Alternative separation steps for monoclonal antibody purification: Combination of centrifugal partitioning chromatography and precipitation

•Using high throughput screening to develop a centrifugal partitioning chromatography process step.•Using high throughput screening to develop a precipitation and resolubilization step.•Combining aqueous two-phase extraction and precipitation to purify a monoclonal antibody.•Using centrifugal partitioning chromatography with citrate-PEG ATPS.•Integration into a traditional protein purification process.

Protein drugs continue to grow both in medicinal importance as in scale of their production. This furthers the interest in separation technologies that have the potential to replace chromatographic steps in a protein purification process. Two such unit operations that are employed in large scale in the chemical industry are extraction and precipitation. Their usefulness for the purification of proteins has been demonstrated, but the integration of such unit operations in a way that generate an output stream of high protein concentration and low process related impurities was missing. In this work, we employ centrifugal partitioning chromatography (‘CPC’) in combination with precipitation of the protein of interest to purify a cell culture supernatant of a monoclonal antibody producing cell line. Centrifugal partitioning chromatography was used as means of multi-step extraction using aqueous two-phase systems and was able to remove up to 88.2% of host cell protein (‘HCP’). The following PEG driven precipitation and resolubilization of the protein of interest was use to condition the CPC output stream to suit subsequent chromatographic steps, to increase mAb concentration, remove the phase forming polymer, further improve HCP clearance, and integrate a low pH hold step for viral clearance. The entire process reduced HCP content by 99.4% while recovering 93% of the protein of interest. High throughput screening techniques were extensively employed during the development of the process.