期刊
JOURNAL OF CHROMATOGRAPHY A
卷 1585, 期 -, 页码 121-130出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2018.11.060
关键词
Hindered diffusion; Flow-through purification; Confocal laser scanning microscopy; Secretory immunoglobulins
资金
- Austrian Research Promotion Agency (FFG) [836504]
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 p,m into the particle. Binding capacities after 24h 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 HQresults in faster transport, but also in more binding of secretory immunoglobulins (or thyroglobulin) compared to smaller protein impurities, preventing effective flow-through purification. (C) 2018 Elsevier B.V. All rights reserved.
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