Developing a stable monoclonal antibody formulation requires a clear understanding of how buffer composition influences protein size, aggregation, intermolecular interactions, and thermal stability. Changes in salt concentration or formulation additives can alter antibody behavior and affect formulation performance.
This application note examines the stability of Atezolizumab across multiple formulation conditions using the Zetasizer Core, which combines dynamic light scattering (DLS), static light scattering (SLS), and intrinsic differential scanning fluorimetry (DSF) in a plate-based workflow.
DLS was used to assess hydrodynamic particle size, particle size distribution, polydispersity, and aggregation. DLS and SLS measurements were also used to evaluate colloidal stability through kD and A2/B22. DSF and SLS thermal ramp measurements provided melting temperature (Tm), onset temperature of unfolding (Ton), and aggregation onset temperature (Tagg). Repeated DLS and SLS measurements were additionally used for isothermal stress testing.
Combining DLS, SLS, and DSF provides complementary information on particle size, aggregation, colloidal interactions, and thermal stability. These measurements can support formulation screening and stability assessment while reducing the need for separate analytical workflows.
Register to access the complete application note, including the experimental methodology, formulation-specific results, figures, comparative DLS measurements, colloidal stability data, and thermal stability analysis.
Monoclonal antibodies are increasingly used in therapeutic development, with a growing number of approved treatments reaching the market. Before these biologics can be commercialized, they must undergo rigorous testing, characterization, process development, and quality control to ensure they maintain the required potency, efficacy, and stability throughout their lifecycle. Any loss of stability can compromise therapeutic performance and may increase the risk of immunogenicity, potentially affecting both patient safety and treatment outcomes.
Formulation buffers play a critical role in maintaining the stability of monoclonal antibodies. Even small changes in parameters such as salt concentration, pH, and excipient composition can have significant downstream effects on protein stability. During the formulation and early discovery stages of development, the ability to rapidly screen these variables using high-throughput approaches with low sample requirements can significantly accelerate candidate selection and optimization. This reduces a major bottleneck in development workflows, enabling researchers to identify stable formulations more efficiently while minimizing material consumption and experimental time.
The Zetasizer Core combines three analytical techniques within a single plate reader instrument: dynamic light scattering (DLS), static light scattering (SLS), and intrinsic differential scanning fluorimetry (DSF). Together, these detectors provide comprehensive information on protein and colloidal stability, enabling the measurement of:
By combining these measurements in a plate reader-based format, the Zetasizer Core enables high-throughput sample analysis while simplifying experimental workflows and reducing sample consumption.
This study explores how the Zetasizer Core can be used to characterize the stability of the monoclonal antibody Atezolizumab across a range of formulation buffers. By assessing parameters related to protein size, aggregation behavior, thermal stability, and intermolecular interactions, the impact of formulation composition on antibody stability can be evaluated using a single integrated platform.
Formulated Atezolizumab (7.6 mg/mL in PBS; Evitria) was diluted and buffer-exchanged into a range of formulation buffers to investigate the effects of salt type, salt concentration, and formulation additives on mAb stability. Buffers evaluated were: PBS, PBS supplemented with 10% sucrose, PBS supplemented with 100 mM arginine, 25 mM NaCl, and sodium acetate (NaAc) at concentrations of 50 mM, 100 mM, 200 mM, and 300 mM.
The Zetasizer Core DLS detector was used to determine the hydrodynamic size and polydispersity of Atezolizumab formulations and to assess the presence of aggregates. Atezolizumab samples were prepared at a concentration of 0.5 mg/mL, and eight replicate measurements of 20 µL were analyzed in individual wells of a 384-well Aurora microplate with round-bottom wells. Each measurement consisted of ten 1-second acquisitions.
For comparison, Atezolizumab samples were also analyzed using the Zetasizer Advance. Samples were prepared at a concentration of 0.5 mg/mL, with 1 mL loaded into individual disposable cuvettes. Measurements were performed using five acquisitions per sample, with adaptive correlation employed to automatically determine the number and duration of sub-runs.
