Buffer optimization is an important part of biologics formulation because the solution environment can influence protein folding, aggregation, homogeneity, thermal stability, shelf life, and overall formulation performance. Screening candidate buffers early in development can help researchers identify conditions that maintain protein quality while reducing the time required to establish a suitable formulation.
This application note examines lysozyme as a model protein and compares its behavior in several commonly used solution conditions. Lysozyme from hen egg white was prepared at 1 mg/ml in acetate, HEPES, Tris, sodium chloride, and phosphate-buffered saline formulations. The study applies complementary light-scattering and fluorescence techniques to evaluate both the initial condition of the protein and its response to controlled thermal stress.
Dynamic Light Scattering, or DLS, measures the hydrodynamic size and polydispersity of particles in solution. For protein formulations, these measurements can indicate whether a sample is predominantly monomeric and homogeneous or contains a broader distribution of particle sizes. A low polydispersity index is associated with a relatively narrow size distribution, while a higher value indicates greater sample heterogeneity.
Differential Scanning Fluorimetry, or DSF, assesses thermal stability by monitoring changes in intrinsic tryptophan fluorescence as a protein unfolds during heating. The resulting thermal profile can be used to determine parameters such as the onset temperature of unfolding and the melting temperature. Static Light Scattering, or SLS, can be recorded at the same time to monitor the development of thermally induced aggregation.
Using these methods together provides a broader picture than any single measurement alone. DLS characterizes protein size and homogeneity before thermal stressing, DSF tracks structural unfolding, and SLS provides information about aggregation behavior. This orthogonal approach is useful because protein unfolding and aggregation do not necessarily occur at the same temperature or follow the same pattern.
The initial DLS measurements showed that lysozyme remained predominantly monomeric and monodisperse in all investigated formulations. Average hydrodynamic diameters were around 3.5 nm, although the HEPES formulation produced a broader particle size distribution and the highest polydispersity index among the tested conditions.
Thermal measurements revealed clearer differences between formulations. Acetate buffer at pH 4 provided the greatest thermal stabilization of lysozyme and produced the highest unfolding onset and melting temperatures in the study. Tris and sodium chloride also improved thermal stability relative to phosphate-buffered saline. HEPES showed lower thermal stability and greater sample heterogeneity under the investigated conditions.
The full application note provides practical information for scientists involved in protein characterization, formulation development, analytical development, and biopharmaceutical quality assessment. Readers can explore:
The results demonstrate how high-throughput biophysical screening can distinguish formulation conditions even when initial protein size measurements appear broadly similar. Register for full access to review the complete experimental method, quantitative tables, thermal transition data, figures, and conclusions from the lysozyme buffer comparison.
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Protein stability is a critical consideration throughout biopharmaceutical development, formulation optimization, and quality control. Buffer composition can significantly influence protein folding, aggregation, shelf life, and overall performance. Rapid screening of buffer systems enables researchers to identify conditions that maximize protein stability while minimizing formulation development time.
Dynamic Light Scattering (DLS) is widely used to assess protein size, homogeneity, and the presence of aggregates in solution. By measuring the hydrodynamic diameter and polydispersity of protein formulations, DLS can provide early indications of changes formulation quality.
Differential Scanning Fluorimetry (DSF) provides a label-free approach for assessing protein thermal stability by monitoring changes in intrinsic tryptophan fluorescence during thermal unfolding. Simultaneously, aggregation onset can be detected through Static Light Scattering (SLS), providing a comprehensive assessment of protein behavior under stress.
In this study, DLS was used to characterize the size distribution and homogeneity of lysozyme in different buffer systems prior to thermal analysis, while DSF and SLS were employed to evaluate thermal unfolding and aggregation behavior. The combined approach enables a comprehensive assessment of protein stability and formulation performance. This study investigates the effect of four commonly used buffer systems and a sodium chloride solution on the stability of lysozyme at a concentration of 1 mg/ml.
The following buffers were prepared using analytical-grade reagents and deionized water: acetate buffer (pH 4.0), 50 mM HEPES buffer (pH 7.0), and 10 mM Tris-HCl buffer (pH 7.0). A 10 mM NaCl solution was also prepared. Buffer pH values were adjusted using HCl or NaOH as appropriate and verified using a calibrated pH meter prior to use. All solutions were prepared fresh or filtered through a 0.02 μm membrane before use when required. Lysozyme from hen egg white was prepared at a concentration of 1 mg/ml in all the buffers as well as phosphate-buffered saline (PBS).
Each formulation was prepared in eight replicate wells to enable assessment of measurement repeatability. Aliquots of 20 μL were dispensed into individual wells of a 384-well Aurora microplate with round-bottom wells.
DLS measurements were performed in backscatter detection angle (158 °). Measurements were acquired using the recommended settings of three acquisitions per well, with an acquisition time of 1 s per acquisition.
