This assay format was adapted from EMA’s Immunological guidelines for this purpose, given the lack of specific guidelines for the determination of enzyme activity. Enzyme-based drugs or drugs involved in enzymatic reactions are quantified by means of their biocatalytic activity in two different ways:
- Endpoint measurements: The enzyme reaction is stopped after a defined time period
- Real-time enzyme kinetics: The enzyme reaction is monitored per time unit
Both assay formats can use the signal of the produced product or substrate digestion for quantification. Calibration curves with the product or substrate or the enzyme velocity can be used for determination of the enzyme activity. The enzyme activity is divided into the specific activity (units/mg) and volume activity (units/ml). One unit is in general defined as 1 µmol substrate turn over per minute.