JOURNAL OF APPLIED BIOANALYSIS, July 2016, p. 90-102. http://dx.doi.org/10.17145/jab.16.013 (ISSN 2405-710X) Vol. 2, No. 3 Biophysical characterization of antibodies with isothermal titration calorimetry Verna Frasca Malvern Instruments, Northampton, MA 01060, USA (Received: 3 June 2016, Revised 30 June 2016, Accepted 5 July 2016 ) Correspondence: Malvern Instruments, 22 Industrial Drive East, Northampton, MA 01060, USA. Phone: +1-4135701515. E-mail: Verna.Frasca@malvern.com 90 REVIEW Antibodies play a key role in the immune response. Since antibodies bind antigens with high speciicity and tight afinity, antibodies are an important reagent in experimental biology, assay development, biomedical research and diagnostics. Monoclonal antibodies are therapeutic drugs and used for vaccine development. Antibody engineering, biophysical characterization, and struc- tural data have provided a deeper understanding of how antibodies function, and how to make better drugs. Isothermal titration calorimetry (ITC) is a label-free binding assay, which measures afinity, stoichiometry, and binding thermodynamics for biomolecular interactions. When ther- modynamic data are used together with structural and kinetic data from other assays, a complete structure-activity-thermodynamics proile can be constructed. This review article describes ITC, and discusses several applications on how data from ITC provides insights into how antibodies function, guide antibody engineering, and aid design of new therapeutic drugs. Keywords: protein engineering, biopharmaceutical, thermodynamics, isothermal titration calorimetry, antibodies. Introduction Antibodies, also known as immunoglobulins, are naturally produced glycoproteins, secreted by plasma cells derived from B cells. Antibodies have two distinct functions: to speciically bind to their target antigens, and to elicit an immune response against the bound antigen by recruit- ing other cells and molecules. Antibodies are classically “Y-shaped” and each tip of the Y contains a binding re- gion that is speciic for an antigen (antigen-binding frag- ment or Fab). The stem of the Y is the fragment crystal- lization (Fc) region, which interacts with Fc receptors to initiate effector function. The association between an antibody and antigen involves a network of non-covalent interactions between the epi- tope (the binding site on the antigen), and the paratope (the binding site on the antibody). The ability of anti- bodies to bind antigens and receptors with a high degree of speciicity and tight afinity is the key to the immune response. Speciicity and afinity are qualities that make antibodies an important tool in experimental biology, as- say development, biomedical research, diagnostics, vac- cine production, and as therapeutic drugs. The diversity of antibody-binding capabilities is striking, since there is a high degree of structural similarity between antibod- ies. Recombinant antibodies, antibody engineering, ther- modynamics and structural data have provided a deeper understanding of how antibodies function and recognize antigens and receptors. The development of monoclonal antibody technolo- gy in the late 1970s and early 1980s opened a new era in therapeutics through the production of monoclonal antibodies, which are speciic for a single epitope of an antigen [1]. Antibodies are attractive as drugs due to their high degree of target speciicity, as well as the antibody’s distinct structural and functional domains. Early engi- neering work concentrated primarily on “humanizing” the antibody, making it tolerable to the human immune system. The modiication of antibodies via engineering is of major interest by biopharmaceutical companies since changes in antibody functionality and physicochemical