(802f) Separation of Monoclonal Antibodies From Chinese Hamster Ovary Cell Supernatant With Aqueous Two-Phase System

The pharmaceutical and biotechnological industries are investing substantial resources in the development of antibody-based therapeutic products, especially for monoclonal antibodies (mAbs).  Currently, the antibody expression of mammalian cells with titer of 5 g/L becomes common and the bottleneck of antibody production has shifted from antibody expression to downstream processing. Protein A affinity chromatography has been the important platform for the purification of antibodies in large-scale production, but it still has some limitations. Some alternative techniques have been developed to improve the downstream processes, including the extraction with aqueous two-phase system (ATPS).

In the present work, the ATPS with polyethylene glycol (PEG) and hydroxypropyl starch (HPS) was used to separate mAbs from Chinese hamster ovary (CHO) cell supernatant. Firstly, the phase diagrams of PEG/HPS ATPS were investigated and the effects of NaCl addition on the phase diagrams were determined. The results indicated that the addition of NaCl could lead to a shift on the binodal curve and the phase separation would occur at higher PEG and HPS concentrations. The effects of pH, NaCl addition and the load of CHO cell supernatant on the partitioning of mAbs were investigated. It was found that with 6% CHO cell supernatant and 15% NaCl addition at pH 6.0 the yield of mAbs in the top phase was 97.6% with high purity of 96.0%. The back extractions of mAbs into a phosphate-rich bottom phase with the PEG/phosphate ATPS were also investigated. After two-step extraction with ATPSs the purity of mAb could reach 97.6%±0.5 with the yield of 86.8±1.0%, which was comparable to the separation with Protein A chromatography. The results indicated that the extraction with ATPSs might be a promising alternative for the purification of mAbs from the cells supernatant.

* This work was supported by National Natural Science Foundation of China and Zhejiang Provincial Natural Science Foundation of China.