Continuous separation of protein loaded nanoparticles by simulated moving bed chromatography.
Bottom Line: In the case of beta casein where no multimers are present we achieved 89% purity and 90% recovery of loaded nanoparticles in the Raffinate and an extract free of particles (92% purity).Using a tangential flow filtration unit with 5kDa cutoff membrane we proved that the extract can be concentrated for recycling of protein and buffer.The calculated space-time-yield for loaded nanoparticles was 0.25g of loaded nanoparticles per hour and liter of used resin.
Affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), Muthgasse 18, 1190 Vienna, Austria.Show MeSH
Related in: MedlinePlus
Mentions: To select model nanoparticles, two important factors have to be considered. The particle has to bind enough protein to be detectable by our analytical methods, and the size should be as close as possible to the size of a virus (roughly 70–150 nm). Additionally, the process parameters to ensure completely covered nanoparticles have to be found. The saturation range of protein loaded nanoparticles can be found by adsorption isotherms. To find optimal conditions for perfectly loaded nanoparticles for different sized nanoparticles, silica nanoparticles with amide functionalization in the size of 30 nm, 70 nm, 200 nm and 1000 nm were studied together with two model proteins: BSA and beta-casein (Fig. 3). The surface modification of these particles allows proteins to not only adsorb to the negatively charged silica surface, but provide additional positively charged binding opportunities. For both proteins the adsorbed amount of protein decreased with increasing particle size because of changed surface to volume ratio. This data show that our intended model of 70 nm particles adsorbs enough protein (8.14 ng/mm2 for BSA and 3.11 ng/mm2 of Beta Casein) to be detectable for our analytical system and is therefore a suitable model system.
Affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), Muthgasse 18, 1190 Vienna, Austria.