Isothermal titration calorimetry (ITC), reaction calorimetry and differential scanning calorimetry (DSC) are the three main types of calorimetry, which passively measures changes of state or the formation of aqueous solutions. ITC currently accounts for more than a quarter of the calorimetry market. ITC initial system sales grew an estimated 11% in 2009. Its sales are projected to overtake the largest segment of the market, reaction calorimetry.

ITC’s growth is driven by the biotechnology and pharmaceutical industries. For example, in the biotech industry, ITC is useful for examining the interaction and stability of active biopharmaceutical ingredients. Since almost all binding events involve heat being absorbed or generated, ITC is used for the study of biomolecular interactions. It is also used to measure interactions between ligands such as proteins and peptides, or proteins and other ligands.

Since it applies to native, unmodified proteins in solution, ITC is useful for measuring proteins that lose or change functional behavior when chemically modified or attached to a surface. ITC is utilized for qualitative applications, such as determining whether a proposed binding interaction actually occurred, and for quantitative applications, such as measuring the concentration of a functionally active protein.

ITC is most widely used in drug discovery and development applications for lead selection and optimization. Demand is growing in both applications, according to Mary Jo Wojtusik, product marketing manager for GE Healthcare’s MicroCal product portfolio, because of ITC’s ability to provide insights into the mechanism of binding, to eliminate false positives and to identify potentially successful drug candidates. “When confirmation of small molecule interactions with target proteins is a key step, the use of ITC yields important information on the binding interaction, giving confidence that medicinal chemistry efforts are focused on the appropriate compounds. ITC can provide information on binding affinity, and more important, insights into the binding mechanism.”

“In a single experiment, ITC provides a quick and easy biological activity monitor for measuring binding affinity and stoichiomentry,” said Dile Holton, TA Instruments’ Microcalorimetry product manager, adding that such an experiment can provide rich, quantitative data about binding reactions at the molecular level. This information is used to characterize the structure, activity and functions of proteins, nucleic acids, lipids and other biomolecules.

Another major application in drug discovery and development in which ITC is used is monitoring biological activity during any purification process, Mr. Holton added. “ITC is a rapid and easy way to monitor the biological activity of biotherapeutic drugs during purification development without requiring chemical alterations such as labeling or immobilization. In a single experiment, ITC provides a quick and easy biological activity monitor for measuring binding affinity and stoichiomentry.”

ITC is also used for resolving stability issues of protein drugs. For example, it allows scientists to optimize the stability of various products, which helps to achieve the necessary shelf life. Protein instabilities can take the form of physical instabilities or chemical degradation. Currently, research is being conducted to determine the level of excipients necessary to benefit the stability of a protein drug during storage. Such investigations are becoming more frequent toward the design and development of biopharmaceuticals, according to MicroCal.

The two main companies that participate in the ITC market are MicroCal, a GE Healthcare business, and TA Instruments, a subsidiary of Waters. MicroCal is the market leader in ITC sales, and has experienced the fastest growth in its vendor share due primarily to its strong presence in the fast-growing biotech industry.

TA Instruments grew its ITC business by acquiring Calorimetry Sciences Corporation (CSC) and Thermometric AB. Since acquiring CSC in 2007 (see IBO 8/15/07), TA Instruments has expanded the business’s physical plant in Lindon, Utah, from 6,000 square feet to more than 21,000 square feet. According to Mr. Holton, the ensuing Microcalorimetry Center of Excellence provides state-of-the-art R&D labs, manufacturing facilities, applications labs and worldwide customer training facilities. As a result of the investment in facilities and personnel, as well as advances in instrument technology, the microcalorimetry product line has been TA Instruments’ fastest growing for the past two years. The company has also expanded the microcalorimetry sales and service organization from less than 10 to more than 200 people worldwide. As a result of the expansion, Mr. Holton told IBO, the number of employees at the Lindon site has increased by more than 30%. Additional microcalorimetry application scientists were also added at strategic sites in Sweden, Germany, China, Japan and Taiwan.

