Differential scanning calorimetry: your guide to analyzing proteins for vaccine stability

Introduction 

We’re living in exciting times with the speed of vaccine development now faster than it’s ever been before. Breakthrough developments in nanotechnology and nucleic acid chemistry have turbocharged development pipelines. Yet, despite the emergence of new vaccine technologies, protein subunit-based vaccines remain a popular choice for development. 

No matter which modality you’re working with, the goal is the same: decrease the time it takes to bring your vaccines to the people who need them. 

Whether you are working with protein subunits, viral particles, or virus-like particle vaccine candidates, differential scanning calorimetry (DSC) delivers the highest standard of stability and characterization data. This guide explains how DSC can help optimize your process to speed up the selection of vaccine candidates. 

Fingerprinting vaccine candidates for optimal candidate selection

From March 2020 to September 2021, we saw huge leaps in vaccine development, with COVID-19 providing the catalyst for accelerated programs. Pharmaceutical companies collaborated in new ways to maximize the impact of their expertise and regulators worked with the industry to expedite approval processes. As a result, mRNA and adenovirus vector vaccines, which have shown promise for so long, finally became a medical reality. But vaccines like these present a new set of challenges. mRNA vaccines are composed of biomolecular assemblies and, just as sub-strains of the same virus can have very different structures (leading to variations in vaccine stability in viral vector and virus-like particle candidates) a change in the production processes can generate a range of mRNA-lipid assemblies of various sizes and structures. Variabilities like these can lead to delays and increased costs during development if the optimal strain or assembly is not chosen from the outset. 

Determining the ‘fingerprint’ of the higher-order structure of a vaccine candidate is critical for product characterization, formulation development and comparability analyses; areas that are increasingly important as developers explore novel candidates. 

Despite the emergence of new vaccine technologies, the older second-generation protein subunit vaccines continue to be a popular choice for development as the vaccine market continues under this new head of steam. Based on tried and tested technology, protein subunit vaccines are used for a wide range of diseases, such as Hepatitis B, Tetanus and Typhoid and characterization of these vaccines is equally important. As protein sub-unit vaccines continue to evolve, it can be challenging to create the most effective antigen combination from increasingly complex structural molecules.

Clinical trials form a large part of the vaccine development timescale, and a considerable part of the financial outlay. A robust quality-by-design (QbD) program¹ enables you to ‘home in’ on candidates that are most likely to perform well in clinical trials. In order to achieve this, vaccine developers must gather as much information as possible about the protein structure of the vaccine candidates. 

Revealing the true value of thermal stability testing 

Since cells are used to manufacture protein and biomolecular products and the mechanisms of this rely largely on a black-box process that is not fully defined, it is impossible to create products that are 100% active. However, vaccine researchers need to know how much active substance is present within a batch, the consistency between batches, and how the active biomolecules may change over time. 

Similarly, when working with viral-vector and virus-like particles, you need a detailed understanding of the protein structure of the particle or virus capsid. Protein structure has a direct impact on the stability of the resulting vaccine in different environmental conditions or formulations. Understanding and optimizing protein stability early in vaccine development has a direct correlation with the success of a clinical trial and the shelf-life of the final product. 

DSC is increasingly relied on to measure the thermal stability of a protein, a critical parameter that will determine the stability of the protein and its unique fingerprint. DSC achieves this by measuring the protein’s melting point (T_m). Conversely, to what the name suggests, the T_m does not measure melting but rather the point at which half of the protein unfolds – that irreversible stage at which denaturation takes place. This unfolding gives a relative measure of the protein’s stability. 

The following guide focuses on how DSC can be used to determine stability and characterization data for proteins, but this can be extended to all biomolecules – including the mRNA-lipid assemblies so important in mRNA vaccine development. 

How does DSC work?

DSC is a powerful tool: fully automated, it gives a direct readout without further analysis or interpretation, and it works across a broad temperature range (from 2°C - 130°C), making it suitable for the measurement of nearly every type of protein. The combination of these factors means that DSC is highly sensitive to thermally induced unfolding and can give a ‘fingerprint’ style indication of the nature of a protein, even when used to compare very similar proteins or sub-species of a virus. 

A protein or vector is added to a sample cell along with a buffer solution. A reference cell, containing only the buffer, is then introduced alongside the sample cell in an insulated box. 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 (T_onset), the temperature of the sample cell begins to lag and 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 T_m. The temperature and energy graphs automatically generated by the DSC shows the unique profile of a protein (see figure 1). 

Figure 1: Energy graphs showing the unique profiles of four different proteins according to the onset of thermal unfolding, the peak at the melting point and the change in apparent heat capacity.

