Chromatographic purification method and uses thereof

A chromatographic purification method for the isolation of a desired product from a feed mixture consisting of the desired product and at least two further components, at least one component with impurities more weakly adsorbing than the desired product and at least one component with impurities more strongly adsorbing than the desired product,

• said method using two or more chromatographic columns,
• wherein a first upstream column has a first column inlet and a first column outlet, and a second downstream column has a second column inlet and a second column outlet,
• wherein in one step b) the first upstream column is loaded with feed via the first column inlet,
• followed by at least one interconnected step c) in which the columns are interconnected in series for passing eluate containing desired product via the first column outlet to the second column inlet,
• wherein in said at least one interconnected step c) the first upstream column is fed with eluent with a gradient in the form of a temporally changing modifier concentration, and
• wherein in said at least one interconnected step c) the stream exiting the first column outlet is adjusted in line with inline adjustment eluent before entering the second column inlet at least during a period of gradient elution, and
• wherein the inline adjustment eluent is as the eluent fed at the first column inlet but controlled to have a higher or lower modifier concentration than the eluent exiting at the first column outlet, and wherein the modifier difference of the inline adjustment eluent is chosen such that the adherence of the desired product to the stationary phase of the second column (2) is higher than without that difference
• wherein
• either it involves only two columns, and wherein in said at least one interconnected step c) desired product is collected at the second column outlet;
• or it involves said first and said second column and at least one further column, fulfilling, downstream of said second column, the equivalent function of said second column, in that the stream exiting the second column is adjusted in line with inline adjustment solvent before entering the further column inlet at least during the period of gradient elution, and wherein the corresponding inline adjustment solvent is the same as the solvent fed at the first column inlet but controlled to have a different modifier concentration than the solvent exiting at the second column outlet, and wherein the modifier difference of the inline adjustment solvent is chosen such that the adherence of the desired product to the stationary phase of the further column is higher than without that difference, and wherein in said at least one interconnected step c) desired product (P) is collected at the further column outlet.