Table of Contents
Preamble
In preclinical drug development, everything revolves around understanding molecular interactions. Whether protein-ligand complexes, antibody-antigen binding or enzyme-inhibitor interactions – for the selection and optimization of biopharmaceutical active ingredients, it is not just whether a molecule binds, but how well, how strongly and why it binds.
This is exactly where isothermal titration calorimetry (ITC) comes into play: a label-free method that works in solution to determine binding affinity, enthalpy (ΔH)entropy (ΔS), free energy (ΔG) and stoichiometry can be determined in a single experiment. It makes it possible to characterize interactions not only qualitatively, but also completely thermodynamically – and under almost physiological conditions.
For laboratory staff involved in the characterization of biologics, peptide therapeutics or recombinant proteins, ITC is a powerful tool. In early screening phases, it allows the rapid identification of binding-active molecules. Later, it helps to optimize formulations, validate buffer systems or evaluate critical stability parameters.
This article is aimed at anyone working on biopharmaceutical active ingredients in the laboratory – whether in universities, start-ups or established pharmaceutical companies. We show how ITC works, when it is superior to other methods and what you need to look out for to get the best out of every experiment.
How ITC works - and why it is crucial
Isothermal titration calorimetry (ITC) is one of the few biophysical methods that directly measures the heat released or absorbed when two molecules bind. In contrast to spectroscopic or label-based methods such as SPR (surface plasmon resonance spectroscopy) or MST (microscale thermophoresis), ITC works completely label-free and in solution, which makes it particularly attractive for sensitive or native samples.
What happens in the measuring cylinder?
The principle is simple but powerful: the analyte molecule (e.g. a protein) is dissolved in a highly sensitive calorimeter cell. The ligand molecule (e.g. an inhibitor or antigen) is titrated via a fine syringe – usually in 10-20 small injections. Each time molecules bind, heat is released or absorbed. This heat is measured by the device as a thermal pulse.
What does the ITC actually deliver?
A binding curve is calculated from the heat flows for each injection. This results in
- Binding constant (K): Indicates how strong the bond is.
- Enthalpy change (ΔH): Shows whether the bond is exothermic or endothermic.
- Entropy change (ΔS): Provides information about order changes in the system.
- Free energy (ΔG): The combined thermodynamic expression of the bond.
- Stoichiometry parameter (n): How many ligands bind per target molecule?
These parameters not only help to identify hit or lead molecules, but also provide information on whether the bond is entropy or enthalpy driven, which is crucial for subsequent optimization. In its combination of informative value, simplicity and physical directness, ITC is one of the most robust methods for characterizing molecular binding – especially where understanding the energetic basis counts.
ITC in use: typical applications in biopharmaceuticals
For many in the laboratory, the question arises: When is the use of ITC really worthwhile?
The answer: Whenever the aim is not only to detect bonds, but to understand them in detail. Isothermal titration calorimetry not only provides values for the binding constants for biopharmaceutical issues, but also offers deep thermodynamic insights that are crucial for the characterization and optimization of active ingredients.
Protein-ligand interactions: The classic
In early drug research in particular, the aim is to identify binding partners, quantify their affinity and decide whether a lead structure has potential. The ITC can be used to:
- Test small molecules (e.g. inhibitors) against enzymes or transport proteins
- Investigating peptide active ingredients and their target binding
- Detecting conformational changes during binding energetically
Antibody-antigen interactions: Understanding affinity, not just measuring it
The method delivers results for solids and liquids as well as powders and pastes with high measurement accuracy, which makes it particularly valuable for the development of innovative electrode materials.
Material-specific considerations and ageing effects
ITC is a valuable addition to the development of antibody-based therapeutics:
- Precise measurement of bond enthalpy and entropy (→ thermodynamic fingerprints)
- Assessment of stoichiometry (e.g. monovalent vs. bivalent binding)
- Comparison of native and modified antibody variants in buffer or serum
This not only enables the selection of the best binding candidates, but also allows conclusions to be drawn about epitopes and binding mechanisms.
Formulation development: buffer screening and stability analyses
ITC can also be informative without ligand binding – for example for analysis:
- Buffer selection (e.g. whether a buffer itself interacts with the protein)
- of thermal stability via so-called “heat of dilution” experiments
- of self-associating systems (e.g. aggregation, dimerization).
An example from practice: In one study, ITC was used to test recombinant proteins in different formulations. This revealed not only differences in binding affinity, but also indications of structural instabilities – long before visible aggregations occurred.
Conclusion
ITC provides precise, robust and in-depth data that traditional methods often do not offer, or only indirectly. For laboratory users, this means: a tool that saves time, secures experiments and enables well-founded decisions.
Bibliography
[1] V. Linkuviene, G. Krainer, W.-Y. Chen, D. Matulis, Isothermal titration calorimetry for drug design: Precision of the enthalpy and binding constant measurements and comparison of the instruments, Analytical Biochemistry 515, 61-64, 2016
[2] H. Su, Y. Xu, Application of ITC-Based Characterization of Thermodynamic and Kinetic Association of Ligands With Proteins in Drug Design, Frontiers in Pharmacology 9, 1133, 2018
[3] L. Baranauskiene, T.-C. Kuo, W.-Y. Chen, D. Matulis, Isothermal titration calorimetry for characterization of recombinant proteins, Current Opinion in Biotechnology 55, 9-15, 2019
[4] N. L. Traulsen, Thermodynamic analysis of supramolecules by isothermal titration calorimetry, dissertation, 2015, Freie Universität Berlin
