SEPARATION METHOD

The invention relates to a method of isolating an immunoglobulin, comprising the steps of: a) providing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, b) contacting a liquid sample comprising an immunoglobulin with the separation matrix, c) washing said separation matrix with a washing liquid, d) eluting the immunoglobulin from the separation matrix with an elution liquid, and e) cleaning the separation matrix with a cleaning liquid, wherein the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined bySEQ ID NO 51 or SEQ ID NO 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, iso leucine, tryptophan, methionine, valine, alanine, histidine and arginine.