The biopharmaceutical landscape is evolving fast, with innovation in proteins, lipids, and RNA therapeutics. As these molecules move through the development pipeline, one challenge looms large: ensuring their stability. Selecting the right stability measurement platform is therefore a mission-critical decision.
That’s why we’ve created A Buyer’s Guide to Stability Measurement Platforms for Biopharmaceuticals. This comprehensive resource outlines what you to know about the latest technologies, key considerations, and best practices to simplify your decision-making process.
Stability analysis is the foundation of success in biopharmaceuticals. Whether it’s monoclonal antibodies (mAbs), RNA-based therapeutics, or lipid nanoparticles, understanding how these molecules behave under varying conditions is essential for:
But not all stability measurement tools are equal. The guide explores why Differential Scanning Calorimetry (DSC) stands out as the gold standard for comprehensive, reliable, and reproducible data.
Here’s a sneak peek at what you’ll discover:
This guide is tailored for pharmaceutical scientists, R&D professionals, and decision-makers who want to:
Making the wrong choice in stability measurement can cost your team time, money, and credibility. With our Buyer’s Guide, you’ll gain the clarity and confidence to invest in a platform that aligns with your goals and delivers measurable results.
Please login or register for free to download A Buyer’s Guide to Stability Measurement Platforms for Biopharmaceuticals today and discover how tools like the MicroCal PEAQ-DSC can revolutionize your approach to stability analysis.
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 are needed to inform selection of candidates with favorable stability profiles and to ensure effective manufacturing and storage of these products over long periods.
Real-time stability assays, required to assess the shelf life of biomolecules in solution, are 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 or other biomolecules undergo thermal transitions, providing valuable data for comparative studies.
By generating unfolding or thermal stability profiles, researchers can compare the intrinsic stability of biomolecules under a given set of conditions during protein engineering and candidate selection. Further, thermal stability profiles can be generated for any candidate molecule in a range of buffers and co-solutes to help identify stabilizing/destabilizing conditions during pre/formulation development.
In process development thermal stability assays are used to identify purification strategies that will maximize yields by determining stable loading and elution buffers for chromatographic processes. The more quantitative analytical technologies, such as Differential Scanning Calorimetry (DSC), are also used for biosimilarity and comparability studies.
It is generally assumed that molecules undergoing thermally induced transitions at temperatures close to ambient conditions are intrinsically unstable while the molecules requiring higher temperatures to induce conformational changes may have longer shelf lives. For example, proteins requiring higher temperatures to induce thermal transitions are less prone to degradation pathways linked to partially/unfolded protein states. However, reliability and effectiveness of thermal stability analysis of biomolecules depends on the choice of stability assay platform.
There are several technologies on the market for measuring stability of proteins and other biomolecules. 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’.
While alternative technologies can generate good data in certain contexts, they can fall short in terms of repeatability, specificity, versatility, information content and accuracy. These limitations, which are rarely publicized, highlight the importance of choosing the most effective measurement platform.
