Design and characterization of a microfluidic packed bed system for protein breakthrough and dynamic binding capacity determination

Abstract

A 1.5 microL ion exchange chromatography column to accommodate resins used for biopharmaceutical 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 mum 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.

Growth and productivity impacts of periplasmic nuclease expression in an Escherichia coli Fab' fragment production strain

“…Basic research insights now frequently inform host cell modifications for boosting titre (Tan et al, 2008) and efficiency of downstream processing (DSP) steps such as capture (Clemmitt and Chase, 2000; Nesbeth et al, 2006) and purification (Humphreys et al, 2004). Combining these cell‐engineering approaches with ultra scale down approaches (Chan et al, 2006; Shapiro et al, 2009) and modeling techniques (Chhatre et al, 2008; Edwards‐Parton et al, 2008), provides a powerful means of optimizing and integrating unit operations to improve whole bioprocesses.…”

Flocculation on a chip: a novel screening approach to determine floc growth rates and select flocculating agents

“…[33][34][35][36] These systems have been used extensively to study upstream bioprocessing steps, [37][38][39][40][41] but the number of reports of microfluidic devices developed to study process optimisation of downstream processing steps, such as purification and separation of biomolecules, is still limited. 27,[42][43][44][45][46][47][48] In downstream bioprocessing, flocculation has attracted renewed interest but suitable analytical tools to comprehensively understand the flocculation mechanism have been absent. The precise control over the microenvironment afforded by microfluidic devices opened up an opportunity to investigate, for the first time, the formation and growth of flocs independently from floc breakage and ageing phases.…”

A review of advanced small‐scale parallel bioreactor technology for accelerated process development: Current state and future need

“…The longer term (10–15 years) will likely bring further throughput improvements from the introduction of microfluidic approaches into the automated development workflow. Early work has demonstrated feasibility of microfluidic approaches to ion exchange chromatography at the 1.5 μL scale 74. It is also anticipated that standard LC column methods will be replaced by microfluidic chip analysis, such as recently demonstrated for DNA and RNA analysis 75, 76…”