Hindered diffusion of proteins in mixture adsorption on porous anion exchangers and impact on flow-through purification of large proteins

• Published in: Journal of Chromatography A 2019-01, Vol.1585, p.121-130
• Main Authors: Matlschweiger, Alexander, Fuks, Preston, Carta, Giorgio, Hahn, Rainer
• Format: Article
• Language: English
• Subjects: Confocal laser scanning microscopy; Flow-through purification; Hindered diffusion; Secretory immunoglobulins
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2018.11.060

•Large proteins bind in a layer close to particle surface when pore size is not significantly larger than protein size.•Binding stops after a few μm distance into the particle in batch adsorption.•Bound protein layer significantly hinders diffusion of smaller proteins in mixture.•Flow-through purification of large proteins is possible in small-pore resins with negligible binding of the larger protein. Complex adsorption kinetics behaviors of proteins in mixtures hampers chromatographic process development and complicates model-based prediction of separation. We investigated the adsorption characteristics of mixtures comprised of a larger protein (secretory immunoglobulins or thyroglobulin) and a smaller protein (serum albumin or green fluorescence protein) on the small-pore anion exchanger Q Sepharose FF. Confocal laser scanning microscopy measurements revealed that binding of the large protein was extremely slow and eventually stopped completely after the adsorption front penetrated just a few μm into the particle. Binding capacities after 24 h of incubation were nevertheless around 35 mg/mL of particle which is relatively high when considering that only a fraction of the particle was saturated, suggesting that locally-high bound protein concentrations are attained in a layer close to the particle surface. During mixture adsorption, the bound protein layer also significantly hindered diffusion of the smaller proteins into the particles resulting in about three times slower adsorption kinetics compared to single component adsorption. The combined effects of restricted diffusion and protein binding explain why flow-through purification of these mixtures with the small-pore resin Q Sepharose FF is effective under practical conditions. In this resin, diffusion of secretory immunoglobulins (or thyroglobulin) is restricted in the small pores so that despite their intrinsically greater affinity for the resin, much less binds compared to small proteins. Using the large-pore resin POROS 50 HQ results in faster transport, but also in more binding of secretory immunoglobulins (or thyroglobulin) compared to smaller protein impurities, preventing effective flow-through purification.