| A
S K Y O U R S E L F | Do I need to invest in the current and future project needs and consider a versatile tool capable of assessing stability of proteins and other biomolecules and their assemblies? |
|---|---|
| Is it necessary to characterize the stability of individual domains in a multi-domain protein? | |
| Do I require data to be free of experimental artifacts that could affect results? | |
| Is consistent, reproducible data a priority? | |
| Do I need to measure stability across a wide range of buffers or co-solutes? | |
| Do I need to characterize thermophilic proteins or biomolecules undergoing transitions at sub-ambient or high temperatures? | |
| Do I want simple-to-analyze data? | |
| Do I need to detect subtle changes in the stability of my protein? | |
| What is the added cost of running the thermal stability technology? | |
| What level of service and support do I need for my instrument? |
Selecting the optimal biomolecular 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 position their products as direct comparison to Differential Scanning Calorimetry (DSC). 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. 1). The perceived benefit of saving sample material becomes negligible if the resulting data is misleading, potentially resulting in poor decision-making or if 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 WP160725stabilitybuyers - A Buyer's Guide to Stability Measurement Platforms for Biopharmaceuticals.png] Figure 1 WP160725stabilitybuyers.jpg](https://dam.malvernpanalytical.com/64a0e17a-164a-4749-b195-b2d800eddd60/Figure%201%20WP160725stabilitybuyers_Original%20file.jpg)
Figure 1. (A) Thermal stability of a mAb sample presented through primary readout of three techniques: DSC and extrinsic and intrinsic DSF. Note that DSC directly reports on incremental progression of thermal transitions, while there is nothing differential about the technical principle and primary readout of DSF - DSF signal is cumulative. The amplitude and the area of the DSC peaks are directly related to the amount of the material undergoing unfolding. This is not the case for DSF, where the amplitude of the primary signal depends on the fluorescence characteristics of fluorophores and the differences in the total fluorescence of folded and unfolded species at every temperature. DSC unfolding profiles of mAb samples (B) neat and (C) after oxidative stress overlayed with the corresponding 1st derivative traces of DSF data. Note that reliable assignment of Fab unfolding transitions is not possible based on intrinsic- and extrinsic-DSF data and requires complementary DSC data.
DSC is a truly versatile and direct thermal stability assay well-suited to the fast development pace of bioscience. It is equally well applicable to the analysis of proteins1, lipids1, nucleic acids2, and their assemblies2,3. It enables analysis of biomolecules as they are, without a need for additional reagents and independent of the number and locations of intrinsic fluorophores. Technologies that measure intrinsic fluorescence, despite being label-free, are only indirect assays limited to assessment of the stability of proteins and only through changes in the polarity of environment in the vicinity of tryptophan residues 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 necessarily the general case for a large and evolving space of proteins in development.
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 the characterization of other types of biomolecules, such as nucleic acids, lipids and their assemblies which are readily amenable to analysis by DSC. Fluorescence-based thermal stability analysis of biomolecules other than proteins requires the use of dedicated fluorescent dyes and reagents, often associated with measurement artifacts.
When throughput is essential and identifying and deselecting very unstable samples is your primary aim, DSF may offer a value, however, it is wise to confirm the findings with DSC whenever possible.
DSC’s high reproducibility and its ability to monitor structural changes of the entire protein make it ideal for measuring and comparing the stability and cooperativity of transitions of individual subdomains. Menzen and Friess reported “..high reproducibility of DSC ..justifies the validity of single measurement for the sake of time and material”7. While spectroscopic techniques may generate information about subdomains, there are also many instances where they are blind to certain structural changes or bring in artefacts (Fig. 1B & C). 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 not well-resolved or undetected, as this can lead to the selection of an unsuitable candidate or incorrect formulation choices (Fig. 2B. DSC is a generic, global, high-resolution technique for measuring biomolecular 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 (Figs. 1 & 2). 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. 2A & B), and DSC data would be required to resolve the issue. 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 2 v2 WP160725stabilitybuyers - A Buyer's Guide to Stability Measurement Platforms for Biopharmaceuticals.png] Figure 2 WP160725stabilitybuyers .jpg](https://dam.malvernpanalytical.com/d5b85314-324a-4789-9454-b2d800eead88/Figure%202%20WP160725stabilitybuyers%20_Original%20file.jpg)
Figure 2: DSC thermograms and 1st derivative of intrinsic DSF data for two mAb candidates 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 for both mAbs, 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 and early transitions 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.
