Throughout the 18^th century, many scientists questioned the nature of heat. Isaac Newton thought that heat was transferred by the vibrations of particles, while Robert Hooke believed heat was a property of the body that arose from the motion of its parts.¹ However, the first well known contributor to the history of the measurement of heat is Joseph Black, a Scottish physician and chemist.¹ In 1761, he discovered using precise measurements that the addition of heat to ice at its melting point or to water at its boiling point does not result in a change in temperature.¹ His observations led him to be the first scientist to distinguish between temperature and heat, signaling the beginning of thermodynamics.

Following in Joseph Black’s footsteps were many important scientists who aided in the discovery of calorimetry and microcalorimetry. Antoine Lavoisier built the first calorimeter in 1789, and used the data collected to determine that the respiration process was a combustion reaction.¹ However, James Prescott Joule is credited with taking the first exact measurement of heat. In 1841 he determined the mechanical equivalent of heat, 4.184 J per calorie of work, to raise the temperature of 1 lb. of water by one degree Fahrenheit.¹ Joule’s work proved that heat was a measurable form of energy. Overall, these significant milestones led us to the development of modern calorimetry and microcalorimetry.

The first major contribution to modern calorimetry was on a Kawakami calorimeter in the late 1920s, used to measure the heat of mixing of alloys.¹ Nearly ten years later, the first adiabatic reaction calorimeter was constructed to measure the direct synthesis processes of intermetallic compounds.¹ As technology advanced, the ability to measure nano scale reactions developed and was coined microcalorimetry.

Microcalorimetry became critical to evaluate and derive the heat transfer associated with changes of a body’s state due to, for example, a chemical reaction or physical change. In recent times, this technique has become especially useful in the biophysical characterization of molecular interactions to understand structure-function relationships. These measured characteristics can include:

• Enthalpy
• The equilibrium constant
• Entropy
• Gibbs free energy
• The binding stoichiometry

Combined with structural information, microcalorimetry has provided a large variety of key information essential for rational drug designs.

Currently, highly sensitive microcalorimeters such as isothermal titration calorimetry (ITC) and nano differential scanning calorimetry (DSC) are the most popular to use. ITC has emerged as the primary tool for measuring the amount of heat released or consumed during a reaction, and its popularity comes from its ability to determine all thermodynamic parameters directly and quickly without the need for chemical tagging or immobilization.² This makes ITC a great in-solution method that can be used with turbid, colored solutions, or particular suspensions in a variety of applications.²

Nano DSC has been deemed one of the most important methods used in thermal analysis, which includes analyzing material thermal properties and melting temperature of biomolecules.³ The data collected is very beneficial in the design and development of drugs.

The ease of use of these calorimeters, alongside different data analysis software, has helped to create more accurate measurements that aid in overall analysis.

During drug development, it is essential to understand how the drug will interact with its target. This is characterized in the late stages of discovery into pre-formulation using ITC to get a full understanding of the driving forces behind the interaction before proceeding to further development. In most cases, the goal is to have a drug that binds specifically to its target. This will help to decrease any unwanted side-effects.

Binding specificity can be determined from the thermodynamic profile of the characterized interaction. The free energy of binding (∆G) is composed of two parts enthalpy (∆H) and entropy (∆S). Interactions that are driven by an enthalpic contribution will be more specific because this heat is coming from Van der Waals interactions and hydrogen bonds forming in the binding pocket opposed to interactions that are driven by hydrophobic interactions which is part of the entropic contribution. ITC is also used to study enzyme kinetics without the need for any dyes or immobilization.

Enzymes are a special class of therapeutics because not only do they bind to their target, but they also convert that target into a product. During an enzymatic experiment, ITC is used to monitor the heat generated by the enzyme during this process. Through analysis one can determine the V_max, the maximum rate of catalysis, and K_m, the concentration of substrate required to achieve half-maximal velocity. These values can be used during development to produce enzymes with enhanced potency and specificity.

After a pool of candidates have been selected to move forward in the development process, it is important to understand the molecule’s conformational stability and higher order structure. This will serve as a baseline as the molecule goes through different stages of development. The standard way of measuring this is to determine its T_M, or melting temperature, from Nano DSC. The T_M of a biomolecule is dependent on the formulation buffer. It is common practice to test different pHs, salt concentrations, excipients, and surfactants, so a screen will be performed to see if the molecule is stabilized or destabilized under certain conditions. The formulation will be modified until reaching a desirable T_M, while minimizing aggregation. It is also critical to monitor the higher order structure during the manufacturing process to minimize any lot-to-lot variations and to ensure the desired product is produced.

In review, microcalorimetry has a long-standing history of innovation to advance the understanding of materials and biological processes. The microcalorimetry tools and techniques used today in biological drug development have enabled researchers to detect and develop lifesaving medicine. ITC and Nano DSC are two measurement methods that enable the understanding of how a biomolecule is interacting with its exposed environment and provide key insights during the drug development cycle.