Active concentration measurements of recombinant biomolecules using biosensor technology

Whereas the concentration of a biomolecule simply refers to the amount of chemical substance per unit of volume, its active concentration refers to a relational parameter that has meaning only with respect to the molecule's ability to interact specifically with one particular ligand. When proteins are studied in a biological context, it is the biologically active concentration that is relevant, and not the total concentration of correctly and incorrectly folded molecules. Using a biosensor instrument the concentration of active biomolecules in a preparation can be measured by injecting the preparation at different flow rates onto a sensor chip surface presenting a high concentration of a specific ligand. The method can be used under conditions of partial mass transport limitation and does not require a pre-established standard curve. When the method was used to measure the active concentration of several recombinant proteins it was found that the active concentration was much lower than the nominal concentration determined by conventional methods. The active concentration also depended on the ligand used in the binding assay, reflecting the fact that active concentration can only be defined with respect to one specific probe. Such discrepancies in concentration values, if undetected, may lead to erroneous conclusions regarding the properties and behaviour of recombinant proteins tested in different assays.