The fled of biologics, i.e. proteins, particularly monoclonal antibodies (mAbs), as pharmaceutical drugs, is the fastest growing segment in the biopharmaceutical industry. Therapeutic formulations of monoclonal antibodies usually require concentrations on the order of 100 mg/mL or more, a condition that exacerbates protein denaturation and aggregation tendencies. Selecting the monoclonal antibody with the best denaturation/aggregation profile and identifying the solution conditions (formulation) that maximize the long term stability of the antibody are major goals in the development process. To accomplish these goals, researchers routinely measure different aspects of protein denaturation and aggregation using various complementary or orthogonal techniques. Some techniques measure the conformational stability of the protein (e.g., differential scanning calorimetry, differential scanning ?uorometry, isothermal chemical denaturation) and other techniques measure aggregation after lengthy sample incubation (e.g., size exclusion chromatography, light scattering). Since the expected long term stability is longer than one year, incubation times are usually reduced by accelerating denaturation/aggregation process by, for example, increasing the incubation temperature or stressing the sample in different ways. It would be highly desirable for a more efficient development of new therapeutic mAbs, to have access to a technique that provides the rate of denaturation/aggregation fast and accurately. In this technical note, we introduce the Thermal Activity Monitor (TAM) as a way to measure the rates of denaturation and aggregation of monoclonal antibodies and other proteins at constant temperature.

Biofilms are defined as “bacteria growing on a surface as single or multi-layered communities”. Over the past several years the importance of biofilms in research has been increasingly growing as scientists realized that many, if not a large majority, of microorganisms exist naturally as biofilms. Indeed, biofilms studies are common in water science (natural waters or waste waters), environmental studies (biofilm formation on rocks), material science (antifouling surfaces) and finally medical or biomedical studies (infections, implantology). The study of biofilms has evolved with the development of many staining, molecular, or microscopy techniques. However, most of these techniques are destructive and therefore only provide endpoint measurements. Consequently, the metabolism and the dynamic behavior of biofilms remains mostly inaccessible to researchers. In this context, isothermal microcalorimetry is a valuable tool enabling investigation of development and metabolic activities of growing or mature biofilms. Several techniques combined with an isothermal calorimeter can be used to study biofilms and this application note aims at summarizing these techniques and providing some tips and tricks.

Isothermal calorimetry is a viable technique for determining the quality of soil through respiration measurements. Substrate induced respiration follows the carbon in the soil and can be used to determine carbon incorporation. Additional experimental conditions can further characterize the soil through the idea of calorespirometry. The findings via calorimetry in the references show a direct correlation between calorimetry results and soil quality.

通过差示扫描量热仪（DSC）使用CSC Nano-DSC III测定蛋白质的热力学参数，需要大量相同的蛋白质做表面等离子体共振或荧光研究。由于Nano-DSC III 高度的敏感性和基线重复性，以及样品池的小体积(300 μL)和毛细管配置（能够延迟不可逆的蛋白质聚合和沉淀），一个完整的、可判断的、准确的扫描可以得到基本上所有的想要测试的蛋白质。?

通过活体组织提供一个直接的的定量检测同时测量热率产生和氧气利用率，意味着在代谢状态下面对改变生理和环境条件检测细微变化。The ratio of calorimetrically measured heat flux to the respirometrically measured oxygen flux is often called the “calorimetric-respirometric ratio” or CR ratio.

The expression or replication of genes is affected by the binding of small molecule ligands and proteins to nucleic acid sequences. Such binding events are critical for the physiological integrity of organisms and therefore are of fundamental interest to life scientists. Recently, the thermodynamics driving these interactions have also become important to pharmaceutical scientists investigating the anticancer, antibacterial and antiviral potential of nucleic acid/ligand interactions. In addition, as the number of diseases identified as being due to a malfunction of cellular control processes increases, the possibility of treating disorders by manipulating gene expression is further focusing attention on the thermodynamics underlying nucleic acid binding affinity and specificity.

Simultaneous measurements of the rates of heat production and oxygen utilization by living tissues provides a direct quantitative means of detecting subtle changes in metabolic state in the face of altered physiological and environmental conditions. The ratio of calorimetrically measured heat flux to the respirometrically measured oxygen flux is often called the “calorimetric-respirometric ratio” or CR ratio. This can be used to partition total metabolic energy flux into its aerobic and anaerobic components by comparison of the experimental CR ratio with the theoretical “oxycaloric equivalent” for fully aerobic respiration. Such information is most important when evaluating the physiological effect of environmental stress.

