Separation method

A method of isolating an immunoglobulin, comprising the steps of:

• a) providing a separation matrix comprising at least 15 mg/ml multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support,
• b) contacting a liquid sample comprising an immunoglobulin with said 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 comprising at least 0.5 M NaOH, wherein in step b) at least 40 mg immunoglobulin per ml separation matrix is contacted with said separation matrix;
• wherein said alkali-stabilized Protein A domains comprise mutants of a parental Fe-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% identity to, SEQ 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, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.