Continuous matrix assisted refolding of alpha-lactalbumin by ion exchange chromatography with recycling of aggregates combined with ultradiafiltration.
Continuous matrix assisted refolding (MAR) can be achieved on a solid support by using a continuous chromatographic system. Recycling the aggregate fraction, simultaneously formed during a refolding reaction, can further increase the refolding yield. Due to the nature of this reaction, aggregates are the main reason for a refolding yield below stoichiometric conversion. A preparative continuous annular chromatographic system (P-CAC) equipped with an ion exchange resin was used to continuously refold the model protein alpha-lactalbumin. For this purpose, this protein was denatured, reduced and adsorbed on the ion exchange resin. Elution was performed with or without redox reagents in the buffer system permitting fast formation of the native disulfide bonds. In the case redox reagents were present, the protein refolds then during its residence time on the matrix. However, aggregate formation is also increased and refolding yields are lower. Tightly bound aggregates were removed from the column by 2M guanidinium hydrochloride. In order to increase the system yield, this aggregate fraction was recycled after lowering the conductivity by ultradiafiltration and adjustment of the protein concentration by dilution. For on-column refolding, recycling of aggregates at a recycling rate of 0.17 increased the system yield from 25% to 30%. An algorithm was developed to show interdependencies of the single influencing parameters. The operability of the system was demonstrated but limitations due to instability of the P-CAC, especially inhomogeneous flow and peak wobbling, have to be considered.
Machold
Christine
C
Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Schlegl
Robert
R
Buchinger
Wolfgang
W
Jungbauer
Alois
A
D000465:Algorithms; D002852:Chromatography, Ion Exchange / Q000379:methods*; D007768:Lactalbumin / Q000737:chemistry*; D014462:Ultrafiltration / Q000379:methods*
D007768:Lactalbumin