A Buyer's Guide to Stability Measurement Platforms for Biopharmaceuticals

Overview

In the dynamic landscape of biopharmaceuticals, proteins, protein derivatives, lipids and nucleic acids stand at the forefront of innovation. Among these, monoclonal antibodies (mAbs) represent the largest class currently on the market or in development while nucleic acids represent novel modalities of drugs and vaccines with growing presence in academic research and in development pipelines.

Despite the diversity of biopharmaceuticals, a fundamental challenge persists: these products are based on complex molecules and molecular assemblies which are notoriously unstable in solution, and prone to degradation over time. Consequently, methods to effectively manufacture and store these molecules in solution for long periods are needed.

The Critical Role of Protein Stability Assays

Real-time stability assays, required to assess the shelf life of a protein in solution, are often time-consuming. Faster predictive methods have been established to accelerate the process of developing stable biologic drug formulations and process conditions. The most common of these predictive approaches are thermal unfolding methods. These methods determine the temperature at which a protein undergoes conformational changes, providing valuable data for comparative studies.

Thermal stability profiling

By generating unfolding or thermal stability profiles, researchers can compare the intrinsic stability of potential biotherapeutics under a given set of conditions – a process known as ‘Candidate Selection’. Thermal stability profiles can be generated for any candidate molecule in a range of buffers and co-solutes to help identify stabilizing/destabilizing conditions. Typical formulation additives or excipients include amino acids, sugars, polyols, salts and detergents, and care must be taken to ensure that these substances do not interfere with the assay.

In process development, thermal stability assays are used to identify purification strategies that will maximize yields of unit operations by determining stable loading and elution buffers for chromatographic processes and optimizing viral inactivation steps. More quantitative analytical technologies, such as differential scanning calorimetry (DSC), are also used in biosimilarity and comparability studies.

It is generally assumed that molecules requiring higher temperatures to induce conformational changes have longer shelf lives. However, this assumption can be misleading if the chosen technique fails to detect certain conformational events or chemical inactivation processes. Therefore, careful consideration is crucial when selecting a protein stability assay platform.

Differential Scanning Calorimetry (DSC): The Gold Standard

There are several technologies on the market for measuring protein stability. Many manufacturers benchmark these instruments and the data they generate against Differential Scanning Calorimetry (DSC). This underscores the high esteem for DSC data within the biophysical community, and why this technique has earned the title of the ‘Gold Standard’.

Dr Sorina Morar-Mitrica of GlaxoSmithKline summarizes the importance of DSC as follows, “DSC is likely the strongest, most informative and relevant of all biophysical methods currently available.”

While alternative technologies can generate good data in certain contexts, they often fall short in terms of repeatability and accuracy. These limitations, which are rarely publicized, highlight the importance of choosing the most effective measurement platform.

Key considerations when choosing a measurement platform for protein, RNA and lipid stability assessment

What do I need to know about the available technologies?

Selecting the optimal protein stability measurement platform requires a clear grasp of the fundamental differences between available technologies. Vendors often employ their own specialized terminology and present instruments in ways that highlight particular features. However, the starting point for any decision should be a thorough understanding of your specific application requirements and how different product specifications and features align with them.

Many providers of non-calorimetric technologies for protein stability position their products as direct comparison to Differential Scanning Calorimetry (DSC) (Fig.1). While these alternative instruments may offer certain advantages—such as lower sample consumption per run and higher throughput—they often do so at the expense of information richness, data reproducibility, versatility and use of surrogate rather than direct data (Fig. 2). The perceived benefit of saving sample material becomes negligible if the resulting data is misleading, potentially leading to poor decision-making or the technique proves not to be amenable to the sample types of interest. The ramifications of such issues are significant; repeating substantial portions of a development project due to ambiguous protein stability data or a need to develop a new method or procure an alternative technology can be exceedingly costly and time-consuming.

Figure 1. Thermal stability of an mAb sample assessed with three techniques.

1. Universal applicability

Ask yourself: Do I need to invest in the current and future project needs and consider a versatile tool to assess stability of proteins and other biomolecules and their assemblies?

DSC is a truly versatile and direct thermal stability assay well-suited to the fast development pace of bioscience. It is applicable to analysis of proteins, lipids¹, nucleic acids and their assemblies^2,3. It enables analysis of biomolecules as they are without a need for additional reagents and independent of the number and locations of intrinsic fluorophores (Fig. 2). By contrast, technologies which measure intrinsic fluorescence are only indirect assays limited to assessment of protein stability through changes in vicinity of fluorophores. What they measure is the change in polarity of the environment of a tryptophan residue as the protein unfolds.

