Using Microcalorimetry to Accelerate Drug Development

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Calorimetry is the science of measuring heat changes that result from chemical reactions or physical events. The “micro” in microcalorimetry refers to the extremely small scale at which experiments can be conducted, because of its ultrasensitive technology. The ability to make highly precise, information-rich measurements using as little as 10 µg of protein/drug substance (depending on the properties of the sample) makes microcalorimetry a powerful technique for investigating the biochemical interactions that underpin drug efficacy and safety. This article provides an overview of how microcalorimetry works, and the value and application of the data this technique produces.

How does it work?

Microcalorimetry can be subdivided into isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC), both of which are valuable for pharmaceutical/biopharmaceutical applications. These techniques share similar principles of operation but differ in terms of instrumentation and experimental set-up, and as a result, offer different but complementary analytical capabilities.

The thermal core of a microcalorimeter consists of two cells: a reference cell and a sample cell (see Figure 1). When a reaction or binding event takes place in the sample cell, heat is either released or absorbed, inducing a temperature differential (ΔT) between the two cells. This differential is eliminated through automatic control of the energy input to the sample cell. The magnitude of the resulting change in energy input correlates directly with the enthalpy (ΔH) of the interaction that has taken place and can be used to determine a range of parameters, depending on the specific technique applied and the experimental set-up.

Figure 1. An isothermal titration microcalorimeter with an example of the raw data output for a binding experiment, and its conversion to detailed information about the binding reaction.

In an ITC system, the reference cell and sample cell are set to the desired temperature, with one reactant, in solution, loaded into the sample cell. The experiment is performed at constant temperature by titrating the second binding partner or reactant into the solution in the sample cell using an automated syringe capable of injecting precisely metered aliquots. As binding or reaction occurs, temperature changes of a few millionths of a degree Celsius are detected and measured, to determine the heat released or absorbed. Injection continues until the binding or reaction has reached equilibrium, to generate a complete thermodynamic profile for the reaction, including information about binding affinity (i.e., the strength of interaction between the first and second binding partner or reactant).

In a DSC experiment, the reference cell is typically filled with buffer, and the sample cell is filled with a solution of the entity under investigation; both cells are then heated at a constant known rate. Here, it is the sample undergoing a thermally-induced change that creates the temperature difference between the cells. Enthalpy values and changes in specific heat capacity are directly determined from the power drawn to correct this imbalance.

What can ITC measure?

ITC enables a robust, label-free evaluation of the mechanisms of intermolecular interactions between a drug candidate and a target molecule. These interactions are an indicator of bioactivity, a critical determinant of drug efficacy. Quantifying binding affinity is, therefore, an important way of ranking bioactivity. In addition, by providing a complete thermodynamic profile of a molecular interaction, ITC more broadly supports the optimization of a drug candidate(s). Specifically, it can be used to quantify the following parameters:

• Equilibrium dissociation constant (KD): a measure of the strength of bimolecular interactions, with smaller values indicating a stronger affinity between a ligand and a target molecule

• Reaction stoichiometry (n): the ratio of biomolecule to ligand involved in a binding interaction/reaction; this parameter quantifies how many drug molecules can be attached to the target site before saturation is achieved

• Enthalpy (ΔH): the energy released or absorbed per mole of ligand as bonds are broken and created; a measure of the type and strength of bond changes during binding, especially changes in hydrogen and van der Waals bonding

• Entropy (ΔS): the change in degrees of freedom of the interacting species relative to the complex; most usefully indicative of hydrophobic interactions and conformational changes.

What about DSC measurements?

DSC measurements directly address a different but equally important aspect of drug performance: stability, specifically thermal stability, which is a major concern for biopharmaceuticals. DSC is especially useful for the measurement of the following:

• Melting point (T_m): In a protein solution, a native (folded) protein that exhibits two-state reversible unfolding behavior is present in equilibrium with the analogous unfolded protein. T_m is the temperature at which the size of these two populations is identical. A higher T_m is, therefore, indicative of higher stability

• Onset temperature (Tonset): This is the temperature at which substantial unfolding of the protein begins to occur, so as with T_m, a higher value is associated with greater stability

• ΔH of unfolding: This is the enthalpy change associated with breakage of the non-covalent bonds that stabilize the protein and can, therefore, provide insight into unfolding mechanisms.

