Magnetic immunoglobulin-binding particles
The invention discloses an immunoglobulin-binding magnetic bead, comprising a porous matrix and one or more magnetic particles embedded in said matrix, wherein said matrix comprises a porous polymer and at least 10 mg/ml Fc-binding proteinaceous ligands covalently coupled to said porous polymer.
1. A method of separating a target biomolecule from a cell culture, comprising the steps of:
a) providing a plurality of magnetic beads capable of binding said target biomolecule;
b) contacting said plurality of magnetic beads with a cell culture comprising a target biomolecule, to bind said target biomolecule to said beads;
c) retaining said magnetic beads with a magnetic field in a high gradient magnetic field separator (HGMS) and washing said beads with a washing liquid; and
d) eluting said magnetic beads with an eluent to desorb said target biomolecule from said beads and recovering said target biomolecule in an eluate,
wherein said contacting of said plurality of magnetic beads with said cell culture is performed in a flexible bag mounted on a tray adapted to rock around a horizontal axis, wherein step b) is performed in a bioreactor, and wherein said bioreactor is said flexible bag.
2. The method of claim 1 , wherein before step c), said magnetic beads are conveyed to said HGMS.
3. The method of claim 1 , wherein step d) is performed in a second high gradient magnetic field separator (HGMS) or in the same HGMS.
4. The method of claim 3 , wherein both steps c) and d) are performed in the same HGMS.
5. The method of claim 3 , wherein the HGMS of step c) is a washer HGMS and step d) is performed in an elution HGMS.
6. The method of claim 1 , wherein after step d), said magnetic beads are cleaned, reequilibrated and reused in a bioreactor.
7. The method of claim 1 , wherein said cell culture comprises at least 1 mg/ml target biomolecule.
8. The method of claim 1 , wherein said eluate is applied directly to a chromatography column.
9. The method of claim 1 , wherein step c) comprises a sequence of:
i) removing said magnetic field;
ii) resuspending said beads;
iii) contacting said beads with a portion of washing liquid;
iv) retaining said beads with a magnetic field; and
v) removing washing liquid from said retained beads.
10. The method of claim 1 , wherein in step d) said beads are retained with a magnetic field.
11. The method of claim 1 , wherein step c) and optionally step d) are performed in said bioreactor.
12. The method of claim 11 , wherein a portion of said bioreactor is configured for magnetic retention of said magnetic beads.
13. The method of claim 1 , wherein said tray comprises at least one magnet or magnet holder.
14. The method of claim 1 , where said magnetic beads are presterilized.
15. The method of claim 14 , wherein said magnetic beads are supplied dry.
16. The method of claim 14 , wherein said magnetic beads are conveyed from a presterilized bead container via presterilized tubing and one or more aseptic connectors to the bioreactor, to a magnetic separator or to a contactor.
17. The method of claim 1 , wherein after step d), said magnetic beads are regenerated and recirculated to the bioreactor and steps b)-d) are repeated.
18. The method of claim 17 , wherein said magnetic beads are sterilized before recirculation to the bioreactor.
19. The method of claim 1 , wherein cells are cultivated in the bioreactor in the presence of said magnetic beads.
20. The method of claim 1 , wherein in step b) said cell culture is an unclarified or cell-depleted cell culture.
21. The method of claim 20 , wherein said unclarified or cell-depleted cell culture comprises at least 1 mg/ml target biomolecule.
22. The method of claim 1 , wherein said target biomolecule is an immunoglobulin, and wherein said magnetic beads comprise a porous matrix and one or more magnetic particles embedded in said matrix, wherein said matrix comprises a porous polymer and at least 10 mg/ml Fc-binding proteinaceous ligands covalently coupled to said porous polymer.
23. The method of claim 22 , wherein said ligands comprise one or more alkali-stabilized mutants of SpA Fc-binding domains.
24. A method of separating a target biomolecule from a cell culture, comprising the steps of:
a) providing a plurality of magnetic beads capable of binding said target biomolecule;
b) contacting said plurality of magnetic beads with a cell culture comprising a target biomolecule, to bind said target biomolecule to said beads;
c) retaining said magnetic beads with a magnetic field in a high gradient magnetic field separator (HGMS) and washing said beads with a washing liquid; and
d) eluting said magnetic beads with an eluent to desorb said target biomolecule from said beads and recovering said target biomolecule in an eluate,
wherein said contacting of said plurality of magnetic beads with said cell culture is performed in a flexible bag mounted on a tray adapted to rock around a horizontal axis, wherein step b) is performed in a contactor, and wherein said contactor is said flexible bag.
25. The method of claim 24 , wherein, after step d), said beads are recirculated to said contactor.