Advancing Monoclonal Antibody Stability Studies With Mass Photometry

Antibody aggregation can severely impact a biopharmaceutical product’s efficacy and safety. Hence, researchers apply an array of techniques to assess the physiochemical stability of candidate therapeutic antibodies throughout the biopharmaceutical development cycle. Mass photometry is a robust method to characterize the biophysical properties of antibodies. It can also be applied to antibody stability studies and the characterization of antibody–antigen interactions. Biophysical characterization using mass photometry Mass photometry is an innovative biophysical analysis tool that can be used for sample characterization, interaction studies and the analysis of protein complexes. The technique builds on the principles of interference reflection microscopy and interfero-metric scattering microscopy to measure the molecular mass of proteins and protein assemblies in solution.1,2 ® APPLICATION NOTE Advancing monoclonal antibody stability studies with mass photometry DR. AMY CHAU Field Applications Scientist Figure 1. The principles of mass photometry. A detector measures the scattered light and creates an interference pattern that can be visualized and analyzed to identify the species in the sample, based on their mass, and quantify their relative abundance. Biomolecules approaching a glass surface are illuminated by a laser The biomolecules scatter light, which interferes with the light reflected at the interface The resulting interference contrast scales linearly with the mass of the biomolecule A mass histogram is generated from the single molecule measurements Molecules in solution Incident light Scattered light Mass photometry contrast Molecules in solution Expected mass (kDa) 200 400 600 800 1000 400 200 800 1000 600 Mass measured by mass photometry (kDa) Incident light Scattered light Mass photometry contrast Molecules in solution Mass photometry contrast Counts 1 2 3 4 APPLICATION NOTE ADVANCING MONOCLONAL ANTIBODY STABILITY STUDIES WITH MASS PHOTOMETRY 2 Instruments measure the light scattered by a biomolecule in solution, which is linearly scaled with the molecule’s volume and refractive index (Figure 1). The optical properties and density vary by only a few percent within a class of biomolecules (e.g., proteins). This means that the scattering signal directly correlates to the molecule’s mass. Mass photometry enables accurate mass measurements of single molecules in solution without the need for labels. This makes it a rapid and cost-effective method to study the native behavior of biomolecules with minimal sample preparation. Mass photometry can be used to determine complex stoichiometry or oligomeric states and monitor macromolecular assembly or other complex processes. This technique can also be used to characterize and monitor sample heterogeneity – this is particularly important for antibody studies – providing insights into sample purity and allowing the observation of stability under different conditions. Two case studies are presented below to illustrate mass photometry applications in therapeutic antibody development. Characterizing antibody stability Mass photometry is an ideal way to assess sample purity and monitor aggregate formation, owing to its wide mass range and high dynamic range. In this case study, researchers used mass photometry to conduct forced degradation assays to screen candidate therapeutic antibodies and select the right candidates for further development.3 Forced degradation assays monitor how samples react under different stress conditions. This study tested 14 different antibodies to establish whether changes in exposure to light, pH and hydrogen peroxide (oxidative stress) resulted in antibody aggregation or fragmentation. For this reason, the researchers used an automated mass photometer (TwoMP Auto) to expedite their workflow, reduce the amount of hands-on time required and improve the precision of the results by removing human error from the pipetting and mixing steps. For “antibody 1” (Figure 2), the results showed the presence of the intact antibody monomer. However, a low molecular mass species (LMM) and high molecular mass species (HMM) were also identified, representing the fragmented antibody and aggregated antibody respectively. The percentage of abundance for each peak was quantified for each form of antibody 1, indicating that stress condition 2 induces the highest degree of fragmentation, whereas stress condition 3 induces the highest degree of aggregation. For “antibody 2” (Figure 3), the aggregation region was further defined with trimer formation. Comparison of relative abundance showed that stress conditions 1 and 3 promote aggregation (the formation of dimers and trimers). This study demonstrated that analytical tools spanning a wide mass range may be required to capture the complete data set for in-depth characterization of all products or variants in Condition % Fragmented antibody % Intact antibody % Aggregated antibody Control 9% (± 0.7) 90% (± 0.6) 1% (± 0.3) Stress 1 10% (± 1.1) 88% (± 1.1) 2% (± 0.1) Stress 2 27% (± 1.8) 70% (± 1.0) 4% (± 0.9) Stress 3 14% (± 2.2) 81% (± 2.1) 5% (± 0.3) Figure 2. Fragmentation and aggregation products from “antibody 1” upon exposure to three stress conditions. Figure 3. Fragmentation and aggregation products from “antibody 2” upon exposure to three stress conditions. Condition % Fragmented antibody % Intact antibody % Aggregated antibody (dimer) % Aggregated antibody (trimer) Control 4% (± 0.8) 95% (± 0.7) 1% (± 0.1) 0% Stress 1 5% (± 0.4) 85% (± 0.3) 8% (± 0.4) 2% (± 0.2) Stress 2 7% (± 1.5) 91% (± 1.4) 2% (± 0.1) 0% Stress 3 5% (± 0.8) 86% (± 0.5) 8% (± 0.3) 1% (± 0.1) APPLICATION NOTE ADVANCING MONOCLONAL ANTIBODY STABILITY STUDIES WITH MASS PHOTOMETRY 3 degradation analysis. Mass photometry can quickly screen a large number of candidates, supporting