Atezolizumab samples were filtered through 20 nm syringe filters to remove pre-existing aggregates before being prepared at concentrations ranging from 0 to 2 mg/mL. Five replicate measurements of 40 µL were analyzed in individual wells of a 384-well Aurora microplate with square-bottom wells. A clear sealing film was applied to the microplate as an evaporation barrier throughout the experiment.
The ZS Discover software includes a predefined colloidal stability method in which an SLS measurement is first performed over three passes with a measurement time of 10-seconds per pass, followed by a DLS measurement consisting of three passes of ten 1-second acquisitions per sample. SLS detector response was calibrated using BSA at concentrations of 0 to 2 mg/mL. Data was automatically processed using the ZS Discover software.
To characterize the thermal stability of Atezolizumab, samples were prepared at a concentration of 0.5 mg/mL, and eight replicate measurements of 20 µL were analyzed in individual wells of a 384-well Aurora microplate with round-bottom wells. 20 µL of oil was added as an evaporation barrier to each well. Samples were subjected to a thermal ramp from 25 °C to 95 °C at a rate of 1 °C/min with one SLS and DSF measurement acquired from each sample at every 1 °C increment. A buffer-only blank well was included, and all data were blank-subtracted during analysis using ZS Discover software.
Atezolizumab samples were prepared at 0.5 mg/mL, and eight replicate measurements of 20 µL were analyzed in individual wells of a 384-well Aurora microplate with round-bottom wells. 20 µL silicon oil was added to act as an evaporation barrier. Samples were thermally incubated at 65 °C on the Zetasizer Core, with DLS measurements comprising five x 1 second acquisitions and SLS measurements comprising a single 5 second acquisition collected from each well every 5 minutes over 18 hours.
The Zetasizer Core DLS detector was used to determine the hydrodynamic size and polydispersity of Atezolizumab in a range of formulation buffers. The particle size distributions were generally consistent across all formulations, with the highest-intensity particle population observed at approximately 11-13 nm in diameter. However, additional larger particle populations indicative of aggregation was detected in the 50 mM sodium acetate (NaAc) and 25 mM NaCl formulations, whereas there was no aggregation under the other buffer conditions (Figure 1). The PBS formulation supplemented with sucrose exhibited an additional population with an apparent diameter of less than 1 nm. This feature is likely associated with the presence of sucrose in the buffer and contributed to lower Z-average values relative to the other formulations.
The Z-average, peak 1 size (defined as the particle population with the highest scattering intensity), and polydispersity index (PI) values are summarized in Table 1.
![[AN290915-figure1-atezolizumab.png] AN290915-figure1-atezolizumab.png](https://dam.malvernpanalytical.com/46b2ab90-73f4-4116-8a01-b4c2006b19a6/AN290915-figure1-atezolizumab_Original%20file.png)
![[AN290915-figure2-atezolizumab.png] AN290915-figure2-atezolizumab.png](https://dam.malvernpanalytical.com/9177f334-86b5-4081-9310-b4c2006b182b/AN290915-figure2-atezolizumab_Original%20file.png)
| Formulation | Zetasizer Core | Zetasizer Advance | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Z-Average (nm) | Z-Average SD (nm) | Peak 1 diameter (nm) | Peak 1 diameter SD (nm) | PI | PI SD | Z-Average (nm) | Z-Average SD (nm) | Peak 1 diameter (nm) | Peak 1 SD (nm) | PI | PI SD | |
| PBS | 11.0 | 0.1 | 12.0 | 0.2 | 0.03 | 0.02 | 11.6 | 0.1 | 12.7 | 0.5 | 0.1 | 0.02 |
