Purification of proteins and nanoparticles by continuous field-flow fractionation

Biotechnology and nanotechnology have enabled the production of biomolecules and nanoparticles but their purification remains a challenge. A narrow size distribution is often required for the proper function of the final product. For instance, therapeutic proteins need to be separated from their aggregates which is typically achieved by using chromatography. However, the chromatographic supports increase substantially the production costs which has led to a quest for alternative purification methods. Asymmetrical flow field-flow fractionation (AF4) is a technique that fractionates nano-sized solutes based on their hydrodynamic size under very mild conditions since the separation takes place inside an open channel. It is mainly used as an analytical technique; the loading capacity is limited because the sample is compressed against a membrane during separation. However, channels with larger dimensions are occasionally used for preparative purposes. The present thesis explores alternative ways to increase throughput in AF4. Using different protein standards, it is revealed that the overloading effect (i.e., peak broadening and shift in retention time) is caused by the sample-dependent viscosity increase close to the membrane where the sample concentration is very high. In addition, in order to increase throughput, a novel continuous two-dimensional asymmetrical flow field-flow fractionation (2D-AF4) is introduced using microstructured membranes with slanted grooves on their surface. The slanted grooves are causing a lateral displacement of the solutes and the deflection angle depends on the hydrodynamic size. The system can be considered as two-dimensional as the continuous separation occurs because of the different selectivity along and across the grooves.