Via measurement of these parameters (see Figure 2), DSC can elucidate the factors that contribute to the folding and stability of native biomolecules, including hydrophobic interactions, hydrogen bonding, conformational entropy, and the nature of the physical environment, for example, pH or exposure to oxidation.

Figure 2. Differential scanning calorimetry measures the temperature at which conformational changes occur, directly supporting assessments of protein stability.

Applications in drug development

Drug candidate choice is often guided from the outset by the affinity between a therapeutic and target molecule, with high bioactivity maximizing efficacy and/or minimizing the amount of drug required to achieve the desired therapeutic effect. While advantageous for all pharmaceuticals, high bioactivity is particularly important for biopharmaceuticals because of its ability to alleviate the difficulties associated with high-concentration drug delivery via injection or infusion. Stability is also a crucial early screen for biopharmaceutical molecules because of the risk of reduced efficacy and/or immunogenicity associated with a compromised protein being delivered to the patient.

Such requirements make the informational output of microcalorimetry closely aligned to the early stages of drug development, to candidate validation, and to early formulation development. The practicalities of the techniques are equally well matched to this stage of the pipeline, with modern systems offering:

• Fully automated operation with the capacity for unattended running of industry standard 4 x 96-well plates

• High signal-to-noise ratios for excellent data quality with minimal sample volume required

• Automated washing for high reproducibility

• Compatibility with a broad range of sample types, solvents and buffers, including systems that are highly concentration, colored, and/or turbid

• Simple assay development.

In terms of specific applications, ITC has become the gold standard technology for studying intermolecular interactions, and its attributes make for a highly efficient screening tool. The level of hydrogen bonding between a drug candidate molecule and its target molecule is directly quantified by ∆H, which can consequently be an effective predictor of efficacy, more so than the hydrophobic interactions quantified by ∆S. Optimizing ∆H is a strategy applied to an increasing extent in candidate validation and early stage formulation.

However, the application of ITC can begin even earlier--in drug discovery--and extend into processing and manufacturing support. At these stages, ITC helps to:

• Quantify binding affinity to support initial candidate selection and optimization

• Confirm intended binding targets in small molecule drug discovery

• Validate IC₅₀ (drug concentration causing 50% inhibition of the desired activity) and EC₅₀ (drug concentration causing 50% of the maximum of a measured biological effect) values during hit-to-lead

• Confirm the bioactivity of an as-manufactured product and/or equivalence in a biosimilar.

The application of DSC to detect and study changes in protein structure, and determine pre-folding events is focused on the early stages of the biopharmaceutical drug pipeline, and for biosimilars, in the area of biocomparability studies. In all of these applications, the ability to study formulations without dilution to realistically explore the mechanisms of oligomerization and aggregation is particularly valuable. However, the use of DSC also extends into process development and manufacturing support, where it may be applied:

• To optimize purification and manufacturing conditions

• For lot release and/or to compare the consistency of lots produced at, for example, different manufacturing sites.

Conclusion

Microcalorimetry technology has developed considerably in recent years, and the resulting instrumentation is particularly valuable in the early stages of drug development. By providing a complete thermodynamic profile of a molecular interaction, ITC goes beyond binding affinities to provide elucidation of the mechanisms responsible for the interactions that underpin drug activity. Such insight supports the rational design and optimization of both small and large candidate molecules, to ensure a highly efficacious product. DSC is an efficient tool for stability detection and elucidation, an important activity in biopharmaceutical candidate validation, and is similarly useful during manufacturing support for lot release. Powerful and easy-to-use, both techniques boost the analytical capability accessible to drug developers helping to accelerate their work to a commercially successful conclusion.

Article Details

Pharmaceutical Technology
Vol. 41, No. 1
Pages: 57–59

Citation

When referring to this article, please cite it as N. Markova, "Using Microcalorimetry to Accelerate Drug Development,” Pharmaceutical Technology 41 (1) 2017.