| PBS+10 % sucrose | 5.2 | 0.1 | 10.7 | 0.3 | 0.5 | 0.01 | 6.3 | 0.1 | 11.2 | 0.3 | 0.4 | 0.04 |
| PBS + 100 mM Arginine | 10.6 | 0.1 | 12.6 | 0.3 | 0.2 | 0.02 | 10.9 | 0.1 | 12.8 | 0.3 | 0.2 | 0.02 |
| 25 mM NaCl | 14.4 | 1.1 | 12.7 | 0.7 | 0.4 | 0.07 | 13.3 | 0.1 | 12.4 | 0.5 | 0.3 | 0.02 |
| 50 mM NaAc | 13.9 | 1.3 | 12.3 | 0.6 | 0.4 | 0.09 | 13.8 | 0.1 | 12.9 | 0.4 | 0.3 | 0.02 |
| 100 mM NaAc | 11.9 | 0.3 | 14.0 | 0.6 | 0.2 | 0.03 | 13.1 | 1.2 | 12.6 | 0.5 | 0.2 | 0.05 |
| 200 mM NaAc | 11.6 | 0.1 | 12.9 | 0.1 | 0.1 | 0.02 | 12.4 | 0.2 | 13.9 | 0.2 | 0.1 | 0.03 |
| 300 mM NaAc | 11.9 | 0.1 | 13.2 | 0.2 | 0.1 | 0.02 | 14.5 | 1.1 | 12.9 | 0.5 | 0.2 | 0.03 |
For comparison, samples were also analyzed using the Zetasizer Advance (Figure 2, Table 1). This allowed assessment of the formulation conditions using a batch DLS platform and comparison with the high-throughput measurements obtained using the Zetasizer Core. Comparable results and trends were observed between the Zetasizer Core and Zetasizer Advance measurements across all formulation conditions.
The Zetasizer Advance showed greater sensitivity to high molecular weight species, as indicated by fluctuations in the correlogram baseline and the detection of larger particle populations in the size distribution plots for the 25 mM NaCl, and 50, 100, and 300 mM NaAc formulations.
The comparable results and trends observed between the Zetasizer Core and Zetasizer Advance measurements provide confidence in the robustness and reliability of the results obtained from both platforms. While the Zetasizer Advance displayed slightly higher sensitivity to high molecular weight species, the Zetasizer Core offered substantially faster, higher-throughput analysis. All eight formulations, each measured in eight replicates, were analyzed in a plate-based format, with the complete dataset acquired in just 13 minutes.
A key advantage of combining independent DLS and SLS detectors is the ability to rapidly assess the colloidal stability of protein formulations using a simple and automated workflow to generate kD and A2/B22 values. The resulting atezolizumab kD and A2/B22 values in different formulation buffers are presented in table 2.
Analysis of the A2/B22 values indicated that all formulations, except for the 25 mM NaCl formulation, exhibited predominantly attractive protein-protein interactions. In contrast, the 25 mM NaCl formulation displayed a slightly positive A2/B22 value, indicative of weakly repulsive intermolecular interactions. kD results followed the same trend as the A2/B22 results where the 25 mM NaCl formulation was the only one to indicate a repulsive diffusion interaction. Increasing NaAc concentration resulted in progressively lower A2/B22 values, indicating that protein-protein interactions became increasingly attractive as the NaAc concentration increased.
The kD analysis is dependent on high-quality DLS data and can be strongly influenced by aggregates or particle populations outside the main sample peak. Samples were therefore filtered prior to analysis to minimize the impact of these species. However, a small secondary population remained present in the PBS supplemented with sucrose formulation following filtration. As a result, the kD data obtained for this formulation are considered less reliable and should be interpreted with caution.
Repulsive interactions are generally associated with improved colloidal stability, as they reduce the likelihood of protein self-association and aggregation at elevated concentrations. Conversely, attractive interactions can promote self-association, increasing the risk of aggregation or precipitation during high-concentration formulation.
