A Pulsed Tangential-Flow Ultrafiltration Technique for Studying Protein-Drug Binding

We describe a pulsed tangential-flow ultrafiltration technique for rapid analysis of protein-drug binding. A protein-drug pulse was injected into a tangential-flow membrane device and made to flow parallel to the surface of a protein-retaining ultrafiltration membrane. The protein and protein-drug complexes were flushed out of the device in the retentate stream, whereas the free drug present in the permeate stream was quantified using on-line UV detector. The height of the permeate drug peak and its area under the curve were both found to be proportional to the free drug concentration in the injected sample. The fraction of bound drug was determined by comparison with peak obtained with protein-free drug sample. The characteristics of the permeate drug peak such as residence time, peak width, and peak height depended on both feed and permeate flow rates. The proposed technique in addition to being fast was "self-priming" in nature because the injected samples were flushed out of the module along with the retentate and permeate. This feature makes this technique particularly suitable for automated sample analysis. The technique was validated using three-model protein-drug combinations: bovine serum albumin (BSA)-antipyrine, BSA-tryptophan, and BSA-aspirin.