Design and Characterization of a Microfluidic Packed Bed System for Protein Breakthrough and Dynamic Binding Capacity Determination

摘要

A 1.5 mu L ion exchange chromatography column to accommodate resins used for biophar-maceutical processing has been designed to produce breakthrough curves and to quantify dynamic and maximum protein binding capacities.Channels within a glass chip were fabricated using photolithography and isotropic etching.The design includes a 1 cm long microfluidic column in which compressible,polydispersed porous agarose beads(70 pm mean diameter)were packed using a keystone method where particles aggregate in a narrow channel.The depth of the column is such that two bead layers exist.The fabrication technique used forms Cartesian geometries as opposed to circular cross sections found in standard columns.The voidage was therefore higher than standard values when measured by 3D confocal microscopy.In conjunction with microscopic techniques,the column allows visualization of events within the bed such as adsorption profiles that would otherwise be difficult to observe.In this work,the binding of fluorescently labeled protein during isocratic loading was used to generate breakthrough from the microcolumn.Useful breakthrough curves were achieved using mobile phase velocities from 60 to 270 cm h~(-1).Calculated dynamic binding capacities were compared well with previously published data on conventional scale columns.The microfluidic chromatography column described here thus allows study of process scale chromatography behavior at scales 20,000 times smaller than in current practice.The work described in this article is representative of the proof of principle of a potentially powerful tool for the generation of microfluidic process bed data for the biopharmaceutical industry.