There are several problems with this approach. The change in the environment of this particular amino acid residue needs to be representative of the entire molecule unfolding. That is, the tryptophan residue needs to be buried in the core of the protein and present in all the domains of a multi-domain protein. In reality, this is not likely to be the case.

Figure 2: Stress stability study mAbs at 4 and 40ºC, after 2, 3, 9, 16, 22, and 30 days of incubation.

The structural stability of a subdomain that does not include a tryptophan residue may not be assessed, which could result in incorrect formulation or candidate selection. This will impact on the next stage in the development pipeline.

Intrinsic fluorescence methods are ineffective with some proteins that do not include tryptophan residues and cannot be generally applied to characterization of other types of biomolecules, such as nucleic acids, lipids and their assemblies. The analysis of the latter would require the use of dedicated fluorescent dyes and reagents often associated with measurement artifacts.

2. Domain-specific stability characterization 

Ask yourself: Is it necessary to characterize the stability of individual domains in a multi-domain protein?

“This confirms the reported high reproducibility of DSC and justifies the validity of single measurements for the sake of time and material.”   Menzen, T. and Friess W.⁷

DSC’s high reproducibility and ability to monitor structural changes of the entire protein make it ideal for measuring the stability and cooperativity of transitions of individual subdomains. While spectroscopic techniques may generate information about subdomains, there are also many instances where they are blind to certain structural changes. This is because tryptophan residues are not distributed throughout the protein in an ideal way for fluorescence detection; some domains will have buried tryptophan residues, and some will not. This limitation is particularly problematic when early transitions are undetected as this can lead to the selection of an unsuitable candidate or incorrect formulation choices. DSC is a generic, global, high-resolution technique for measuring protein stability and does not suffer from these types of issues.

Another advantage of using DSC to monitor the subdomain stability of antibodies is that the area of the transition of the Fab binding domain is typically larger than that of the CH2 and CH3 domains, making it easier to identify. The amplitude of the signal for most spectroscopic techniques is not domain-specific; therefore, understanding which domain is stabilized or destabilized is far from trivial (Figs. 2 & 3). This is particularly important for protein engineering and candidate selection where it is vital to understand which part of the biologics is being affected.

Figure 3: DSC thermograms and 1st derivative of intrinsic DSF data for two formulations of a mAb candidate from a stress stability study. The profiles show distinct unfolding transitions in (top figure) DSC and in (bottom figure) intrinsic DSF.  Although the peak positions detected with DSC and DSF agree reasonably well, the thermal unfolding profiles recorded directly with DSC showed marked difference from the surrogate DSF data. Based on the DSF data alone the unambiguous assignment of the mAb domains and the effects of stress on the individual domains would be challenging and misleading.  In contrast to DSF, DSC thermograms provide multiple direct descriptors of thermal stability and Higher Order Structure which enables multi-level ranking of protein formulation.⁸

3. Freedom from experimental artifacts

Ask yourself: Do I require data to be free of experimental artifacts that could affect results?

Methods with low sample consumption often use fluorescence as an indirect measurement of protein stability. Unfortunately, fluorescence suffers from artifacts like quenching, aggregation and light scattering. This has two important consequences. Firstly, it makes it very difficult to get reproducible data. Secondly, these artifacts can impact the shape of the unfolding curves, which makes analysis different from measurement to measurement (Fig. 2). These factors make interpretation of data based on fluorescence detection extremely subjective, requiring a great deal of expertise and complementation by DSC data to correctly interpret results.

Some spectroscopic protein stability assays require the use of dyes. These can affect the stability of the protein or interfere with other assay components, resulting in stability profiles that reflect the interaction of the dye with the protein or a buffer component, compromising the integrity of the data.

DSC, as a first principle technique, measures the stability of the entire biomolecule, making it a truly global technique that does not suffer from the limitations outlined above.

Figure 4: The thermal unfolding of a mAb as observed by barycentric mean of intrinsic fluorescence. The profile displays a significant shift towards low wavelength upon heating (so-called blue shift as opposed to the red shift postulated for a typical DSF experiment). The drift in the DSF readout precludes the analysis. Notably the same sample shows a well-resolved unfolding profile when analyzed with DSC (insert).

Figure 5: Comparison between the SUPR-DSF (green), conventional DSF (orange), and DSC (blue) techniques: (Top) normalized first derivative data of native mAb sample, (Bottom) normalized (to max value) first derivative data of oxidized mAb sample. 

