Da Yu Protein Sciences – Functional Characterization
The utility of proteins is based on their function. Da Yu Protein Sciences has the capability of performing several types of non-cell-based in vitro functional assays using multi-titer plates.
Enzyme-Linked Immunosorbent Assays (ELISA) - ELISA are immunological methods that have wide application in the CMC development of Biologics. Many Biologics elicit their biological effects by binding to other proteins and ELISA can characterize these interactions. For monoclonal antibodies, regulatory expectations include characterization of the Biologic’s immunological properties. The characterization should include, at a minimum, the assessment of the Biologics interaction with its antigen and the determination of its binding affinity. Some monoclonal antibodies may also require characterization of Fc receptor binding. Non-antibody therapeutic proteins may bind to other proteins, including cell-surface receptors, which can also be characterized by ELISA. For process characterization, ELISA also have application where the high sensitivity of the method is often utilized to measure the clearance of process-related impurities, such as upstream cell culture medium ingredients or host cell proteins, as the process is being developed. Process improvements can be monitored by applying ELISA to different lots during CMC development and thereby guide process changes leading to manufacturing optimization.
Michaelis-Menten Enzyme Analysis - Two numerical parameters are generally determined to characterize enzyme activity at steady state:
- Km is the substrate concentration at which half-maximal velocity occurs for a defined set of assay conditions.
- kcat is the enzyme turnover number and it measures the number of product molecules produced per molecule of enzyme in a unit of time, generally 1 second. It is calculated by dividing the maximal velocity (Vmax) by the enzyme concentration.
Km and kcat are determined by acquiring steady-state enzyme velocity (v) data at multiple different substrate concentrations and then fitting the data to the Michaelis-Menten equation:
v = (Vmax x S)/(Km + S)
Ligand-Binding Assays - We offer a number of different formats for performing ligand-binding assays to determine binding constants including the following:
- Fluorescence Anisotropy – In fluorescence anisotropy a sample is first excited with polarized light. The resulting emitted light then passes through a polarizer that measures its intensity (I). When the orientation of the emission polarizer is parallel to that of the polarized excitation light the intensity of the emitted light is referred to as I‖, while it is called Iꓕ when the emission polarizer is perpendicular to the polarized excitation light. The fluorescence anisotropy (r) is then calculated as:
r = (I‖ - Iꓕ)/(I‖ + 2Iꓕ)
- Fluorescence anisotropy is dependent on a molecule’s mobility. Large molecules, such as proteins, have increased fluorescence anisotropy compared to small ligands. Thus, when a ligand binds to a protein it becomes part of a larger, less mobile molecular complex and the ligand’s fluorescence anisotropy increases. When the ligand contains a fluorophore it then becomes possible to measure the binding constant using fluorescence anisotropy.
- Biophysical techniques can measure ligand binding constants in several ways. The binding of a ligand to a protein can alter the intrinsic signal of the protein and those changes can be monitored directly to determine a binding constant. Ligand binding to a protein also leads to increased protein stability. This increased stability can be measured either by an increase in the protein’s melting temperature (Tm) or by changes in denaturant-induced unfolding. Either condition can be used to measure the binding constant. At Da Yu Protein Sciences we have a number of biophysical techniques that can be employed to determine binding constants including Differential Scanning Fluorimetry, Circular Dichroism and UV Derivative Spectroscopy.
Clients desiring more information about any of our functional characterization capabilities are invited to Contact Us.