(802b) Multi-Stage Aqueous Two-Phase Extraction of a Monoclonal Antibody in a Mixer-Settler Battery

Monoclonal Antibodies (mAbs) are biologically active proteins produced by plasma cells in response to the presence of foreign substances. During the past years, the application of mAbs for therapeutic treatment has been increasing. Several diseases like cancer, asthma or autoimmune diseases are already treated by mAbs. Due to the fact, that mAb-based pharmaceuticals are mainly administered for chronic diseases at high doses, a large demand of these pharmaceuticals has to be satisfied. Through an intensified research on the field of upstream-processing, the mAb concentration in the fermentation broth has significantly been increased. Still purification of mAbs is a challenging step in the manufacturing process accounting for a major part of the total production costs. Since conventional unit operations like preparative chromatography are often limited by their capacity, buffer consumption and ligand cost, growing interest exists in finding alternative downstream-processing methods.

Aqueous two-phase systems (ATPS) are a promising option for the improvement of mAb purification. By mixing two different phase forming components (PFCs), e.g. two hydrophilic polymers or a hydrophilic polymer and a salt in water at high concentration, two liquid phases can form. One of the phases is rich in PFC 1, the other is rich in PFC 2. Both phases consist of 70-80 % of water and thus have an aqueous character. Purification of the target product is achieved because of a different partitioning of target component and impurities between both phases. Especially for sensitive products ATPS are a promising alternative as they offer mild separation conditions at high, continuous capacity.

In this work, an ATPS consisting of polyethylene glycol, phosphate salt and water is chosen for the purification of mAbs from a Chinese Hamster Ovary cell supernatant (CHO-CS). Essential for the design of an aqueous two-phase extraction (ATPE) is a fundamental knowledge of substance properties, liquid-liquid equilibrium data as well as partitioning of target component and impurities. Several influences on the purification of mAbs like polyethylene glycol molecular weight, pH, initial concentration of CHO-CS and concentration of additional sodium chloride were analyzed in single-stage extraction experiments prior to this work. To further enhance the efficiency of mAb extraction, ATPE is performed in multi-stage experiments in a miniplant mixer-settler battery. The feasibility of multistage ATPE at miniplant scale will be discussed with regard to operational challenges and different operation modes. Those include the position and phase of CS inlet. Furthermore the extraction of mAb from CHO-CS will be evaluated, with regard to purity and recovery of mAb. The influences of stage number, throughput and operation mode will be presented.