(341f) Purification of Human Antibodies by Using Liquid/Liquid Extraction

To our knowledge, two approaches have been followed for the purification of glycoproteins: methods based on the affinity and specificity of lectins for particular carbohydrates bounded to the protein and methods based on the differences in charge of the different proteins as a result of slight differences in the carbohydrate composition (pellicular ion exchange chromatography). Aqueous two-phase extraction is a well-established technique with wide spread use in laboratory separations and some impact in industrial scale separations. For either industrial or recombinant proteins the method offers notable advantages. The principles of aqueous two-phase extraction are quite simple. Mixing two hydrophilic polymers or one hydrophilic polymer and a salt at well-defined concentrations forms two equilibrated liquid phases. Proteins and other biological materials distribute unequally between the two phases. We have investigated the use of aqueous two phase systems for the purification of a human antibody from a cell extract. The antibody was expressed either fully glycosylated or de-glycosylated. We show that by using a poly(ethylene)glycol/phosphate system in combination with an interface precipitation step it is possible to recover 80 percent of the antibody with a 10-fold purity. The method is simple, very robust, and easy to scale up. We showed that a 100-fold increase in scale does not recovery or purity.