Ion exchange resins for bioprocessing are designed to balance a series of properties, which will define their performance. These include particle size, which affects pressure drop and plate number, intra-particle porosity that directly impacts surface area/capacity and the accessibility of binding sites, and ligand density, which is critical to selectivity.<br/>Here, we investigated particle size and ligand density aspects of agarose based anion exchange resins to understand interactions between dynamic binding capacity, protein uptake rate and robustness to fouling. An array of 9 bead types of 45, 65 and 90 µm in diameter and 0.09, 0.17 and 0.24 mmol/mL ionic capacity were generated and studied using bovine serum albumin and immunoglobulin G in chromatographic separations and confocal laser scanning microscopy. Resin with low ligand density (0.09 mmol/mL) provided high binding capacity and a fast protein uptake rate, improved mass transfer and resin lifetime. In contrast, the high ligand density resin (0.24 mmol/mL) suffered from impaired mass transfer effects due to potential steric hindrance and/or electrostatic repulsion as investigated by confocal microscopy. Moreover, this resin lost 20-30 % of its binding capacity after 5 cycles.<br/>A novel technique using a Proteostat dye to investigate the extent of fouling was developed. Fresh and used resin samples were stained with the fluorescent dye and imaged under confocal microscopy to determine the extent of protein aggregation. The results confirmed increased protein aggregation levels for high ionic capacity resin compared to the low ionic capacity resin. The study demonstrates that selecting the appropriate combination of ligand density and particle size is crucial for optimal resin design and performance.

Particle size and ligand density effects on anion exchange chromatography behaviour for therapeutic protein manufacture

255th National Meeting (2018)

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