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, and
• wherein the immunoglobulin-binding alkali-stabilized Protein A domains are, or have at least 80% sequence identity to, a sequence comprising any of SEQ ID NOS:
• 1 or 3-7, wherein N11 is mutated to an amino acid selected from glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, and arginine.