A Comprehensive Guide to Protein Stability Assay Platforms

Most biotherapeutic molecules include either proteins or protein derivatives, with monoclonal antibodies (mAbs) representing the largest group of biopharmaceuticals on the market.

Antibodies have a certain binding nature, which is exploited by the biopharmaceutical industry to adjust the activity of pharmaceutically-related molecules of interest, to prevent or control diseases.

Standard small molecule pharmaceuticals are different from biopharmaceuticals, which have to be formulated and supplied in liquid form. Since proteins are relatively unstable in solution, new techniques have to be developed through which these biotherapeutic molecules can be synthesized and stored for extended period of time in solution without any degradation. In this regard, protein stability assays have been shown to be useful in the development of biotherapeutics.

Real-time stability assays have to be used to evaluate the shelf life/longevity of a protein in solution, but such assays take a considerable amount of time. Therefore, in order to expedite the learning process faster, predictive approaches have been established through which stable process conditions and biologic drug formulations can be devised.

Thermal unfolding methods

Thermal unfolding methods are known to be the most common among these predictive techniques which track the protein’s physical properties as a function of temperature. These data can be used to determine the temperatures at which a protein experiences conformational changes, which can be eventually employed for comparative studies.

It is often believed that molecules requiring higher temperatures to promote conformational changes are more stable or have a longer shelf life, but there are some exceptions to this rule which is described later in the sections.

For various drug candidates, thermal stability or unfolding profiles can be acquired in the same buffer to compare the inherent stability of promising biotherapeutics under specified conditions.

This application is called Candidate Selection. It is possible to create thermal stability profiles for all candidate molecules in different types of co-solutes and buffers to detect stabilizing or destabilizing conditions.

Amino acids, salts, sugars, detergents, and polyols constitute the standard formulation excipients or additives, and these substances should not be allowed to interfere with the test. Process development and pre/formulation groups often perform such types of tests.

The process development group aim to detect purification tactics that will lead to increased biotherapeutic yield by detecting consistent loading and elution buffers for the optimization of viral inactivation steps and for chromatographic procedures. Differential scanning calorimetry (DSC) is also employed for comparability and biosimilarity analyses.

As mentioned earlier, it is believed that molecules that need higher temperatures to cause conformational changes are more stable or have a longer shelf life, but this thumb rule can be ambiguous if the method used to track such changes is ‘blind’ to specific chemical inactivation processes or conformational events. Therefore, extra care should be adopted while procuring protein stability assay platforms.

Many technologies are available on the market for determining protein stability, and the quality of the data produced by these instruments are compared with the results collected with DSC.

DSC is the standard through which many manufacturers qualify their technologies. This demonstrates the usefulness of the DSC data for the biophysical community. This is the reason why DSC is commonly known as the ‘Gold Standard’ for thermal stability analysis.

Though alternative technologies can create good data, there are many examples where these technologies have been found to be less effective, but such examples are seldom reported for obvious reasons.

Main differences between DSC and alternative technologies

Vendors adopt different means to showcase their instruments and specifications, but customers planning to purchase these instruments to determine protein stability should first understand their specific application needs and how features and specifications of a product will affect them.

The majority of vendors dealing in non-calorimetric technologies for protein stability place their products corresponding to DSC. Though some of these systems can provide certain benefits in terms of reduced sample usage per run, reproducibility and information content will be compromised.

Therefore, it is not beneficial to save sample when the data produced can be inaccurate. Also, it becomes costly when most of the development project has to be repeated because of inaccurate protein stability data.

Key questions to ask before purchasing an instrument for protein stability assessment:

Do I want to be able to use the instrument to assess the stability of all proteins/biotherapeutic candidates?

DSC can be used for protein analyses, notwithstanding the position or number of existing tryptophan residues. While other technologies measure the inherent fluorescence, they can only determine the polarity change in a tryptophan residue environment as the protein unfolds and thus, do not provide accurate protein stability.

This method also involves several issues. The environmental change of this specific amino acid residue must indicate the whole molecule unfolding. In other words, the tryptophan residue has to be embedded in the heart of the protein and should also exist in the domains of a multidomain protein.