rapid decision making in biopharmaceutical development. Assessing antibody aggregation When assessing the aggregation levels of a biologic, mass photometry can complement other techniques such as size exclusion chromatography (SEC). A study was designed to assess the aggregation levels of the monoclonal antibody trastuzumab (a biologic used to treat cancer under the commercial name Herceptin) and four biosimilars (clones 3, 8, 9 and 10), using both mass photometry (TwoMP system) and SEC.4 Although SEC separates molecules based on their molecular sizes and mass photometry identifies molecules based on their mass, the results from both techniques should lead to the same conclusion. The SEC chromatogram produced in the study (Figure 4a) showed a small secondary peak – indicating the presence of an antibody dimer – for clones 3 and 10. When the same five samples were measured using mass photometry (Figure 4b), clones 3 and 10 also showed notable secondary peaks, indicating the presence of dimers. SEC results must be quantified appropriately to achieve standardization. Absorbance cannot be directly compared to the number of counts measured in mass photometry (i.e., the height of the histogram bar), because the absorbance data is both a function of the molecule’s concentration and is dependent on the samples’ UV absorbing properties. The specifications of the UV detector itself (e.g., its sensitivity at a particular UV wavelength) also influence the data. Therefore, the Beer–Lambert law must be applied to the absorbance values generated from SEC chromatograms in order to calculate relative abundance values and compare the data to mass photometry results. For this type of experiment, the results generated from SEC and mass photometry are very similar (Table 1). However, the advantage of using mass photometry is that these results can be achieved in a shorter time – 1 minute per measurement and less than 3 mins including analysis. Mass photometry also requires a smaller sample for analysis – only nanograms compared to the micrograms required for SEC. If researchers don’t need sample separation or want to be more strategic in selecting the right candidate for these longer separation experiments, then mass photometry is the right choice. Figure 4. (a) SEC chromatogram showing the elution of molecules over time, where larger molecules elute first. Below is a magnified version highlighting a secondary peak between 7 and 8 minutes. (b) Mass photometry histogram using the same samples. (a) (b) Table 1. Quantified percentage abundance values calculated from SEC-UV (top) and mass photometry (bottom). SEC-UV Monomer 99.9 97.7 99.8 99.2 97.6 Dimer 0.1 2.3 0.2 0.8 2.4 Abundance (%) Sample Trastuzumab Clone 3 Clone 8 Clone 9 Clone 10 originator Mass photometry Monomer 99.3 96.8 98.8 98.7 97.1 Dimer 0.7 3.2 1.2 1.3 2.9 Abundance (%) Sample Trastuzumab Clone 3 Clone 8 Clone 9 Clone 10 originator Mass photometry systems from Refeyn Refeyn is a company specializing in the development and manufacture of instrumentation for the characterization of biomolecules using mass photometry technologies. The case studies highlighted in this application use the TwoMP system offered by Refeyn, which is quick and easy to use; from measurement to analysis, users can obtain the results in less than five minutes. The TwoMP is a mass photometry instrument that requires manual pipetting during sample preparation, and the TwoMP Auto features the same internal system plus a pipetting robot. With the TwoMP Auto, the user can design experimental protocols with the number of repeats and dilutions that are necessary, and the instrument performs the dilutions and transfers samples to the measurement stage. Advantageously, the TwoMP system can be retrofitted with the automation unit to upgrade when the need arises. APPLICATION NOTE ADVANCING MONOCLONAL ANTIBODY STABILITY STUDIES WITH MASS PHOTOMETRY 4 Conclusion Whether it is using a manual or automated system, researchers can gain quick insights by conducting mass photometry on a dedicated instrument. When used as an initial screening tool to streamline analyses, it is an easy and cost-effective way to accelerate biologics development. In the development of therapeutic antibodies, mass photometry is well suited for the characterization of antibody aggregation and fragmentation. It can also be used to characterize antibody– antigen interactions and quantify binding site occupancy under different conditions. Furthermore, binding affinity between proteins can be determined using single-shot Kd measurements.5 Mass photometry is already making a big impact in furthering the understanding of biomolecule behaviors. Since 2018, Refeyn has installed over 350 mass photometers worldwide, with users in both academia and industry, and more than 300 peer reviewed papers have used these techniques to answer pressing biological questions. Advance your research with mass photometry References 1. Verschueren H. Interference reflection microscopy in cell biology: methodology and applications. J Cell Sci. 1985;75:279-301. doi:10.1242/ jcs.75.1.279 2. Ortega-Arroyo J, Kukura P. Interferometric scattering microscopy (iSCAT): new frontiers in ultrafast and ultrasensitive optical microscopy. Phys Chem Chem Phys. 2012;14(45):15625-15636. doi:10.1039/ c2cp41013c 3. Characterization of forced antibody degradation with automated mass photometry. Application Note. Refeyn. https://www.refeyn.com/ characterizing-antibody-aggregation-with-mass-photometry. 4. Verscheure L, De Vos J, Sadowska W, et al. Characterizing antibody aggregation with mass photometry. Technical Note. Refeyn. https:// www.refeyn.com/mass-photometry-vs-sec-in-protein-analysis. 5. Soltermann F, Foley EDB, Pagnoni V, et al. Quantifying Protein-Protein Interactions by Molecular Counting with Mass Photometry.Angew Chem Int Ed Engl. 2020;59(27):10774-10779. doi:10.1002/ anie.202001578