Control and Stabilization of a Free-Flow Counterflow Gradient Electrophoresis Device for Preparative Protein Purification through Coupled Multiphysics Transport

Abstract

Biomolecule purification remains a critical yet resource-demanding component of downstream processing in pharmaceutical and biotechnological applications. Presently, chromatography serves as the gold standard; however, it is constrained by its batch operation, high cost, limited scalability and throughput. Continuous separation techniques, such as Free-Flow Electrophoresis (FFE), offer a promising alternative by enabling gentle, high-throughput separations based on electrophoretic mobility. Despite these advantages, the widespread adoption of FFE has been hindered by challenges related to analyte dispersion, Joule heating, electrochemical instability, and the difficulty of maintaining control over strongly coupled transport variables. Hybrid FFE modalities have been explored to mitigate these limitations. Among them, free-flow Isoelectric Focusing (FF-IEF) has achieved notable success due to its ability to self-focus analytes at their isoelectric points along an imposed pH gradient. However, the pH gradient establishment requires the use of expensive carrier ampholytes – costly reagents that can interact with target biomolecules – introducing complications for downstream processing and limiting practical scalability. This thesis presents the development and stabilization of a preparative-scale Free-Flow Counterflow Gradient Focusing (FF-CGF) system for charge-based biomolecule separation. FF CGF leverages the balance between hydrodynamic counterflow and electrophoretic migration to achieve continuous axial focusing without the need for carrier ampholytes, thereby preserving sample integrity and ensuring compatibility with downstream processes. A theoretical framework is established to describe FFE as a coupled multiphysics system governed by interacting electrical, hydrodynamic, thermal, and chemical fields, providing a unified interpretation of existing FFE modalities as control strategies within a shared parameter space. Guided by this framework, a modular microfluidic platform is designed that integrates optimized flow control, electric field delivery, and ion-exchange membrane interfaces to maximize single analyte focusing and improve voltage efficiency. The system is experimentally validated through the separation of fluorescently labeled monoclonal antibodies, demonstrating stable operation and preparative capability. Overall, this work advances FF-CGF as a viable alternative to chromatographic methods in downstream processing, and establishes a foundation for scalable, continuous biomolecule purification technologies.