Application of Multicolumn Countercurrent Solvent Gradient Purification to the polishing of therapeutic proteins

Abstract

Industrial purification of biopharmaceuticals is usually performed through single-column preparative liquid chromatography. The polishing step, which is required to remove all product-related impurities, represents one of the most critical points of the entire production process of biopharmaceuticals. Indeed, product-related impurities have very similar chemical characteristics to the target compound. The removal of these impurities via chromatography is very challenging, because also their chromatographic behavior is like that of the target. It follows that, despite the use of very shallow gradients to increase resolution of critical impurities, chromatograms are very often characterized by overlapping regions where target and impurities coelute (center-cut separations). This results in an intrinsic limitation of single-column processes that is the so-called yield-purity trade-off. This drawback can be partially overcome thanks to the use of multicolumn continuous (or semicontinuous) techniques. In particular, this chapter is focused on the advantages of Multicolumn Countercurrent Solvent Gradient Purification (MCSGP), one of the most recently developed countercurrent approaches specifically designed for challenging center-cut separations. The automatic internal recycling of the impure fractions inside the chromatographic system results in an increased yield without compromising the purity of the pool besides allowing for the automatization of the process. Each step of the purification process will be illustrated, together with a synthetic overview of most interesting applications of MCSGP.