| Formulation | A2/B22 (mol.mL/g2) | kD (mL/mg) |
|---|---|---|
| PBS | -1.97E-04 | -3.98E-02 |
| PBS+ 10% sucrose | -8.50E-04 | -15.9E-02 |
| PBS + Arginine | -1.29E-04 | -6.73E-02 |
| 25 mM NaCl | 0.309E-04 | 8.56E-02 |
| 50 mM NaAc | -0.73E-04 | -0.904E-02 |
| 100 mM NaAc | -0.96E-04 | -1.43E-02 |
| 200 mM NaAc | -1.78E-04 | -1.15E-02 |
| 300 mM NaAc | -3.51E-04 | -2.67E-02 |
The addition of additives or changes in salt concentration can have significant effect on the thermal stability of antibodies. DSF provides thermal unfolding profiles of proteins and demonstrates that buffer selection has a measurable impact on both the melting temperature (Tm) and the onset temperature of unfolding (Ton). When combined with SLS data, which measures the aggregation onset temperature (Tagg), the Zetasizer Core simultaneously provides a comprehensive assessment of protein thermal stability and aggregation behavior. These results highlight the critical importance of formulation conditions in the development of stable protein preparations. The averaged thermal unfolding and aggregation profiles of Atezolizumab in different formulations are shown in Figure 3, while the calculated Tm, Ton, and Tagg values are summarized in Table 3.
![[AN290915-figure3-atezolizumab.png] AN290915-figure3-atezolizumab.png](https://dam.malvernpanalytical.com/c06f220d-dad7-4a31-a1aa-b4c2006b16d4/AN290915-figure3-atezolizumab_Original%20file.png)
The DSF melt curves obtained for Atezolizumab displayed two distinct thermal transitions, indicating the presence of two melting temperatures (Tm values). The first transition (Tm 1) showed a greater response to formulation changes (ranging from 46 °C - 64 °C) than the second transition (Tm 2) (ranging from 76 °C - 83 °C), suggesting that this domain of the antibody was more sensitive to its buffer environment. From previous studies on different IgG samples, separate melting curves have been associated with the presence of three antibody domains where the order of denaturation is typically CH2, Fab, and CH3, from low to high temperature [1]. The CH2 domain is typically the least stable region of a mAb and is sensitive to buffer composition, suggesting this domain is represented by Tm1 [2].
The addition of sucrose to PBS caused both melting transitions to shift to higher temperatures compared with PBS alone, indicating an increase in the thermal stability of Atezolizumab. This stabilizing effect is consistent with the ability of sucrose to preferentially exclude itself from the protein surface, thereby promoting the native folded state and increasing resistance to thermal unfolding [3]. Conversely, in the presence of NaAc, both transitions shift to lower temperatures as the NaAc concentration increased, indicating that increasing NaAc concentration reduced the thermal stability of Atezolizumab.
Consistent with the DSF results, the SLS measurements showed changes in Tagg in response to the different formulation buffers. The formulation exhibiting the highest Tagg value was PBS supplemented with sucrose, providing further evidence of the stabilizing effect of sucrose on the protein. The elevated aggregation onset temperature indicates improved resistance to thermally induced aggregation, which is consistent with the increased melting temperatures observed in the DSF analysis. In contrast, as the NaAc concentration increased, Tagg decreased, indicating that aggregation occurred at lower temperatures under these conditions. The 50 mM NaAc formulation also exhibited an elevated baseline in the SLS intensity prior to the thermal ramp. This observation is likely attributable to the presence of pre-existing aggregates within the sample, which were independently detected by DLS measurements before thermal analysis (Figure 1).
| Formulation | Tm1 (°C) | Tm1 SD | Tm2 (°C) | Tm2 SD | Ton (°C) | Ton SD | Tagg (°C) | Tagg SD |
|---|---|---|---|---|---|---|---|---|
| PBS | 62.0 | 0.2 | 81.3 | 0.2 | 56.2 | 0.5 | 78.3 | 0.8 |
| PBS + 10 % sucrose | 63.9 | 0.2 | 83.5 | 0.2 | 58.1 | 0.4 | 82.2 | 0.1 |
| PBS + 100 mM Arginine | 54.7 | 0.1 | 77.6 | 0.2 | 49.8 | 0.2 | 78.4 | 0.1 |
| 25 mM NaCl | 63.3 | 0.2 | 82.3 | 0.2 | 57.5 | 0.4 | 78.3 | 1.6 |
| 50 mM NaAc | 51.7 | 0.2 | 82.3 | 0.4 | 46.1 | 0.4 | 79.4 | 1.0 |
| 100 mM NaAc | 50.1 | 0.3 | 80.3 | 0.2 | 44.3 | 0.6 | 76.4 | 0.3 |
| 200 mM NaAc | 47.6 | 0.2 | 77.8 | 0.1 | 43.0 | 0.6 | 74.1 | 0.9 |
| 300 mM NaAc | 46.0 | 0.1 | 76.0 | 0.3 | 42.3 | 0.6 | 71.7 | 0.6 |
Atezolizumab was analyzed by DLS and SLS every 5 minutes over an 18-hour period at 65 °C to monitor changes in particle size and aggregation over time. Figure 4 shows the changes in the hydrodynamic diameter and SLS intensity of Atezolizumab in different buffer formulations. This enabled the rate and extent of aggregate formation to be observed and allowed differences in formulation performance that were not apparent from thermal ramp experiments alone to be identified.