4. Reproducibility 

Ask yourself: Is consistent, reproducible data a priority? 

The exceptional reproducibility makes DSC widely used for biosimilarity and comparability studies. This is why it was a key technology in the approval of the biotherapeutic drug, Remsima, a biosimilar to Remicade. Researchers at Amgen found DSC the best technique for detecting subtle changes in the higher order structure of a multi-domain protein, correlated with the oxidation of the biotherapeutic product.¹⁰

Elsewhere, DSC is being investigated for its utility as a diagnostic tool to identify various forms of cancer¹¹. The reproducibility of DSC data is key for this application. The lead scientist on this study, Dr Adrian Velazquez-Campoy – (Institute BIFI - University of Zaragoza, Spain) highlights the high reproducibility of the DSC data: 

• 'Especially appropriate for running unattended large sets of samples and for minimizing human/accidental mistakes.'
• 'Characterized by a remarkable scan reproducibility and an impressive sensitivity for reducing sample usage.'
• 'Straightforward to program with the control software.'
• 'Easy to maintain and keep in shape by programming periodic cleaning/control scans.'

Professor John Carpenter of the University of Colorado Center for Pharmaceutical Biotechnology, adds to this, saying, “With MicroCal DSC, these types of studies can be performed with minimal hands-on work by the operator. The results obtained are by far the most reproducible of any I have seen from any analytical instrument. Incredibly, thermograms from a half dozen analyses of a given sample can be overlaid and one cannot see anything but a single line. Amazing!”

5. Buffer and co-solute compatibility

Ask yourself: Do I need to measure stability across a wide range of buffers or co-solutes?

As a non-spectroscopic technique, DSC is unaffected by buffers or co-solute interference, unlike circular dichroism and fluorescence-based methods. These spectroscopic techniques require specific buffers. Buffers which absorb light at the same wavelengths as the protein cause saturation of the signal and impede the analysis. What’s more, the use of detergents, a common component of formulation buffers, is incompatible with most fluorescence-based technologies.

6. High thermal stability measurement

Ask yourself: Do I need to characterize thermophilic proteins or biomolecules undergoing transitions at sub-ambient or high temperatures?

DSC is designed to accommodate a broad range of thermal stabilities, operating effectively at both sub-ambient and high temperatures. In contrast, spectroscopic techniques cannot typically be used below 20°C or above 90°C. These temperature limitations miss the onset of thermal denaturation in thermally labile proteins, which will often take place below ambient temperatures. At the other end of the spectrum, although many proteins have a Tm of thermal transition lower than 90°C, a temperature of 20°C higher is typically required so that the end point can be determined accurately. This means that the use DSC is preferred if the Tm is ~70°C for any of your proteins. 

7. Ease of data analysis

Ask yourself: Do I want simple-to-analyze data?

The Malvern Panalytical PEAQ-DSC (a MicroCal technology) comes with automatic analysis software, removing subjectivity and reducing the need for expertise. Competing technologies often produce data that is difficult to analyze and that can be very subjective, particularly when analyzing multi-domain proteins such as antibodies.

”The high throughput is supported by the analysis software, which is easy to use and requires no more manual calculations, therefore saving us hours. These time savings have really improved our workflow” (Katherine Bowers - Fujifilm Diosynth Biotechnologies).

8. Sensitivity to subtle changes

Ask yourself: Do I need to detect subtle changes in the stability of my protein?

DSC excels at detecting changes at all levels of protein structure (primary, secondary, tertiary and quaternary) with exceptional reproducibility. This sensitivity enables the detection of minor variations in Tₘ and higher-order structures, which is crucial for ensuring product consistency and efficacy.

A 2015 study⁵ demonstrated DSC's superiority in detecting low levels (<5%) of oxidized biotherapeutic products compared to a range of spectroscopic techniques.

9. Ongoing costs

An often overlooked aspect when choosing a protein stability measurement platform is the ongoing running costs and the need for consumables. Non-calorimetric methods, particularly those that rely on fluorescence techniques require use of pricey disposable optical units and reagents. All of these can incur substantial expenses over time. These reagents and consumables not only add to the operational costs but can also introduce variability and potential interference in assays, affecting the reliability of your data.

In contrast, DSC instruments typically require no additional reagents or consumables for routine operation. This absence of consumable requirements translates to lower running costs and minimizes variables that could compromise data integrity. Furthermore, the robust design of DSC systems often reduces maintenance expenses and extends instrument longevity, enhancing their long-term cost-effectiveness.

