Column ageing of agarose-based chromatography in industrial lactoferrin purification.

Commercial lactoferrin is extracted from bovine milk using cation exchange chromatography with agarose-based Sepharose SP Big Beads. After over a thousand cycles, the column must be replaced due to backpressure exceeding operational limits. This increase in backpressure was not caused by fouling as previously thought but structural degradation of the agarose beads. Internal porosity increased from 0.78 to 0.86, indicating backbone depletion. The beads lose mechanical strength and compress during operation, which increases the backpressure and ultimately cause bed collapse. This structural damage was clearly visible in scanning electron microscopy. Compared to fresh resin, used beads showed a decrease in median particle size (175.20 ± 6.56 µm to 170.88 ± 0.16 µm) and pore size (peak location of log normal pore size distribution: 9.24 ± 0.091 nm to 8.43 ± 0.100 nm). While equilibrium binding capacity remained stable, ionic capacity declined by 23%, and dynamic binding capacity increased by 31%. This increase in dynamic binding capacity can be explained by the smaller particle size and higher intraparticle porosity, which both enhance mass transfer, while the slight reduction in pore size has a negligible effect. FTIR and Raman spectroscopy confirmed chemical changes in the agarose structure. No agarose-degrading enzymes were detected in milk, but exposing fresh beads to cleaning agents (NaOH, acetic acid, NaClO) replicated the chemical changes observed in used resin, demonstrating that the cleaning-in-place (CIP) treatment degrades the agarose backbone over time, leading to a loss in ligands, leaching of the backbone, and ultimately a mechanically weaker resin.