In all sample cases the effect of the SLS intensity was matched by the effect on hydrodynamic diameter. In PBS and PBS supplemented with sucrose, both the particle size and SLS intensity remained relatively stable throughout the 18 hours, although a slight increase in diameter was observed in the PBS supplemented with sucrose formulation. In contrast, samples formulated in 100 mM NaAc and PBS supplemented with 100 mM arginine exhibited substantial increases in average particle diameter, rising from approximately 10 nm to over 30 nm and 40 nm, respectively. These increases indicate the formation of larger species, suggesting protein aggregation under these formulation conditions. However, the kinetics of aggregation were noticeably different, with 100 mM NaAc formulation aggregating to a greater extent earlier on before plateauing, suggesting stabilization of the aggregate population. In contrast PBS supplemented with arginine showed a slower initial increase in SLS intensity and diameter however it didn’t show indication to stabilize and sustained a continued increase in both SLS intensity and diameter over time.
![[AN290915-figure5-atezolizumab.png] AN290915-figure5-atezolizumab.png](https://dam.malvernpanalytical.com/12b989c9-6cf8-4113-8d41-b4c2006b14b3/AN290915-figure5-atezolizumab_Original%20file.png)
The Zetasizer Core enables comprehensive characterization of antibody formulations through a single plate-based workflow. In this study, the monoclonal antibody Atezolizumab formulated in eight different buffer conditions was analyzed by DLS to determine particle size and aggregation state. Subsequent thermal ramp experiments using the SLS and DSF detectors enabled the determination of key stability parameters, including Tm, Ton, and Tagg. Together, these measurements provided a comprehensive assessment of both the colloidal and thermal stability of the antibody formulations within a single high-throughput experiment.
When combined with orthogonal technologies such as the Zetasizer Advance, these measurements provide increased confidence in the overall characterization of therapeutic proteins. Together, the techniques offer a robust and scientifically relevant approach for evaluating formulation performance.
Additional applications of the Zetasizer Core include colloidal stability analysis workflows, which enable rapid determination of kD and A2/B22 parameters across multiple formulation conditions using DLS and SLS measurements acquired over a range of sample concentrations. Furthermore, the ability to perform repeated measurements in a high-throughput plate-based format enables long-term stability and aggregation studies to be conducted on the same platform. This provides valuable insights into protein-protein interactions, colloidal stability, and aggregation behavior under accelerated stress conditions, while significantly reducing the time and sample consumption associated with traditional batch-based measurements.
[1] Akazawa-Ogawa Y, Nagai H, Hagihara Y. Heat denaturation of the antibody, a multi-domain protein. Biophys Rev. 2018 Apr;10(2):255-258. doi: 10.1007/s12551-017-0361-8. Epub 2017 Dec 18. PMID: 29256117; PMCID: PMC5899721.
[2] Xi Z, Liu X, Lin R, et al. The reduced form of the antibody CH2 domain. Protein Science. 2021; 30: 1895–1903. https://doi.org/10.1002/pro.4142
[3] Lee JC, Timasheff SN. The stabilization of proteins by sucrose. The Journal of Biological Chemistry. 1981;256(14):7193–201.