Highly Carboxylated Cellulose Microspheres via Alginate Pre-Grafting for High-Resolution and High-Capacity Weak Cation Exchange Chromatography

Abstract Weak cation-exchange (WCX) chromatography is a core technique for protein purification, and developing WCX packings with both high capacity and high resolution is of great significance for modern biopharmaceuticals and protein research. Crosslinked cellulose microspheres (CCMs, 10−25 μm) were verified in our previous work as a promising matrix for high-performance packings. Herein, highly carboxylated CCMs (CA-SACMs) for high-capacity and high-resolution WCX packings were fabricated via alginate pre-grafting followed by deep carboxylation. This alginate pre-grafting strategy achieves a “one-arrow-three-birds” effect: increasing the carboxyl content, augmenting the number of hydroxyl groups for further carboxylation, and providing a flexible long-chain layer that facilitates protein adsorption. The CA-SACMs exhibited a dynamic binding capacity (DBC) of 115 mg/mL for lysozyme (Lyz), nearly double that of chloroacetate-only modified CCMs (CACMs, 67 mg/mL). Meanwhile, both CA-SACMs and CACMs enabled high-resolution separation of four standard proteins, and CA-SACMs exhibited stronger retention. Notably, CA-SACMs exhibited excellent capture ability and stability, and were successfully used for Lyz purification from egg white and acidic variant removal from monoclonal antibodies, confirming their practical application potential. This work presents high-performance cellulose-based WCX packings derived from natural materials via a full-aqueous synthesis route, holding broad application prospects in protein purification.