Separation matrix

Patent metadata

Field	Value
Publication number	US-10654887-B2
Application number	US-201615282367-A
Country	US
Kind code	B2
Filing date	Sep 30, 2016
Priority date	May 11, 2016
Publication date	May 19, 2020
Grant date	May 19, 2020

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 Fc-binding ligands covalently coupled to a porous support having a ligand content of at least 11 mg/ml, 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 56-70 micrometers, a dry solids weight of 55-80 mg/ml, and a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.69-0.85 for dextran of Mw 110 kDa,
wherein the stability of the separation matrix increases with increasing ligand content, and
wherein said alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% identity to, SEQ ID NO: 53,

(SEQ ID NO: 53)

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

X 10 X 11 X 12 LAEAKX 13 X 14 NX 15 AQ

wherein individually of each other:
X 1 =A, 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, V, or is deleted
X 10 =K, R, A, or is deleted
X 11 =A, E, 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.

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

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

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

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

6. 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 or a 10% breakthrough dynamic binding capacity for IgG of at least 60 mg/ml at 6 min residence time.

7. The separation matrix of claim 6 , wherein the10% breakthrough dynamic binding capacity for IgG at 2.4 min or 6 min residence time is reduced by less than 20% after incubation 31h in 1.0 M aqueous NaOH at 22+/−2 C.

8. 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.

9. The separation matrix of claim 1 , wherein said alkali-stabilized Protein A domain has a sequence defined by SEQ ID NO: 11 without linker region amino acids 1-8 and 56-58.

10. The separation matrix of claim 1 , wherein said alkali-stabilized Protein A has a sequence defined by SEQ ID NO:4-7, without linker region amino acids 1-8 and 56-58 wherein the amino acid residue at the position corresponding to position 11 in SEQ ID NO:4-7 is, or has been mutated to, a glutamic acid or a lysine.

11. The separation matrix of claim 1 , wherein said alkali-stabilized Protein A has a sequence defined by SEQ ID NO:4-7, without linker region amino acids 1-8 and 56-58 wherein the amino acid residue at the position corresponding to position 40 in SEQ ID NO:4-7 is, or has been mutated to, a valine.

12. 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, X 14 =L.

13. 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.

14. The method of claim 13 , wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH.

15. The method of claim 14 , wherein steps a)-d) are repeated at least 10 times.

16. A separation matrix comprising Fc-binding ligands covalently coupled to a porous support, having ligand content of at least 15 mg/mL and a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.69-0.85 for dextran of Mw 110 kDa,
wherein the stability of the separation matrix increases with increasing ligand content,
wherein said ligands comprise multimers of alkali-stabilized Protein A domains, and
wherein said alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% identity to, SEQ ID NO: 53,

(SEQ ID NO: 53)

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

X 10 X 11 X 12 LAEAKX 13 X 14 NX 15 AQ

wherein individually of each other:
X 1 =A, 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, V, or is deleted
X 10 =K, R, A, or is deleted
X 11 =A, E, 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.

17. The separation matrix of claim 16 , having a sequence defined by SEQ ID NO:33 made up of alkali-stabilized Protein A domain defined by SEQ ID NO:11, without linker region amino acids 1-8 and 56-58.

18. The separation matrix of claim 16 , wherein said cross-linked polymer particles comprise cross-linked agarose particles.

19. The separation matrix of claim 16 , having a 10% breakthrough dynamic binding capacity for IgG of at least 45 mg/ml at 2.4 min residence time or a 10% breakthrough dynamic binding capacity for IgG of at least 60 mg/ml at 6 min residence time.

20. The separation matrix of claim 19 , wherein the 10% breakthrough dynamic binding capacity for IgG at 2.4 min or 6 min residence time is reduced by less than 20% after incubation 31h in 1.0 M aqueous NaOH at 22+/−2 C.

21. The separation matrix of claim 16 , wherein said multimers comprise tetramers, pentamers, hexamers or heptamers of alkali-stabilized Protein A domains.

22. The separation matrix of claim 16 , having a dissociation constant for IgG2 of

Assignees

• Ge Healthcare Bioprocess R&D Ab
• Ge Healthcare Bio Process R&D Ab

Inventors

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

Classifications

• B01J20/286

  Phases chemically bonded to a substrate, e.g. to silica or to polymers · CPC title

• B01J20/267

  Cross-linked polymers · CPC title

• B01J2220/52

  Sorbents specially adapted for preparative chromatography · CPC title

• C07K16/00

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

• C07K1/22 Primary

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title