Separation method
The invention relates to 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 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; b) contacting a liquid sample comprising an immunoglobulin with the separation matrix; c) washing the 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.
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 pore size corresponding to an inverse gel filtration chromatography Kd value of 0.69-0.85 for dextran of Mw 110 kDa;
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 more than 2M NaOH,
wherein in step e) the contact time between the separation matrix and the cleaning liquid is 3 minutes or less.
2. 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.
3. The method of claim 1 , wherein said immunoglobulin comprises an Fc fusion protein.
4. The method of claim 1 , wherein said immunoglobulin has a hydrodynamic radius of at least 6.0 nm.
5. The method of claim 1 , wherein said immunoglobulin comprises a bispecific, trispecific or polyspecific antibody.
6. The method of claim 5 , wherein said method separates half antibodies or homodimeric antibodies from said bispecific, trispecific or polyspecific antibody.
7. The method of claim 1 , wherein said immunoglobulin comprises a conjugated antibody.
8. The method of claim 1 , wherein said immunoglobulin comprises an antibody fragment.
9. The method of claim 1 , wherein the separation matrix is a Protein A chromatography column.
10. The method of claim 1 , wherein steps b)-e) are repeated at least 10 times.
11. The method of claim 1 , wherein in step b) the pH is 6-8.
12. The method of claim 1 , wherein in step b) the residence time of said liquid sample on said separation matrix is 2-20 min.
13. The method of claim 1 , which is performed in a continuous or semicontinuous multicolumn chromatography process.
14. The method of claim 1 , which is performed in a periodic countercurrent chromatography (PCC) process.
15. The method of claim 1 , wherein the separation matrix is an antibody affinity chromatography cross-linked agarose matrix having alkaline-stabilized protein A-derived ligands.