Testing thermal stability of proteins with DSC
In the second of our three-part blog series, we’re going to take a look at how differential scanning calorimetry (DSC) works and how it’s used to understand the thermal stability profile of protein subunits. By the end of the series, you’ll understand how DSC can help you speed up the selection of vaccine candidates.
Read the previous blog in the series here.
DSC and thermal stability testing
In our last blog [link], we showed you how fingerprinting can help speed up vaccine development programs by ruling out unsuitable vaccine candidates at an early stage. DSC can be a useful tool here, as it measures the thermal stability of a protein: a critical parameter to determine its overall stability and its unique fingerprint. But why is thermal stability so important?
Vaccine researchers need to know how much active substance is present in a batch of vaccines. Protein and biomolecular product manufacture can’t ever be 100% active. This is because cells are used in the manufacture, and the mechanisms rely on a black-box process that isn’t fully defined. This complexity means that scientists must also understand how consistent the process is between batches, and how the active biomolecules change over time.
And let’s not forget about viral-vector and virus-like particles. Protein structure has a direct impact on the stability of the resulting vaccine in different environmental conditions or formulations. So, we need a detailed understanding of the protein structure of the particle or virus capsid. If we’re able to understand and optimize protein stability early on, we can increase the chance of the clinical trials being successful and improve the overall shelf-life of the final product. That’s where DSC comes in. MicroCal PEAQ-DSC measures the protein’s melting point (Tm). Tm doesn’t measure melting, but the point at which half of the protein unfolds – that irreversible stage at which denaturation takes place. It’s this unfolding that gives a relative measure of the protein’s stability.
How does DSC work?
MicroCal PEAQ-DSC is a flexible instrument, allowing for flexibility in your process, and it can be fully automated. It gives a direct readout without needing further analysis or interpretation. Additionally, MicroCal works across a broad temperature range (from 2°C – 130°C), meaning you can measure nearly every type of protein.
These features make MicroCal highly sensitive to thermally induced unfolding so it gives a ‘fingerprint’ indication of the nature of a protein. It’s effective even when comparing very similar proteins or sub-species of a virus. So, how is DSC used?
First, a protein or vector is added to a sample cell along with a buffer solution. Alongside this sample cell, a reference cell, containing only the buffer, is introduced. Now it’s time to heat things up: the temperature of the box is gradually increased and the temperatures of the two cells are recorded.
When the protein in the sample cell starts to unfold (Tonset), the temperature of the sample cell begins to lag. More energy is needed to maintain the equivalent temperature rise in the buffer cell. The additional heat enthalpy indicates the change in apparent heat capacity, and this reaches a peak at Tm – what DSC measures. The DSC generates temperature and energy graphs automatically, showing the unique profile of a protein:
Sleeker model, better results
MicroCal has high system sensitivity and accuracy, thanks to a nifty capillary design. This capillary structure has increased surface area, providing higher sensitivity while maintaining the temperature range and automation benefits. It also helps to prevent protein aggregation and dropping out of solution, meaning more accurate results.
By understanding the thermal stability profile of different protein subunits or viral vector or virus-like particles, unstable vaccine candidates can be quickly identified and screened. The stability profile of potential vaccine can be assessed too, by changing pH conditions or adding ligands. And, parameters can be set to control storage conditions, protecting candidates with strong potential.
This can help get effective vaccines to those who need them, quicker. In the next blog, we’ll cover how MicroCal PEAQ-DSC can be used as a screening tool for other protein stability techniques.
Download the full guide here to find out how DSC can improve your vaccine development program.