10. Post-sales support and service

Ask yourself: What level of service and support do I need for my instrument?

Investing in a DSC instrument from a reputable, technology-leading company ensures high manufacturing standards and reliable, publication-quality results. Choose a manufacturer with a proven track record at the forefront of developing and optimizing these technologies. These companies produce instruments with good machine-to-machine variability and are more likely to provide ongoing innovations and robust support.

Purchasing an instrument initiates a long-term relationship with the vendor. Choosing a company that offers extensive service options—telephone, in-person, email and training opportunities, field service, and expert-level assistance—ensures that you receive the necessary support throughout the instrument's lifecycle.

Comparing biomolecular stability analysis platforms

The table below highlights why DSC is widely regarded as the Gold Standard for protein stability measurement in the biopharmaceutical industry.

Table 1: DSC applications and requirements summary

Differential Scanning Calorimetry (DSC) emerges as the most universally appropriate technique for assessing the stability of biologics:

• Comprehensive assessment: Offers a global view of protein stability and is sensitive to all structural levels.
  
  
• High reproducibility: Provides consistent data, which is essential for regulatory submissions and comparability studies.
  
  
• Versatility: Proven track record of applications across broad range of biomolecules and their assemblies including proteins, nucleic acids and lipids. Compatible with a wide range of buffers and co-solutes.
  
  
• Quantitative precision: Quantitative Precision: Delivers precise thermodynamic data, aiding in detailed stability characterization.

Conclusion

While spectroscopic techniques have their merits, they come with significant limitations. These include limited applicability across ever expanding range of sample types, susceptibility to artifacts, indirect measurements, and compatibility issues, which can lead to less reliable data and potential misinterpretation.

Investing in a high-quality DSC platform, like Malvern Panalytical's PEAQ-DSC, equips researchers and developers with an unparalleled tool for protein and other biomolecular stability analysis. Its comprehensive capabilities not only streamline the development process but also enhance the quality and efficacy of biotherapeutic and vaccine products, ultimately benefiting patients worldwide.

Endnotes

*Tm (thermal transition midpoint): This is the so-called melting temperature of the sample, denoted by an endothermic peak in the DSC thermogram. The higher the thermal transition midpoint, the more stable the sample. Shifts in Tm can indicate structural heterogeneity of the sample, or degradation.

** T1/2 (the width of thermal transition at half-height): The T1/2 reflects the extent of cooperativity of the thermal transition. The narrower the transition, the more cooperative it is.

References

• 1. Willis, L F et al, Rationalizing mAb Candidate Screening Using a Single Holistic Developability Parameter |Mol. Pharmaceutics 2025, 22, 1, 181–195, https://doi.org/10.1021/acs.molpharmaceut.4c00829
• 2. Characterizing Thermotropic Phase Transitions | Malvern Panalytical, 2016
• 3. Markova, N et al. Biophysical Characterization of Viral and Lipid-Based Vectors for Vaccines and Therapeutics with Light Scattering and Calorimetric Techniques | Vaccines (Basel). 2021 Dec 30;10(1):49. doi: 10.3390/vaccines10010049
• 4. Markova, N et al. Biophysical Characterization of Viral and Lipid-Based Vectors for Vaccines and Therapeutics with Light Scattering and Calorimetric Techniques | Vaccines (Basel). 2021 Dec 30;10(1):49. doi: 10.3390/vaccines10010049
• 5. How to run mRNA-LNP measurements on MicroCal PEAQ-DSC | Malvern Panalytical
• 8. Bowers, K., Markova, N. (2019) Value of DSC in Characterization and Optimization of Protein Stability | Humana Press, New York, NY. 
• 10. Arthur, Dinh, and Gabrielson, Technical Decision Making with Higher Order Structure Data: Utilization of Differential Scanning Calorimetry to Elucidate Critical Protein Structural Changes Resulting from Oxidation |JOURNAL OF PHARMACEUTICAL SCIENCES 104:1548–1554, 2015
• 11. Differential scanning calorimetry as a diagnostic tool for cancer patients, Malvern Panalytical 2017
• 12. Arthur, Dinh, and Gabrielson, Technical Decision Making with Higher Order Structure Data: Utilization of Differential Scanning Calorimetry to Elucidate Critical Protein Structural Changes Resulting from Oxidation |JOURNAL OF PHARMACEUTICAL SCIENCES 104:1548–1554, 2015