Separation matrix

Patent metadata

Field	Value
Publication number	US-12134633-B2
Application number	US-202318298857-A
Country	US
Kind code	B2
Filing date	Apr 11, 2023
Priority date	May 11, 2016
Publication date	Nov 5, 2024
Grant date	Nov 5, 2024

Abstract

The invention relates to a separation matrix comprising at least 11 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein:
a) the ligands comprise multimers of alkali-stabilized Protein A domains, and b) the 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.

First claim

The invention claimed is:

1. A separation matrix comprising at least 15 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein:
the ligands comprise multimers of alkali-stabilized Protein A domains;
the porous support comprises cross-linked polymer particles having a volume-weighted median diameter (d50,v) of from 56 μm to 70 μm;
the 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; and
the Protein A domains comprise an Fc-binding polypeptide defined by, or having at least 80% identity to SEQ ID NO: 53 and

X 1 QX 2 AFYEILX 3 LPNLTEEQRX 4 X 5 FIX 6 X 7 LKDX 8 PSX 9 SX 10 X 11 X 12 LAEAKX 13 X 14 N X 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 =W 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; and
X 15 =D, F, Y, W, K, or R.

2. The separation matrix of claim 1 , wherein the cross-linked polymer particles have a dry solids weight of from 55 mg/ml to 80 mg/ml.

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

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

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

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

8. The separation matrix of claim 1 , wherein the 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 Mx 100 kDa.

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

10. The separation matrix of claim 1 , wherein the 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 minutes residence time.

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

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

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

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

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

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

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

19. 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.

20. 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.

21. The separation matrix of claim 1 , 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; and
X 14 =L.

22. The separation matrix of claim 1 , wherein the amino acid sequence has at least 90% identity to SEQ ID NO: 53.

23. The separation matrix of claim 1 , wherein the amino acid sequence has at least 95% identity to SEQ ID NO: 53.

24. The separation matrix of claim 1 , wherein the amino acid sequence has at least a 98% identity to SEQ ID NO: 53.

25. The separation matrix of claim 1 , wherein the amino acid sequence is identical to SEQ ID NO: 53.

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

27. The separation matrix of claim 1 , wherein at least two polypeptides are linked by likers 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).

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

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

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

31. A method of isolating an immunoglobulin, comprising the steps of:
a) contacting an immunoglobulin with a separation matrix according to claim 1 ;
b) washing the 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.

32. The method of claim 31 , wherein the cleaning liquid comprises from 0.1 to 1.0 M NaOH or KOH.

33. The method of claim 32 , wherein steps a)-d) are repeated at least 10 times.

34. The method of claim 32 , wherein in step c) the elution liquid has a pH of from 2.5 to 4.5.

35. The method of claim 32 , wherein in step b) the washing liquid has a pH of from 6 to 8.

36. The method of claim 32 , wherein in step b) residence time of the liquid on the separation matrix is from 2 minutes to 20 minutes.

37. The method of claim 32 , wherein the cleaning liquid comprises at least 0.5 M NaOH.

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

Assignees

• Cytiva Bioprocess R & D Ab

Inventors

• Rodrigo Gustav José
• Bjorkman Tomas
• Ander Mats
• Hansson Jesper Ulf

Classifications

• C07K17/10

  the carrier being a carbohydrate · CPC title

• C07K16/065

  Purification, fragmentation · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K14/31

  from Staphylococcus (G) · CPC title

• B01J20/3274

  Proteins, nucleic acids, polysaccharides, antibodies or antigens · CPC title