Development and application of centrifugal precipitation chromatography has been continued. The system internally generates a concentration gradient of ammonium sulfate (AS) along a long channel to fractionate proteins according to their solubility in AS solution. The separation column consists of a pair of disks with mutually mirror-imaged spiral channels that are separated by a semipermeable membrane. The disk assembly is mounted on a sealless continuous flow centrifuge. Concentrated As solution is introduced into the upper channel while a water solution is passed through the lower channel in the opposite direction in a rotating column. A mixture of proteins injected into the water channel moves along an AS gradient of increasing concentration that has been established in the water solution. Each protein species precipitates at a different AS concentration along the gradient. The eluate is continuously monitored and collected using a fraction collector. A series of basic experiments was performed to study the rates of AS transfer and osmosis through the membrane, and the operational parameters including elution time,revolution speed, inclination of the gradient and sample size were optimized using stable protein samples. A new set of columns with different channel configuration was made to improve the efficiency of separation. Preliminary applications were successful in purification of monoclonal antibodies from cell culture supernatant and ascitic fluid, and affinity separation of recombinant ketosteroid isomerase from a crude E. coli lysate. The method was also successfully applied to fractionation of other biopolymers such as hyaluronic acid, chondroitin sulfate, polymerized catechins, and dextran using a gradient between suitable organic solvents and water.
