Advances in Free-Flow Isoelectric Focusing Electrophoresis Purification Technology and Its Microchip Implementation: With a Discussion on Its Application in Recombinant Collagen Production

Abstract Free-Flow Isoelectric Focusing Electrophoresis (FF-IEF) is a continuous liquid-phase purification technique based on the isoelectric point (pI) of proteins as the core separation criterion. It has core advantages, such as no need for solid-phase carriers, high sample recovery rate, continuous processing capability, and excellent retention of biological activity. The micro-chip FF-IEF (μFF-IEF) further enhances the heat dissipation efficiency, separation resolution, and online detection capabilities, making it an important tool in proteomics analysis, downstream purification of biopharmaceuticals, and biomanufacturing. For recombinant collagen the amino-acid sequence is fully defined, so a baseline pI can be predicted from the sequence and residue dissociation constants and, within limits, shifted by charged-residue engineering; the experimentally measured pI, however, also depends on host-specific post-translational modifications (e.g., hydroxylation and glycosylation) and on terminal processing or degradation, and must therefore be verified experimentally. This combination of a designable pI and a continuous, carrier-free separation mechanism makes recombinant collagen an attractive, although still largely prospective, application scenario for FF-IEF. This article systematically reviews the separation principle and key microchip technologies of FF-IEF, deeply elaborates on the predictive and designability of the pI of recombinant collagen proteins for the underlying empowerment mechanism of FF-IEF technology, and finally looks forward to the core research priorities for the future industrialization transformation of FF-IEF, aiming to provide theoretical references and technical ideas for the development of efficient continuous purification technologies for recombinant collagen proteins and the upgrading of recombinant collagen protein biomanufacturing processes.