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Downstream Antibody Purification using Aqueous Two-Phase Extraction The extraction of antibodies using a polyethyleneglycol(PEG)-citrate aqueous two-phase system(ATPS) was in vestigated.Studies using purified monoclonal antibody(mAb) identified operating ranges for successful phase formation and factors that significantly affected antibody partitioning. The separation of antibody and host cel lprotein(HCP) from clarified cell culture media was examined using statistical design of experiments(DOE). The partitioning of antibody was nearly complete over the entire range of the operating space examined.A model of the HCP partitioning was generated in which both NaCl and citrate concentrations were identified assignificant factors.To achieve the highest purity,the partitioning of HCP from cell culture fluid into the product containing phase was minimized using a Steepest Descent algorithm.An optimal ATPS consisting of 14.0%(w/w) PEG,8.4%(w/w)citrate,and7.2%(w/w)NaCl at pH7.2 resulted in a product yield of 89%,anapproximate 7.6- fold reduction in HCP levels relative to the clarified cell culture fluid before extraction and an overall purity of 70%.A system consisting of 15%(w/w)PEG,8%(w/w)citrate,and15% (w/w) NaCl at pH5.5 reduced product-related impurities(aggregates and low molecular product fragments)from 40% to less than0.5%while achieving 95% product recovery.At the experimental conditions that were optimized in the batch mode,a scale-up model for the use of counter-current extraction technology was developed to identify potential improvements in purity and recovery tha tcould be realized in the continuous operational mode |
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