Separation matrix

1. A separation matrix comprising at least 15 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein:

a) said ligands comprise multimers of alkali-stabilized Protein A domains,

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

2. The separation matrix of claim 1 , wherein said cross-linked polymer particles have a volume-weighted median diameter (d50, v) of from 56 micrometers to 66 micrometers.

3. The separation matrix of claim 1 , wherein said cross-linked polymer particles have a dry solids weight of from 60 mg/ml to 78 mg/ml.

4. The separation matrix of claim 3 , wherein said cross-linked polymer particles have a dry solids weight of from 65 mg/ml to 78 mg/ml.

5. The separation matrix of claim 1 , wherein said separation matrix has a max pressure of at least 0.58 MPa when packed at 300+/−10 mm bed height in a 35 mm separation column.

6. The separation matrix of claim 1 , wherein said cross-linked polymer particles comprise cross-linked polysaccharide particles.

7. The separation matrix of claim 1 , wherein said cross-linked polymer particles comprise cross-linked agarose particles.

8. The separation matrix 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.

9. The separation matrix of claim 1 , wherein said multimers comprise tetramers, pentamers, hexamers or heptamers of alkali-stabilized Protein A domains.

10. The separation matrix of claim 1 , wherein said multimers comprise hexamers of alkali-stabilized Protein A domains.

11. The separation matrix of claim 1 , having a 10% breakthrough dynamic binding capacity for IgG of at least 45 mg/ml at 2.4 min residence time.

12. The separation matrix of claim 1 , having a 10% breakthrough dynamic binding capacity for IgG of at least 60 mg/ml at 6 min residence time.

13. The separation matrix of claim 11 , wherein the 10% breakthrough dynamic binding capacity for IgG at 2.4 min residence time is reduced by less than 20% after incubation 31 h in 1.0 M aqueous NaOH at 22+/−2 C.

14. The separation matrix of claim 1 , having a dissociation constant for IgG2 of below 0.2 mg/ml in 20 mM phosphate buffer, 180 mM NaCl, pH 7.5.

15. The separation matrix of claim 1 , wherein said alkali-stabilized Protein A domains comprise an Fc-binding polypeptide having an amino acid sequence as defined by, or having at least 80% identity to SEQ ID NO:53 and

X 1 Q X 2 AFYEILX 3 LP NLTEEQRX 4 X 5 F IX 6 X 7 LKDX 8 PSX 9

SX 10 X 11 X 12 LAEAKX 13 X 14 NX 15 AQ (SEQ ID NO:53)

wherein individually of each other:

X 1 =A or Q or is deleted

X 2 =E, K, Y, T, F, L, W, I, M, V, A, H or R

X 3 =H or K

X 4 =A or N

X 5 =A, G, S, Y, Q, T, N, F, L, W, I, M, V, D, E, H, R or K

X 6 =Q or E

X 7 =S or K

X 8 =E or D

X 9 =Q or V or is deleted

X 10 =K, R or A or is deleted

X 11 =A, E or N or is deleted

X 12 =I or L

X 13 =K or R

X 14 =L or Y

X 15 =D, F, Y, W, K or R.

16. The separation matrix of claim 15 , wherein individually of each other:

X 1 =A or is deleted, X 2 =E, X 3 =H, X 4 =N, X 6 =Q, X 7 =S, X 8 =D, X 9 =V or is deleted, X 10 =K or is deleted, X 11 =A or is deleted, X 12 =I, X 13 =K, X 14 =L.

17. The separation matrix of claim 15 , wherein said multimers comprise hexamers of alkali-stabilized Protein A domains.

18. The separation matrix of claim 15 , wherein the amino acid sequence has at least 90% identity to SEQ ID NO:53.

19. The separation matrix of claim 15 , wherein the amino acid sequence has at least 95% identity to SEQ ID NO:53.

20. The separation matrix of claim 15 , wherein the amino acid sequence has at least 98% identity to SEQ ID NO:53.

21. The separation matrix of claim 15 , wherein the amino acid sequence is identical to SEQ ID NO:53.

22. The separation matrix of claim 1 , wherein the polypeptides are linked by linkers comprising up to 25 amino acids.

23. The separation matrix of claim 1 , wherein at least two polypeptides are linked by linkers comprising or consisting essentially of a sequence having at least 90% identity with an amino acid sequence selected from the group consisting of APKVDAKFDKE (SEQ ID NO:96, APKVDNKFNKE (SEQ ID NO:97), APKADNKFNKE (SEQ ID NO:98), APKVFDKE (SEQ ID NO:99), APAKFDKE (SEQ ID NO:100), AKFDKE (SEQ ID NO:101), APKVDA (SEQ ID NO:102), VDAKFDKE (SEQ ID NO:103), APKKFDKE (SEQ ID NO:104), APK, APKYEDGVDAKFDKE (SEQ ID NO:105) and YEDG (SEQ ID NO:106).

24. The separation matrix of claim 1 , comprising from 15 mg/ml to 21 mg/ml Fc-binding ligands covalently coupled to the porous support.

25. The separation matrix of claim 1 , comprising from 17 mg/ml to 21 mg/ml Fc-binding ligands covalently coupled to the porous support.

26. The separation matrix of claim 1 , comprising from 18 mg/ml to 20 mg/ml Fc-binding ligands covalently coupled to the porous support.

27. The separation matrix of claim 1 having a 10% breakthrough dynamic binding capacity for IgG of at least 50 mg/ml at 2.4 min residence time.

28. The separation matrix of claim 1 having a 10% breakthrough dynamic binding capacity for IgG of at least 55 mg/ml at 2.4 min residence time.

29. The separation matrix of claim 1 , having a 10% breakthrough dynamic binding capacity for IgG of at least 65 mg/ml at 6 min residence time.

30. The separation matrix of claim 1 , having a 10% breakthrough dynamic binding capacity for IgG of at least 70 mg/ml at 6 min residence time.

31. The separation matrix of claim 1 , having a 10% breakthrough dynamic binding capacity for IgG of at least 75 mg/ml at 6 min residence time.

32. The separation matrix of claim 1 having a dissociation constant for IgG2 of below 0.1 mg/ml in 20 mM phosphate buffer, 180 mM NaCl, pH 7.5.

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

a) contacting a liquid sample comprising an immunoglobulin with a separation matrix according to claim 1 ,

b) washing said separation matrix with a washing liquid,

c) eluting the immunoglobulin from the separation matrix with an elution liquid, and

d) cleaning the separation matrix with a cleaning liquid.

34. The method of claim 33 , wherein the cleaning liquid comprises from 0.1 to 1.0 M NaOH or KOH.

35. The method of claim 33 , wherein steps a)-d) are repeated at least 10 times.

36. The method of claim 33 , wherein in step c) said elution liquid has a pH of from 2.5 to 4.5.

37. The method of claim 33 , wherein in step a) the pH is from 6 to 8.

38. The method of claim 33 , wherein in step a) the residence time of said liquid sample on said separation matrix is from 2 minutes to 20 minutes.

39. The method of claim 33 , wherein the cleaning liquid comprises at least 0.5 M NaOH.

40. The method of claim 33 , wherein in step d) the contact time between the separation matrix and the cleaning liquid is less than 10 min.