Investigating Thermal Stability with the NanoSight Pro

Introduction

Therapeutic Proteins are foundations of many therapy products that are widely accepted and used in clinics. During the development of protein-based therapeutics, it is critical to produce high quality products with minimal heterogeneity and contamination to ensure their safety. Intensive tests are carried out to mimic various stress conditions to understand the triggers of protein aggregation, and its consequences for the immune response (immunogenicity). One of the catalysts for protein aggregation is thermal reactions.

Understanding the thermal stability of proteins provides insight into their lifespan, how they should be stored, and how they should be transported. An easy way to investigate protein thermal stability is by measuring protein size and monitoring its change (which indicates the beginning of denaturation and aggregation) while increasing the measurement temperature. One of the techniques that can be used to study protein aggregation is Nanoparticle Tracking Analysis (NTA) using NanoSight. NTA not only yields information of early stage formed aggregates and their concentration, but also provides a visual assessment of the aggregation state.  

Previously, NTA was only able to control measured temperatures up to 50 °C, which for most proteins is below the thermal aggregation point. However, the new NanoSight Pro comes with extended temperature ranges up to 70 °C. Thermal ramps can be scheduled in the new NS Xplorer software to support protein aggregation studies as demonstrated using Bovine Serum Albumin (BSA) protein.

Method

BSA (10 mg/ml) was prepared in phosphate-buffered saline (PBS) and then filtered using a 0.1 µm filter to remove naturally formed aggregates. The sample was loaded into the NanoSight Pro system equipped with 488 nm laser and Low Volume Flow Cell (LVFC). The sample was then heated to and measured at temperatures (°C) of 25, 55, 60, 61, 62, 63, 64 and 65. 65°C was selected as the final temperature as BSA is expected to fully aggregate at 65 °C allowing aggregation initiation to be studied.1 The temperature ramps method (figure 1) was used to collect data with defined properties including the selected temperatures for each ramp. All measurements were made in flow at a speed of 1.5 µL/min consisting of 5 individual videos of 1500 frames.

The defined method was automatically applied through the analysis (capture and data processing) without additional input from the user. 

Figure 1. Image of NS Xplorer Method Builder used to run the BSA thermal ramp. The method consists of multiple size and concentration measurements with defined temperature and capture properties.

Results and Discussion

Filtration of the 10 mg/ml proteins was used to remove the majority of naturally formed aggregates which comprises protein monomers, dimers and trimers. These aggregates are too small to be resolved by NTA with their high concentration usually being visible as a high intensity background (figure 2). However, early-state aggregates from ~30nm can be individually detected and characterized with NanoSight Pro. 

Figure 2. Screenshot of a video of 10 mg/mL BSA recorded at 25 °C.  High concentration protein monomers (high background) and very few protein aggregates were detected. 

When looking at measurements at lower temperatures around 25 °C, very few aggregates were present in the recorded videos, as expected. This can be seen in Figure 2, where only one particle aggregate can be seen clearly (white spot). The horizontal bands present in the background of the video are caused by high protein monomer concentrations, with these bands being more or less intense depending on the protein concentration and their aggregation state.

When the temperature was increased up to 55 °C, the size and number of particle aggregates began to increase, with this increase in size continuing up to 63°C as shown by Figure 3. The increase in size was gradual up to 60 °C, but after, the size exponentially increased. 

Figure 3. Average Mode size measured at each temperature calculated from all 5 captures recorded for each temperature. Error bars are standard deviation values calculated from the modes of the 5 captures. A positive correlation can be seen from temperate increases above 63 °C

To gain more insight into the early-stage aggregation profile, assessment of size distribution is very helpful. In figure 4, size distributions before (25 °C) and during aggregation (55 °C) show clear difference in both the aggregates size as well as concentration. 

Figure 4. Average size distributions of aggregated BSA during NTA thermal ramp measurements at 25 °C and 55 °C . Larger aggregate size and concentrations are shown at 55 °C average when compared to 25 °C averages

Proof of protein aggregation can also be observed in the recorded videos as shown in Figure 5. The background seen at 25°C grows in brightness as the temperature increases due to more aggregate formation. The increase in background noise in the images from figure 5 also continues throughout the temperature ramp as depicted by increased image banding.

Figure 5. Example screenshots of NTA images of aggregated BSA protein at various temperatures. Background noise is shown to increase as temperature also increases.

The increase in noise is the reason for the large error bars in Figure 3 at 63 °C and the size appearing to drop at 64°C. At these temperatures, the noise increase was enough to begin to impact the ability of the measurement to accurately identify and track the individual aggregates of the high concentration aggregated BSA. These results and images however clearly show that the aggregation of the BSA sample already starts at 55°C and aggregation accelerates from 60 °C.

Conclusion

In this application note, we have demonstrated how NanoSight Pro can be used to investigate the thermal stability of proteins. The powerful combination of visual assessment and early aggregate size measurements helps to identify at what point a protein begins to denature and aggregate. The NanoSight Pro NS Xplorer software makes it quick and easy to set up thermal ramps, and the auto set-up functions can help inexperienced users achieve accurate results, even with a changing sample.

References

1: Borzova et al, Kinetics of Thermal denaturation and Aggregation of Bovine Serum Albumin, PLoS one, 2016, 11(4), e0153495