - HER2-hOKT3 bsAb-F, 95 - Heterodimeric IgG-scFv (type 4), cat. no. bAb0520 - HER2-hOKT3 bsAb-H, 167 - Heterodimeric Fab-scFv-Fc (type 3), cat. no. bAb0522 - HER2-hOKT3 bsAb-I, 41 - Heterodimeric Fab/scFv-Fc, cat. no. bAb0523 - HER2-hOKT3 bsAb-R, 44 - Tandem scFv, cat. no. bAb0532 All samples were provided by Absolute Antibody. Any requests for samples can be directed to support@absoluteantibody.com Crosslinking methodology For analyzing bsAb-HER2 complexes, bsAb and HER2 were incubated at room temperature for 45 min at nM concentrations. Chemical crosslinking and mass photometry measurements were performed according to Gizardin-Fredon et al.3 This rapid crosslinking protocol enables detection of the low-affinity binding complex under just one hour. For bsAb or OKT-3 and CD3, samples were incubated at micromolar (μM) concentration, at 1:4 or 1:5 molar ratio, respectively. The antibody and ligand mix was incubated 10 min at room temperature. Samples were then crosslinked with disuccinimidyl dibutyric urea (DBSU) in 400x molar excess. The optimal crosslinking time is 45 min at room temperature. The reaction was quenched by adding 25 mM Tris, for 5 min. Finally, samples were diluted to nM concentration for mass photometry measurements. V2-Jan 26 ©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, MassGlass and Refeyn are trademarks of Refeyn Ltd. orders@refeyn.com refeyn.com Refeyn @Refeyn 20Testimonials“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 ChemUnit 9, Trade City, Sandy Lane West, Oxford OX4 6FF, United Kingdom©2024 Refeyn LtdFor information on products, demos and ordering, write to info@refeyn.comSamux and Refeyn are registered trademarks of Refeyn Ltd.refeyn.com @refeynitRefeynRefeynAbout RefeynRefeyn 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.20Testimonials“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 ChemUnit 9, Trade City, Sandy Lane West, Oxford OX4 6FF, United Kingdom©2024 Refeyn LtdFor information on products, demos and ordering, write to info@refeyn.comSamux and Refeyn are registered trademarks of Refeyn Ltd.refeyn.com @refeynitRefeynRefeynAbout RefeynRefeyn 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.