Separation method

1. 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, wherein said porous support comprises cross-linked polymer particles having a volume-weighted median diameter (d50,v) of 56-70 micrometers and a dry solids weight of 55-80 mg/ml;

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.

2. The method of claim 1 , wherein said cross-linked polymer particles have a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.69-0.85 for dextran of Mw 110 kDa.

3. The method of claim 1 , wherein said separation matrix has a maximum pressure of at least 0.58 MPa when packed at 300+−10 mm bed height in a chromatography column having a diameter of 35 mm.

4. The method of claim 1 , wherein said immunoglobulin comprises an Fc fusion protein.

5. The method of claim 1 , wherein said immunoglobulin has a hydrodynamic radius of at least 6.0 nm.

6. The method of claim 1 , wherein said immunoglobulin comprises a bispecific, trispecific or polyspecific antibody.

7. The method of claim 6 , wherein said method separates half antibodies or homodimeric antibodies from said bispecific, trispecific or polyspecific antibody.

8. The method of claim 1 , wherein said immunoglobulin comprises a conjugated antibody.

9. The method of claim 1 , wherein said immunoglobulin comprises an antibody fragment.

10. The method of claim 1 , wherein the separation matrix is a Protein A chromatography column.

11. 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 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 1.5 M NaOH,

wherein said alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by 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 SEQ ID NO: 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO: 51 or SEQ ID NO: 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.

12. The method of claim 11 , wherein the mutants comprise further mutations in one or more of positions 1, 2, 7, 10, 15, 20, 21, 24, 25, 28, 29, 32, 34, 35, 36, 39, 42 and 43 in SEQ ID NO: 51 or SEQ ID NO: 52.

13. The method of claim 11 , wherein in step e) the contact time between the separation matrix and the cleaning liquid is less than 10 min.

14. The method of claim 11 , wherein in step e) the contact time between the separation matrix and the cleaning liquid is 5 min or less.

15. The method of claim 11 , wherein in step e) the contact time between the separation matrix and the cleaning liquid is 3 min or less.

16. The method of claim 13 , which is performed in a continuous or semicontinuous multicolumn chromatography process.

17. The method of claim 13 , which is performed in a periodic countercurrent chromatography (PCC) process.

18. The method of claim 11 , wherein in step e) the cleaning liquid comprises at least 2 M NaOH.

19. The method of claim 11 , wherein steps b)-e) are repeated at least 10 times.

20. The method of claim 11 , wherein the separation matrix is an antibody affinity chromatography cross-linked agarose matrix having alkaline-stabilized protein A-derived ligands.

21. The method of claim 1 , wherein in step e) said cleaning liquid comprises at least 1 M NaOH.

22. The method of claim 1 , wherein in step b) the pH is 6-8.

23. The method of claim 1 , wherein in step b) the residence time of said liquid sample on said separation matrix is 2-20 min.