1 Purification of human antibodies by using liquid/liquid extraction J-W Lee and D Forciniti Chemical and Biological Engineering Department University of Missouri-Rolla Rolla MO 65409 USA Summary Aqueous two-phase extraction is a well-established technique with wide spread use in laboratory separations and some impact in industrial scale separations. We have investigated the use of aqueous two phase systems for the purification of a human antibody expressed in corn. The antibody was expressed fully non-glycosylated. We show that by using a poly(ethylene)glycol/phosphate or poly(ethylene)glycol/citrate systems in combination with an interface precipitation step it is possible to recover 80 percent of the antibody with a 10-fold pu- rity. The method is simple, very robust, and easy to scale up. Introduction Mixing two hydrophilic polymers or one hydrophilic polymer and a salt at well-defined concentrations forms two liquid phases at equilibrium (Forciniti, 1999b; Forciniti et al. 1991). These systems are called aqueous two-phase systems (ATPS). ATPS can be used for the pu- rification of biological products because proteins and other biological materials distribute un- equally between the two phases. Aqueous two-phase extraction is a well-established tech- nique with wide spread use in laboratory separations and some impact in industrial scale sepa- rations. For either industrial or recombinant proteins ATPS offer notable advantages (Winter et al. 1999; Lorch, 1999). For industrial enzymes, the method’s utility stems from the ease with which it can be adapted to continuous production and scaled up to meet industrial needs. For example, Genencor International (Winter et al. 1999) currently uses aqueous two-phase ex- traction coupled with ion exchange chromatography for the purification of chymosin. The most common system is made up by poly(ethylene)glycol (PEG) and dextran. However, PEG/salt systems are particularly attractive from an industrial view point because of their low cost. Common salts used include sodium or potassium phosphate, ammonium sulfate, sodium chlo- ride and sodium citrate. The selectivity of the technique can be improved by adding affinity ligands, by operating it in liquid chromatography mode (one phase is immobilized and the other is used as the eluent (Walsdorf, 1990; Huang and Forciniti, 2002)), or by a combination of both. Whether the technique is used in batch or in chromatography modes, all the know-how that exists for liquid/liquid extraction using organic solvents and for chromatography is available. The transgenic production of antibodies in plants needs to be followed by a robust sepa- ration process. Otherwise, the cost advantages associated with the production of the antibody in plants are lost. Because the use of plants for the production of human proteins is relatively knew, little is known about the downstream processing units that would present an economic advantage. Kusnadi et al. (Kusnadi et al. 1997) have recently pointed out that there is a lack of data in the downstream processing of proteins from transgenic plants. They also pointed out that most of the published work is at laboratory scale using techniques of questionable scaling up procedure. Our research helps to fill this gap. We have developed a new process by which human antibodies expressed in corn are isolated to high purity and yield using aqueous two-phase extraction. Different modes of op-