Inês F. Pinto

High-Throughput Nanoliter-Scale Analysis and Optimization of Multimodal Chromatography for the Capture of Monoclonal Antibodies

Multimodal ligands are synthetic molecules comprising multiple types of interactions that have been increasingly used for the capture of different biopharmaceutical compounds within complex biological mixtures. For monoclonal antibodies (mAbs) in particular, these ligands have shown the possibility of direct capture from cell culture supernatants in native conditions, as well as enhanced selectivity and affinity compared to traditional single-mode ligands. However, performing the capture of a target mAb using multimodal chromatography comes with the need for extensive optimization of the operating conditions, due to the multitude of interactions that can be promoted in parallel. In this work, a high-throughput microfluidic platform was developed for the optimization of chromatographic conditions regarding the capture of an anti-interleukin 8 mAb, using a multimodal ligand (2-benzamido-4-mercaptobutanoic acid), under a wide range of buffer pH and conductivities. The interaction of the ligand with the fluorescently labeled target mAb was also analyzed with respect to the individual contribution of the hydrophobic (phenyl) and electrostatic (carboxyl) moieties using fluorescence microscopy. The results were further validated at the macroscale using prepacked columns in standard chromatography assays, and recovery yield values of 94.6% ± 5.2% and 97.7% ± 1.5% were obtained under optimal conditions for the miniaturized and conventional approaches, respectively. In summary, this study highlights that a microfluidic-based approach is a powerful analytical tool to expedite the optimization process while using reduced reagent volumes (<50 μL), less resin (∼70 nL), and delivering results in less than 1 min per assay condition.

The application of microbeads to microfluidic systems for enhanced detection and purification of biomolecules

This paper describes microbead-based microfluidic systems. Several aspects of bead assays in microfluidics make them advantageous for bioassays in simple microchannels, including enhanced surface-to-volume ratio, improved molecular recognition reaction efficiency, and the wide range of surface functionalization available with commercial microbeads. Two-level SU-8 molds are used to fabricate PDMS microchannels that can hydrodynamically trap different types of microbeads, with characteristic dimensions of tens of microns. The use of these microbead-based microfluidic systems in the biosensing of antibodies, toxins and nucleic acids, as well as in antibody purification will be presented and discussed in this paper.