Optimized Design And Applications Of Chromatographic Cake In Bioengineering

The thesis includes three parts.1. According to the method for evaluating the resolution of small solutes, the ;haracters of biopolymer separation hydrophobic interaction chromatographic media racked in chromatographic cake was tested and was found the role for the former Iocs not follow to the latter. Based on the short column theory of biopolymer leparation, the original liquid distributor which is simply called as distributor was mproved and tested with various methods. When the composition of mobile phase vas selected as the obtained capacity factor k of BSA about 10, the totally adsorbed imount of BSA by the chromatographic cake with size of 10mm in thickness and 10mm in diameter companying with improved distributor was 4.43mg. or 33.8% more nan from the original one. In addition, two new methods for packing the ;hromatographic media in the chromatographic cake were also suggested, and with me of them, the totally adsorbed amount of BSA was found to raise 8.36mg. or 57.5% more than the original method.2.A method for renaturation with simultaneous purification of rhG-CSF by weak mion exchange chromatography (WAX) is presented in this study. The rhG-CSF as nclusion bodies which was expressed by E.coli was dissolved and reduced by high concentration of urea and ?-mercaptoethanol ( ?-ME), was directly injected into a WAX cake and renatured with simultaneously purified in the presence of a suitable concentration of urea and reduced glutathione (GSH) and oxidized glutathione (GSSG). The chromatographic conditions were also optimized. The obtained 昲G-CSF with specific activity of 1.9108U/mg and purity of above 95% was accomplished in 30min by only one step. The method can normally work when arecipitates form during sample injection. The precipitates which deposit on the frit can be redissolved periodically and refolded again by the WAX chromatographic cake, and the mass and bioactivity recoveries of rhG-CSF can be improved greatly.3. A method for renaturation with simultaneous purification of rhIFN- Y by WCX chromatographic cake is presented in this study. The rhIFN- y as inclusion bodies which was expressed by E.coli was dissolved and reduced by high concentration of urea, the chromatographic conditions were also optimized. The obtained rhIFN-Y with specific activity of 4.3 X 107IU/mg and purity of above 95% was accomplished in 30min also by one step. Compared with the method reported by reference, this method is fast and operation is simple.