GE acquired MicroCal in early 2008 (see IBO 9/30/08). “GE Healthcare’s plan is to continue to develop MicroCal into a center of excellence for microcalorimetry by investing in additional R&D resources and expanding marketing and applications support at the Northampton, Massachusetts, site,” said Ms. Wojtusik. “Because microcalorimetry is complementary to GE Healthcare’s Biacore systems, based on surface plasmon resonance (SPR) technology, the company is able to offer a complete portfolio for information-rich, label-free biomolecular interactions and biomolecular stability analysis.”

MicroCal’s latest developments in ITC, the MicroCal iTC200 system and the automated AutoiTC200 version, have made advances in the speed of measurement, throughput and protein consumption compared with earlier ITC systems, according to Ms. Wojtusik. The system conducts measurements three times faster, and has a throughput of up to 75 samples per day as well as the capacity to run 384 samples unattended. A typical lab would run 30 samples per day. Protein consumption is up to seven times less.

For the steps in drug discovery and development where sample availability may be limited, TA Instruments has introduced a new low-volume instrument, the Nano ITC Low Volume, said Mr. Holton. This offers a fixed-in-place sample cell made of chemically inert 99.999% gold with an active volume of 190 µLs. Heat-sensing and temperature-control architecture allow the Nano ITC LV to deliver a baseline stability of ±0.02 µWatts per hour and a minimum detectable heat of µJoules. When compared with the Nano ITC Standard Volume instrument with a 1.0 mL cell volume, the Low Volume version uses 80% less sample, is twice as sensitive and, for many applications, the total time for an incremental titration experiment can be up to 50% shorter, according to Mr. Holton.

Among ITC’s advantages is its sensitivity. Researchers use ITC’s higher sensitivity to detect trace levels of heat that are generated (or consumed) by even the slowest physical or chemical interactions under isothermal conditions at lower temperatures. ITC also provides the ability to load larger samples and expose them to different humidity conditions than with DSC. It allows it to perform various experiments in solid-state characterization and formulation screening in pharmaceutical development.

Mr. Holton told IBO that ITC is the only assay technique that can give a direct quantitative measurement and full characterization of the thermodynamics of a binding reaction. “Most competitive assay techniques require the use of reaction-altering chemistry such as labeling or immobilization or specialized optical or radiation detection systems.”

For hit validation, Ms. Wojtusik said, other label-free methods for determining affinity include SPR, MS and NMR. “ITC and SPR biophysical technologies are complementary and together provide a more complete picture of the biomolecular interaction,” said Ms. Wojtusik.

“There is a growing demand for the thermodynamic data that only ITC can provide,” Ms. Wojtusik told IBO. “There are examples in the literature showing that medicinal chemists could produce better drugs by including ITC data (enthalpy and entropy) in the optimization process.” She cited the example of Johns Hopkins University Professor Ernesto Freire, “who shows that improvements in statin drugs over a 12-year period have been mirrored by a parallel change in the enthalpy, leading him to conclude that optimizing the enthalpy will result in ‘best in class’ drugs later on. There is also a growing body of evidence suggesting that ITC enthalpy data can be used to ‘design in’ greater specificity to small molecule drugs.”

ITC demand should continue to grow due to its applications within the biotech and pharmaceutical industries. “The ITC applications that are growing the fastest are: one, the rank order of small molecule hits, and two, characterization of qualified candidates for further evaluation and testing,” said Mr. Holton. “Most drug discovery applications that depend on a detailed understanding of the structural activity relationship have shown more frequent use of ITC.”

Also growing rapidly, Mr. Holton added, is the number of applications in which ITC is being used to characterize molecular binding reactions in life science applications. “As more and more biopharmaceuticals are being developed, ITC is becoming the gold standard for characterizing any binding or enzymatic activity. As with most analytical assay techniques, the overall sensitivity, sample volume requirements and throughput capabilities will need to continue to improve to keep up with rapidly changing research, sample validation and quality control specifications in the drug discovery and development applications.”

Greater ease of use could also impact ITC’s growth. “Enabling higher throughput with less sample consumption will continue to be desirable as the study of molecular interactions becomes more important for structure-based drug design and development,” said Ms. Wojtusik.

Bar Graph: ITC Initial Systems Sales (millions)

2009 2010 2011

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