The latest DSC models benefit from a capillary design that increases the system’s sensitivity and accuracy. The increased surface area provided by the capillary structure gives even higher sensitivity while maintaining the temperature range and automation benefits. The capillary also helps to prevent the protein from aggregating and dropping out of the solution which improves the accuracy of the results. 

By understanding the thermal stability profile of different protein subunits or viral vector or virus-like particles, unstable vaccine candidates can be identified and screened out early in the development process. By changing pH conditions or adding ligands, the stability profile of a potential vaccine can be assessed, and parameters set to control storage conditions, protecting those candidates that show strong potential. 

Screening and fingerprints 

One of the key strengths of DSC is its ability to work at a high thermal range. This makes it a useful screening tool for other protein stability techniques, such as differential scanning fluorimetry (DSF) or circular dichroism (CD) that provide higher throughput or work with smaller sample sizes. 

In DSF, protein samples are added to a well plate and their thermal unfolding is followed by an intrinsic fluorescence signal. As the protein starts to unfold intrinsically, fluorescent groups in the protein structure get exposed to the aqueous solution and their fluorescence spectra typically experience redshift to higher wavelength positions for the spectral maxima. 

Alternatively, an extrinsic fluorescence detection can be employed in DSF and a fluorescent dye is added to protein samples on a well plate in a real-time PCR machine. When the protein is in its folded state, the dye remains quenched by the water solution, but as the temperature increases and the protein begins to unfold, the fluorescent dye is exposed to the protein’s hydrophobic core and begins to bind. As the dye binds, its fluorescence intensifies, providing a profile of the protein’s unfolding as the temperature increases. 

DSF has many benefits: it requires a small sample and can run up to 96 tests simultaneously. Nevertheless, it is important to validate the applicability of the DSF assay using DSC first, due to DSF’s limited temperature range and the fact that the intrinsic fluorescence signal might be complex or the dye may alter the molecules. With the DSC profile established, DSF tests can be compared against the DSC standard. Where protein unfolding temperatures are outside of the range of DSF, DSC can be a viable alternative. 

Similarly, DSC works as an orthogonal technique for CD. CD is a highly sensitive technique that uses a wide range of UV light to analyze the secondary structure of proteins. Although CD is also a high-throughput technique, it is limited by buffer conditions. Since many molecules are absorbed in UV light, several standard buffers are unsuitable and require additives to maintain stability. By pre-screening with DSC, any anomalies generated by the CD technique are identified. 

DSC is a powerful tool for characterization and profiling during the assay development stage, alongside high-throughput, small sample volume techniques. It offers wide operating temperatures, ultra-clear pinpointing of the transition stage and high resolution of domains or protein components offered by the technique. DSC does this without the use of dyes or buffer conditions that could compromise protein stability, providing a stable reference point for further analysis techniques. 

DSC scans can therefore be used to provide the precise and detailed characterization of protein structures and their thermal stability parameters. High throughput methods, such as DSF and CD, can then be used for wider testing on multiple batches and as a way of confirming the critical material attributes (CMAs) for the quality target product profile (QTPP), as part of the QbD process. 

In addition to screening, DSC provides precise thermostability profiles, or ‘fingerprints’, of the protein, making it useful in the first principle profiling used in the early stages of discovery. This fingerprint can be used later in the clinical development process as a way of establishing the QTPP. 

Setting a stable base for accelerated vaccine development 

DSC is a trusted and proven technique for protein thermal stability screening in vaccine development. Due to the wide operating temperatures, clear indication of the transition stage and straightforward operating conditions that do not compromise protein stability, DSC can achieve the highest standards of screening. 

By accurately determining the T_m and exact protein stability profile, DSC is fast becoming the method of choice to identify the most stable protein sub-units, viral vectors and virus-like particles. By direct analysis and by defining the parameters for high throughput methods (such as DSF and CD) DSC can help to determine the ideal pH and temperature parameters to extend vaccine shelf-life and ensure safe storage.

Since DSC can provide an unparalleled level of detail for protein profiles, it is an important part of the QbD process, particularly when determining the QTPP of a vaccine candidate. As part of the vaccine development lifecycle, it will continue to help developers save time and money, both by screening out unsuitable candidates and helping to set the parameters for success. 

As researchers push boundaries and move into uncharted territory, developing vaccines for novel viruses and finding new ways to prevent disease, DSC is set to help developers reach these previously unobtainable goals. By increasing the accuracy and speed of characterization and screening the most promising and stable candidates using DSC, researchers can now decrease the time it takes to bring lifesaving vaccines to the people who need them.

References

1. https://www.ema.europa.eu/en/human-regulatory/research-development/quality-design