Methods with low sample consumption often use fluorescence as an indirect measurement of protein stability. Unfortunately, fluorescence suffers from artifacts due to quenching and light scattering (Fig. 3). This has two important consequences. Firstly, it makes it difficult to get reproducible data. Secondly, these artifacts can impact the shape of the unfolding curves, which makes analysis challenging and different from measurement to measurement. 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 stability assays require the use of dyes. These can affect the stability of biomolecules or interfere with other assay components, resulting in stability profiles that reflect the interaction of the dye with the biomolecules 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 3 WP160725stabilitybuyers - A Buyer's Guide to Stability Measurement Platforms for Biopharmaceuticals.jpg] Figure 3 v3 WP160725stabilitybuyers.jpg](https://dam.malvernpanalytical.com/81d9a4c1-f4fb-4fdb-bf69-b2ea008b736c/Figure%203%20v3%20WP160725stabilitybuyers_Original%20file.jpg)
Figure 3: The thermal unfolding of a mAb as observed as 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).
DSC is renowned for the highly reproducible data it provides.
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!”
![[Figure 4 WP160725stabilitybuyers - A Buyer's Guide to Stability Measurement Platforms for Biopharmaceuticals.jpg] Figure 4 v3 WP160725stabilitybuyers.png](https://dam.malvernpanalytical.com/0f7feb06-6794-4aac-bbe4-b2ea008bcd71/Figure%204%20v3%20WP160725stabilitybuyers_Original%20file.png)
Figure 4: Overlays of blank-corrected and baseline-subtracted DSC traces of RNA, mAb, mRNA LNP and DPPC samples
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 product5.
Elsewhere, DSC is being investigated for its utility as a diagnostic tool to identify various forms of cancer6. The reproducibility of DSC data is key for this application. One of the lead scientists on this study, Dr Adrian Velazquez-Campoy – (Institute BIFI - University of Zaragoza, Spain) highlights the high reproducibility of the DSC data:
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 special considerations for the choice of buffers. Buffers which absorb and emit light at the same wavelengths as the protein sample interfere with the signal and impede the analysis. What’s more, the use of detergents, a common component of formulation buffers, is often incompatible with fluorescence-based technologies.
DSC is designed to accommodate a broad range of temperatures, 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.
The MicroCal PEAQ-DSC range comes with automatic analysis software, removing subjectivity and reducing the need for expertise. Alternative technologies often produce data that requires extensive processing and derivation 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
DSC excels at detecting changes in protein structure with exceptional reproducibility. This sensitivity enables the detection of minor variations in Tm and unfolding profile linked to higher-order structure, which is crucial for ensuring product consistency and efficacy.
Athur et al.7 demonstrated DSC's superiority in detecting low levels (<5 %) of oxidized biotherapeutic products compared to a range of spectroscopic techniques.
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.
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.
The table below highlights why DSC is widely regarded as the Gold Standard for protein stability measurement in the biopharmaceutical industry.
| Application/Requirement | MicroCal PEAQ-DSC | Circular Dichroism | Intrinsic Fluorescence | Extrinsic Fluorescence |
|---|---|---|---|---|
| Proteins, nucleic acids, lipids & biomolecular assemblies measured as they are | Yes | Yes (excluding lipids) | No | No |
| Sensitive to all levels of protein and nucleic acid structure | Yes | Yes (primarily secondary structure) | No | No |
| Quantitative readout directly proportional to amount folded material | Yes | Yes | No | No |
| Global, high resolution protein stability assay | Yes | No | No | No |
| Multiple and direct metrics of protein stability (Tm, Tonset. T1/2, DH, unfolding profile, Cp) | Yes | No | No | No |
| Fingerprint nature of unfolding profile | Yes | Yes | No | No |
| Free of optical artifacts | Yes | No | No | No |
| Compatible with most buffers, excipients and complex biofluids | Yes | No | No | No |
| Highly reproducible | Yes | No | No | No |
| No need for dyes, labels or chemical additives | Yes | Yes | Yes | No |
| Free from buffer and co-solute interference | Yes | No | No | No |
| Measure Tms above 70°C | Yes | No | No (not in a typical case) | No |
| Gold standard TM measurements | Yes | No | No | No |
Table 1: DSC applications and requirements summary
Differential Scanning Calorimetry (DSC) emerges as the most universally appropriate technique for assessing the stability of biologics:
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 the Malvern Panalytical 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.