However, this does not appear to be the case because the structural stability of a subdomain, which lacks a tryptophan residue will not be evaluated. This could lead to wrong candidate selection or formulation, which will subsequently affect the corresponding department in the development pipeline.

There are certain proteins that do not even have tryptophan residues and thus, intrinsic fluorescence techniques cannot be applied to assess those proteins.

Do I want to be able to characterize the stability of all the individual domains in a multi-domain protein?

Since DSC provides high reproducibility and is also able to track structural changes of the whole protein, it can be employed to determine the stability of separate subdomains. Spectroscopic methods that create protein stability profiles include data regarding subdomains, but there are many situations where these methods fail to furnish this information and overlook specific structural changes.

This can be attributed to the fact that tryptophan residues are not ideally distributed across the protein for fluorescence detection; while some domains will bury tryptophan residues, others will not.

This is perhaps imperative if an instrument overlooks the initial transition and thus wrongly chooses a formulation buffer or candidate; as structural changes have taken place in the protein that were invisible to the method used for measuring them. DSC is a high-resolution, universal technique method, is suitable for determining protein stability, and does not experience such problems.

DSC can also be used to track the subdomain stability of antibodies, where the Fab binding domain size is often larger when compared to the CH3 and CH2 domains, thus making it relatively easy to locate.

In most spectroscopic methods, the signal amplitude is not domain-specific and therefore, it is very important to understand which domain is actually stabilized or destabilized. This is critical for candidate selection and protein engineering projects, where it is essential to know about the affected part of the biologic.

Do I want my data to be free of experimental artifacts that might affect my results?

Fluorescence is used in techniques with lower sample consumption to indirectly measure protein stability. However, fluorescence suffers from specific artifacts, including inner filtering, quenching, light scattering, and aggregation, which change as a function of the concentration of the sample. This leads to two major outcomes:

• It is not easy to obtain reproducible data when comparing various days and in various laboratories because it is difficult and time-intensive to ensure that all the co-solutes are at analogous concentrations.
• The shape of the unfolding curves can be changed by the artifacts, and this makes analysis different from one run to another, highly subjective, and requires an enormous expertise to understand properly.

In certain spectroscopic protein stability assays, dyes are used to track the stability of protein. However, these tests experience the same problems as inherent fluorescence and the dye could impact the protein stability itself, or a buffer constituent could impact the level of interaction between the protein and the dye.

All these can lead to stability profiles that mirror the protein-dye interaction that is more complicated by a buffer component and which does not have much to do with protein stability. This also holds true for those applications where stability profiles are employed as an indirect measure to screen tiny molecules attached to potential drug targets.

Since DSC is a global, first line method that determines the entire protein stability, it does not have the issues described above.

Do I want reproducible data?

As mentioned above, DSC is also known as the "Gold Standard" of protein stability tests, because it offers a highly reproducible data. Therefore, DSC is applied in biocomparability and biosimilarity studies.

In yet another case, an Amgen group performed a study, where DSC was found to be the best method for detecting an oxidized biotherapeutic product. The highly reproducible DSC data along with the fact that DSC is able to identify even slight changes in a multi-domain protein structure contributed to the success of this experimental application.

Further, DSC is being studied to diagnose patients with different forms of cancer. Obviously, the high reproducibility provided by the DSC method is critical for this application.

Do I want to measure the stability of my biologic in a broad range of buffers or co-solutes?

There are certain technologies that have particular needs for compatible co-solutes or buffers or both. Here, circular dichroism can be considered as a good example of this constraint, as specific buffers like those employed in formulation studies are capable of absorbing light at the same wavelengths just like the protein, which leads to signal saturation.

Further, using even small quantities of detergents is incompatible with the majority of fluorescence-based technologies. These issues do not affect the non-spectroscopic DSC technique.

Do I need to characterize proteins with high thermal stability?

DSC has been developed to determine various thermal stabilities of proteins and can operate at high as well as subambient temperatures. In comparison, spectroscopic techniques methods cannot be employed below 20°C or over 90°C.

