![]() This situation results in a variety of protein–ligand interactions and often leads to unique selectivity, which in some cases includes pseudo-affinity. ![]() 1 The target protein itself is also a multimodal molecule. The ligands of mixed-mode materials typically contain a combination of multiple binding modes like ion exchange, hydrogen bonding, and hydrophobic interactions. Mixed-mode resins may help to overcome these problems because multimodal ligands offer excellent selectivity in combination with a salt-tolerant adsorption of the target protein. ![]() In these cases, single-mode chromatography materials like ion exchange or hydrophobic resins often fail, in particular if the feedstock contains large amounts of complex colored impurities like melanoidins in combination with a relatively high conductivity of 15–30 mS/cm. coli lysates are used as starting materials. However, this strategy significantly increases the risk that contaminants are not completely removed during the purification process, especially if complex microbial feedstocks or E. Modern downstream processes often consist of only two or three separation steps, and usually avoid conditioning steps like ultrafiltration/diafiltration (UF/DF) for buffer exchange to reduce the total number of process steps. This leads to a variety of process- and product-related impurities which must be removed by an efficient, orthogonal, and robust purification process. The impurity profile of a starting material for downstream processing depends mainly on the conditions of the fermentation process, such as the expression system, medium composition, fermentation regimen, time point of harvest, or shear forces during isolation. Because these purification protocols can be scaled up easily to production scale, mixed-mode materials are being considered as potential elements of a general purification platform for recombinant therapeutic proteins produced in various expression systems. Product-related impurities as well as process-related impurities from fermentation media were efficiently removed while the desired product was bound with high selectivity. We used mixed-mode materials for capturing and intermediate purification of several recombinant therapeutic proteins from various expression systems like yeast, Escherichia coli, and mammalian cells. Complex mixtures like fermentation supernatants or cell lysates can be applied directly at relatively high conductivity, and elution is usually achieved by electrostatic charge repulsion. Mixed-mode chromatography materials contain ligands of multimodal functionality that allow protein adsorption by a combination of ionic interactions, hydrogen bonds, and/or hydrophobic interactions.
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