Thermal stability measurements were performed using a temperature ramp from 25 °C to 85 °C at a heating rate of 1 °C/min, corresponding to a temperature increment of 1 °C between successive measurements. Intrinsic fluorescence measurements were collected using an excitation wavelength of 280 nm at 100% UV intensity. Simultaneously, Static Light Scattering (SLS) was measured at 830 nm using a laser intensity of 100%.
DLS measurements were initially performed to characterize the size and homogeneity of lysozyme in the different buffer systems. The results presented in Table 1 showed excellent repeatability, with all formulations exhibiting average hydrodynamic diameters at around 3.5 nm and low polydispersity indices. These data indicate that lysozyme remained predominantly monomeric and monodisperse under all investigated conditions.
| Buffer | Average diameter, nm | PI |
|---|---|---|
| PBS | 3.89 ± 0.08 | 0.07 |
| Acetate buffer | 3.76 ± 0.09 | 0.04 |
| HEPES | 3.16 ± 0.05 | 0.19 |
| Tris | 3.67 ± 0.07 | 0.03 |
| NaCl | 3.70 ± 0.08 | 0.07 |
Among the tested formulations, the HEPES sample exhibited the smallest average protein size (3.16 ± 0.05 nm) but also displayed the highest polydispersity index (0.19) suggesting a broader particle size distribution than the other buffer systems.
![[an260915-lysozyme-buffer-stability-case-study figure1.jpg] an260915-lysozyme-buffer-stability-case-study figure1.jpg](https://dam.malvernpanalytical.com/322934e8-4235-4ee9-8bf5-b4c100ec344e/an260915-lysozyme-buffer-stability-case-study%20figure1_Original%20file.jpg)
As it can be seen the size distribution of lysozyme in HEPES buffer is broader compared to the others as it was also reflected by the PI. In contrast, acetate, Tris, PBS and NaCl formulations exhibited narrow distributions and low PI values (< 0.1). This is an early indication that lysozyme is less stable in 50 mM HEPES compared to the other buffers.
To further evaluate formulation stability, thermal unfolding was measured using Differential Scanning Fluorimetry (DSF). Representative thermal unfolding profiles are shown in Figure 2, while the extracted thermal transition temperatures are summarized in Table 2.
| Buffer | Tm1 (°C) | Ton1 (°C) | Tagg SLS (°C) |
|---|---|---|---|
| PBS | 72.1 ± 0.3 | 67.7 ± 0.2 | 69.6 ± 1.0 |
| Acetate buffer | 77.2 ± 0.3 | 72.8 ± 0.1 | - |
| HEPES | 73.8 ± 0.2 | 69.5 ± 0.1 | - |
| Tris | 75.2 ± 0.3 | 70.6 ± 0.1 | - |
| NaCl | 76.0 ± 0.2 | 71.5 ± 0.3 | - |
![[AN260915-figure2.png] AN260915-figure2.png](https://dam.malvernpanalytical.com/dba002ea-afb6-44f9-b88f-b4b8002b58fa/AN260915-figure2_Original%20file.png)
It can be seen from Table 2 that the onset temperature of unfolding (Ton1) and melting temperature (Tm1) were found to be highly reproducible across all replicate measurements, with standard deviations below 0.3 °C.
Significant differences in thermal stability were observed between the buffer systems. Lysozyme formulated in acetate buffer exhibited the highest thermal stability, with a Tm1 of 77.2 ± 0.3 °C and a Ton1 of 72.8 ± 0.1 °C. These values were approximately 5 °C higher than those measured in PBS, indicating a substantial stabilizing effect of the acetate formulation. Since lysozyme is known to be stable under mildly acidic conditions, the enhanced thermal stability observed in acetate buffer is consistent with its expected solution behavior.
The NaCl and Tris formulations also improved stability relative to PBS. The lower thermal stability was observed in HEPES and combined with the broader size distribution measured by DLS, suggests that this formulation was less favorable for maintaining lysozyme stability under the conditions investigated.
Static light scattering measurements were acquired simultaneously with DSF to monitor thermally induced aggregation. However, clear aggregation transitions were not consistently observed for all formulations, preventing reliable determination of aggregation temperatures, apart from lysozyme in PBS. This behavior suggests that unfolding and aggregation were not strongly coupled under the investigated conditions and highlights the importance of combining multiple orthogonal techniques when assessing protein stability
The combined DLS and DSF measurements demonstrated that buffer composition significantly influences lysozyme stability. Among the formulations investigated, acetate buffer (pH 4) provided the greatest thermal stabilization, resulting in the highest unfolding onset and melting temperatures. Tris and NaCl formulations also improved stability relative to PBS, whereas HEPES showed the lowest thermal stability and exhibited a broader particle size distribution, indicating increased sample heterogeneity.
DLS measurements confirmed that all formulations remained predominantly monodisperse prior to thermal stressing, while DSF provided highly reproducible measurements of thermal unfolding. Together, these techniques provide complementary information on protein size, thermal stability, and aggregation behavior, enabling rapid identification of optimal formulation conditions. For lysozyme, acetate buffer was identified as the most favorable condition among those tested, demonstrating the value of high-throughput biophysical screening during early-stage formulation development.