Separation method
The invention relates to 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 the 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, wherein the 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% such as at least 90%, 95% or 98% identity to, 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 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 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.
1. 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,
wherein said alkali-stabilized Protein A domains comprise mutants of a parental Fe-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% identity to, 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 52 are asparagines, and wherein at least the asparagine residue at position 3 of SEQ ID NO: 51 or 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.
2. The method of claim 1 , wherein the glutamine residue at position 1 of SEQ ID NO: 51 or 52 has been mutated to an alanine.
3. The method of claim 1 , wherein the asparagine or glutamic acid residue at position 35 of SEQ ID NO: 51 or 52 has been mutated to an alanine.
4. The method of claim 1 , wherein said separation matrix comprises at least 11 mg/ml, of said multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support.
5. The method of claim 1 , wherein in step b) at least 40 mg immunoglobulin per ml separation matrix is contacted with said separation matrix.
6. The method of claim 1 , wherein in step e) said cleaning liquid comprises at least 0.5 M NaOH.
7. The method of claim 6 , wherein steps b)-e) are repeated at least 10 times.
8. The method of claim 1 , wherein in step e) said cleaning liquid comprises at least 1 M NaOH.
9. The method of claim 8 , wherein steps b)-e) are repeated at least 10 times.
10. The method of claim 1 , wherein said liquid sample is a clarified cell broth and wherein in step d) said immunoglobulin is recovered as an eluate comprising less than 2000 ppm host cell proteins.
11. The method of claim 1 , wherein in step d) said elution liquid has a pH of 2.5-4.5.
12. The method of claim 1 , wherein said immunoglobulin comprises IgG1, IgG2 and/or IgG4.
13. The method of claim 1 , wherein said washing liquid has a pH of 5-8 and optionally comprises one or more of a detergent, a water-miscible organic solvent, a chaotrope, arginine or an arginine derivative, calcium ions and tetraalkylammonium ions.
14. The method of claim 1 , wherein said multimers are coupled to said support via thioether links.
15. The method of claim 1 , wherein in step b) the pH is 6-8.
16. The method of claim 1 , wherein in step b) the residence time of said liquid sample on said separation matrix is 2-20 min.
17. The method of claim 1 , 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.
18. The method of claim 17 , 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.
19. 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;
wherein said alkali-stabilized Protein A domains comprise mutants of a parental Fe-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% identity to, 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 52 are asparagines, and wherein at least the asparagine residue at position 3 of SEQ ID NO: 51 or 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.
20. The method of claim 19 , wherein said liquid sample is a clarified cell broth and wherein in step d) said immunoglobulin is recovered as an eluate comprising less than 2000 ppm host cell proteins.
21. The method of claim 19 , wherein steps b)-e) are repeated at least 10 times.