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Microcalorimetry of biopharmaceuticals


We are using calorimetry to probe several key aspects of biopharmaceuticals related to safety and efficacy. Microcalorimetry can be used to examine the overall protein stability by monitoring the unfolding temperature, Tm, of biomolecules. Since the unfolding should be the same for a biomolecule and its biosimilar, one can use the Tm to validate that a protein isolated from one manufacturing process is structurally the same as one from a new manufacturing method. Microcalorimetry can also be used to explore the binding strength of a protein and its target. Biopharmaceuticals and associated biosimilars should have similar binding affinities to the target molecules but differences in expression vectors and manufacturing processes may affect the protein binding strength.



The scope of this project is to use microcalorimetry either directly or indirectly as a tool for quickly and precisely comparing biopharmaceuticals. These comparisons can be made as a biopharmaceutical is moved from one manufacturing process to another, to compare a biopharmaceutical to a biosimilars or as a simple means of quality control. As therapeutic proteins, biopharmaceuticals are subject to a variety of intermolecular forces that can affect the higher order structure of the protein ( i.e. secondary and tertiary structure), and it is the higher order structure that often dictates the overall activity and stability of the molecule. The intended impact of this project is to give both industry and regulatory bodies confidence in methodologies used to assess the structure and function of proteins. The methods will be a combination of traditional calorimetric methods and fluorescence based assays that are suitable for high throughput and smaller sample size


  • Compare the thermal stability and unfolding energy of biological molecules and their biosimilars. Evaluate this as a measure for biosimilars and how thermal stability is related to secondary structure.
  • Compare the activity and/or binding energy of proteins and their biosimilars with the appropriate target molecules.
  • Compare fluorescent based unfolding methods with traditional calorimetric methods in terms of accuracy and reproducibility.

Technical approach

We are using differential scanning calorimetry (DSC) to determine the unfolding properties of proteins and their biosimilars that are commercially available. DSC allows us to directly monitor the unfolding of biomolecules and to determine the unfolding temperature, Tm, and the total energy required to unfold the protein, ultimately giving information about the molecules inherent stability.  Furthermore, we are using an Isothermal Titration Calorimeter (ITC) to measure the binding strength of a protein to its target molecule or drug. The ITC not only allows for determination of binding strength, but also binding stoichiometry, which is useful over other methods of determining binding affinity since it does not matter if some of the binding sites are blocked. We have used DSC and ITC to determine unfolding and binding of several drugs to commercial products of human serum albumin (HSA) isolated from pooled human blood, and recombinantly expressed HSA from transgenic yeast and transgenic rice.

We are also investigating a relatively new method to measure the melting temperatures of proteins, Differential Scanning Fluorimetry (DSF), which  relies on specific dyes that have different fluorescent properties in hydrophilic versus hydrophobic environments. As the protein unfolds, its hydrophobic regions are exposed. This allows for a greater interaction of the reporter dye with this region of the protein, thus changing the fluorescence of the dye. The advantage of this method over DSC is that it requires much less sample than DSC and it allows for a much higher throughput than the traditional DSC system. However, this method has not been thoroughly validated with respect to the various dyes that may be used. Nor has the accuracy of the measurements with respect to the DSC been sufficiently investigated, especially with regard to scan-rate. We are making direct comparisons of DSC and DSF with various dye/protein combinations, and are attempting to develop reference system(s) that may be used as a validation measurement for DSF. 


Excess heat capacity curves of various Human Serum Albumin (HSA) samples as measured on the DSC.  While ostensibly the same protein, the source of the protein and purification methods have induced subtle changes to the protein which are reflected in the melting profile and the unfolding temperature. The HSA is from the follow sources: a) and b) recombinant HSA expressed in rice; c) and d)  recombinant HSA expressed in yeast; and e) and f)  HSA from pooled human blood. 

End Date:


Lead Organizational Unit:




Brian Lang
100 Bureau Drive, MS 8312
Gaithersburg, MD 20899