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
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Mentions: Fig. 5 shows the time trace of protein concentration and particle concentration in the extract and Raffinate of the SMB separation of 70 nm protein loaded nanoparticles and free bovine serum albumin. It can be seen that the system is stable after approximately 12 switches (or 3 complete cycles). We achieved a good separation of protein and particle as the extract fraction is almost free of particles; however the Raffinate is getting contaminated with BSA multimers after 9 switches (or roughly 2 cycles). This incomplete separation can be explained by the setup of the system which was to separate BSA monomers and particles. We assumed the separation of a binary mixture when setting up the parameters according to the triangle theory, but in fact the BSA multimers add an additional third species to the system, which is not covered by the theory we used. The BSA multimer peak is between these two species and is therefore found in both fractions, extract and Raffinate. Table 3 shows the corresponding composition of feed, extract and Raffinate based on UV adsorption at 280 nm as well as the recoveries for particles and proteins. The extract is sufficiently pure of particles, but because of BSA-multimer problem the Raffinate purity is insufficient.
Affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), Muthgasse 18, 1190 Vienna, Austria.