Thermal denaturation of thermally labile proteins will usually occur below ambient temperatures, and this phenomenon may be overlooked by standard spectroscopic methods.

While most proteins exhibit a mid-point or TM of thermal transition below 90°C at the other end of the spectrum, a 20°C higher temperature is needed so that the end point can be accurately established. In other words, DSC has to be used if the TM is approximately 70°C for the proteins.

Do I want simple-to-analyze data?

The MicroCal VP-Capillary DSC from Malvern Panalytical employs an automatic analysis software that eliminates subjectivity and also reduces the need for know-how.

The results obtained from most contending technologies can be highly subjective and too complicated to understand. This is obvious when multi-domain proteins like antibodies are being studied.

“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.

Do I want to detect subtle changes in the stability of my protein?

In addition to providing reproducible data, DSC technique also detects changes at all levels of protein structure - whether primary, secondary, tertiary and quaternary meaning that it can detect even slight changes in TM and thus higher order structure (HOS).

In another recent example, the DSC thermogram of a multi-domain protein, such as antibodies, was found to be the best method for detecting low levels of biotherapeutic product that had become oxidized as opposed to a wide range of spectroscopic methods (Figure 1).

Figure 1. The thermal unfolding of an antibody as observed by intrinsic fluorescence detection. The profile has no sharp transition and was therefore suboptimal for TM determination.

How good is the manufacturer or vendor?

Manufacturer or vendor’s reputation should be assessed properly to ensure that money is invested in quality products. High standards have to be applied to develop DSC instruments and should be designed only by top technology companies to generate decision-making results and publication-quality.

Manufacturers that have an impressive history of designing, developing, and optimizing such technologies should be selected. This is because such companies develop good machine-to-machine variability instruments and are usually the first to pioneer new developments in the technology and its related applications.

Such manufacturers are also likely to have the required experience, know-how, and support resources to develop and support unique instruments.

What about post-sales service and support?

Buying a system marks the beginning of a long business association with the manufacturer or vendor and therefore, it is important to buy instruments from a company that provides acceptable service and post-sales support.

Therefore, customers should select a company that offers in–person, telephone, and email support as well as continuous training opportunities, expert-level support, and field-based service.

Prior to procuring a system, customers should ask what support is being provided by the company. This way, they can infer the expected depth and quality of the support as soon as the new system has been installed in the laboratory.

Table 1 shows why the DSC method is extensively used across the biopharmaceutical industry and is regarded as the "Gold Standard" for measuring protein stability.

Table 1. Comparing protein stability analysis platforms

This claim has been reinforced in a recent paper, which describes the responses of several professionals in the field when asked to grade the effectiveness of technologies for use in the biopharmaceutical development pipeline (Gabrielson and Weiss IV (2015), Journal of Pharmaceutical Sciences 104:1240–1245).

The applications spanned from formulation development and candidate selection through to biosimilarity and comparability.

Conclusion

The above detailed information shows that DSC is the perfect tool for evaluating biological stability. Though certain non-spectroscopic methods offer benefits in terms of lower sample consumption as opposed to the DSC method, they are not suitable for this type of application, owing to inherent weaknesses including scattering, poorly characterized sub domain structural data, dye interference, poor/no signal, and inner filtering.

Such incompatibilities and faults may lead to costly, suboptimal decisions, resulting in the non-developability of biologic drugs and redevelopment of projects. In contrast, Malvern Panalytical MicroCal DSC system offers gold standard, artifact-free, and reproducible stability data for all proteins in all buffers.

About Malvern Panalytical

Malvern Panalytical provides the materials and biophysical characterization technology and expertise that enable scientists and engineers to understand and control the properties of dispersed systems.

These systems range from proteins and polymers in solution, particle and nanoparticle suspensions and emulsions, through to sprays and aerosols, industrial bulk powders and high concentration slurries.

Used at all stages of research, development and manufacturing, Malvern Panalytical’s materials characterization instruments provide critical information that helps accelerate research and product development, enhance and maintain product quality and optimize process efficiency.

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