Microcalorimetric instruments and methods are now essential tools for the general understanding of binding thermodynamics of biological macromolecules and specific biological systems. Provided that the enthalpy of binding is of appropriate magnitude, it is possible to determine affinities, KD, in the order of 10 nM (K=l08 M-1) for 1:1 complexes. One of the most important issues with ITC is the time needed to obtain a full binding isotherm. ITC inherently suffers from the relatively small number of binding experiments that are possible to perform per day. The focus has so far been on improving calorimetric response time of the instruments by various means, like assigning instrumental time constants and applying Tian’s equation to dynamically correct the raw calorimetric signal from a fast step-wise titration. Notably, nothing has so far been done to improve the experimental procedure. At the moment 2-3 h is needed to complete an ITC binding experiment. The number of data points that can be obtained, 20-30 points, limits the range of equilibrium constants that can be resolved from the data. For ITC this range is 1 ≤ KCM ≤ 1000, where K is the equilibrium constant and CM is the concentration of the reactant in the vessel. In this Application Note we show the possibility to shorten the time of an ITC experiment by slow continuous titration into the calorimetric vessel, cITC.

Proteomics research is uncovering a vast array of new proteins, including enzymes. It is now clear that the old assumption that one gene encodes one protein in incorrect. One gene can encode for several proteins, and many proteins have multiple functions: which function is displayed is controlled by the protein’s molecular environment. The structure and function of a protein are controlled by interactions with macromolecules such as other proteins, nucleic acids and lipids. Studying a protein in isolation (for example, obtaining the crystal or NMR structure of a purified protein) is critical to establishing a structural basis for the protein’s function(s). However, true functional characterization of the protein generally requires manipulation of multi-component systems under stringently-controlled conditions, and evaluating the roles of the physical characteristics of the protein. Proteomics has highlighted the need for a technique that can quantify the interactions between molecules under physiologically-relevant conditions.

When two proteins interact and bind, conformational changes in the proteins, and rearrangement of the solvent in the vicinity of the binding site, result in the absorption or generation of heat. Quantification of this reaction heat by ITC provides a complete thermodynamic description of the binding interaction, the stoichiometry of binding, and the association constant; in addition, if structural information is available, the contributions of specific amino acids mediating the binding event can be identified and their thermodynamic contributions quantified.

Continuous isothermal titration calorimetry (cITC) is ideal for studying rapid binding events typical of many biological recognition and binding reactions. The binding constant, stoichiometry and enthalpy of the reaction are accurately determined in significantly less time than that required using incremental isothermal titration calorimetry.

All enzymatic reactions generate heat, so all enzymatic reactions can in principle be directly studied by calorimetry. Calorimetric experiments do not require extensive protocol development, and measurements are direct, rapid, non-destructive and sensitive. In addition to kinetics data, ITC also generates thermodynamic information which can help correlate enzyme stability to reaction rates and substrate specificity.

Various techniques yield either structural or functional information about a protein, but DSC uniquely provides insights into both the three-dimensional arrangement and the functional properties of a protein’s structural components.

DSC is the most direct and sensitive approach for characterizing the thermodynamic parameters controlling noncovalent bond formation (and therefore stability) in proteins and other macromolecules. In an experiment requiring only a few micrograms of material, the protein is thermally unfolded, allowing the relationship between enthalpy and entropy of the denaturation process to be established in about one hour. Correlating thermodynamic properties to stability is necessary for the rational design of engineered proteins and protein therapeutics.

Although membrane proteins are very difficult to work with, ultra-sensitive calorimetry can over-come the limitations of many other physical characterization techniques to provide insights into the factors driving membrane protein/lipid assembly and controlling the stability of the conplexes.

Although membrane proteins are very difficult to work with, ultra-sensitive calorimetry can overcome the limitations of many other physical characterization techniques to provide insights into the factors driving membrane protein/lipid assembly and controlling the stability of the complexes.

A combination of HDX –MS and DSC is capable of revealing important structural details for biopharmaceuticals that cannot be obtained using other techniques HDX can map changes in binding regions and define structure of weak binding sites DSC characterization ensures highest quality structural data for differentiating weak binding molecular variants HDX-MS & DSC provide powerful, flexible analysis tools for DD, R&D and QC