Two step capture and purification of IgG₂ using multicolumn countercurrent solvent gradient purification (MCSGP)

Abstract

A two-step chromatography process for monoclonal antibody (mAb) purification from clarified cell culture supernatant (cCCS) was developed using cation exchange Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) as a capture step. After an initial characterization of the cell culture supernatant the capture step was designed from a batch gradient elution chromatogram. A variety of chromatographic materials was screened for polishing of the MCSGP-captured material in batch mode. Using multi-modal anion exchange in bind-elute mode, mAb was produced consistently within the purity specification. The benchmark was a state-of-the-art 3-step chromatographic process based on protein A, anion and cation exchange stationary phases. The performance of the developed 2-step process was compared to this process in terms of purity, yield, productivity and buffer consumption. Finally, the potential of the MCSGP process was investigated by comparing its performance to that of a classical batch process that used the same stationary phase.

Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography

“…This integrated system provided 5× higher volumetric productivity than a fed batch process with comparable yield and purity and smaller column size (Warikoo et al, 2012). The design and optimization of a PCC based chromatography system has also been studied by Pollock et al (2013), while Muller-Spath et al (2010) have examined the use of multi-column countercurrent solvent gradient (MCSG) chromatography with anion and cation exchange resins. Although multi column chromatography has been successfully integrated with perfusion bioreactors, these systems require column packing and complex valve switching, causing them to operate in a cyclic rather than true steady state mode.…”

Continuous downstream processing for high value biological products: A Review

“…In contast to SMB, some of the columns in MCSGP are “short‐circuited” to enable gradient elution; MCSGP can also be used for more than just binary separations. Muller‐Spath et al () have demonstrated the use of a 4‐column MCSGP system for the initial capture of an IgG 2 mAb from clarified cell culture fluid using cation exchange resins with a gradient elution. Krättli et al () discussed the use on online control in the operation of a twin‐column MCSGP system.…”

Preparative Purification of Recombinant Proteins: Current Status and Future Trends

“…It is particularly useful for resolving variants of proteins with an intermediate product being accompanied by weakly and strongly adsorbing variants such as mAb variants [91, 92]. It has, however, been successfully applied to purification of mAbs directly from cell culture supernatant as well [93]. …”