Purification of biological molecules
The present invention relates to improved processes and systems for purification of biological molecules, where the processes can be performed in a continuous manner.
1. A continuous process for the purification of a target molecule comprising the steps of:
a) providing a sample comprising the target molecule and one or more impurities;
b) adding at least one precipitant to the sample and removing one or more impurities, thereby to recover a clarified sample;
c) subjecting the clarified sample from step (b) to a bind and elute chromatography step comprising at least two separation units, thereby to obtain an eluate comprising the target molecule; and
d) subjecting the eluate to flow-through purification comprising use of two or more media, one of which is activated carbon and the other(s) are selected from anion-exchange chromatography media and cation-exchange chromatography media;
wherein at least two steps are performed concurrently for at least a portion of their duration, wherein the process comprises only one bind and elute chromatography step, and wherein the process is a continuous process,
wherein the process comprises use of one or more surge tanks and/or one or more static mixers, wherein the flow-through purification is carried out as: activated carbon followed by anion-exchange chromatography media followed by cation-exchange chromatography media, and
wherein the in-line static mixer and/or surge tank is used between anion-exchange chromatography media and cation-exchange chromatography media to change pH.
2. The process of claim 1 , comprising a virus inactivation step between steps (c) and (d).
3. The process of claim 2 , wherein the virus inactivation step comprises use of a virus inactivating agent selected from acid, detergent, solvent and temperature change.
4. The process of claim 2 , wherein virus inactivation step comprises use of one or more in-line static mixers.
5. The process of claim 2 , wherein virus inactivation comprises use of one or more surge tanks.
6. The process of claim 1 , wherein the target molecule is an antibody.
7. The process of claim 6 , wherein the antibody is selected from a monoclonal antibody or a polyclonal antibody.
8. The process of claim 1 , wherein the precipitant in step (b) is a stimulus responsive polymer.
9. The process of claim 8 , wherein the stimulus responsive polymer is a modified polyallylamine polymer.
10. The process of claim 1 , wherein the precipitant in step (b) is selected from the group consisting of an acid, caprylic acid, a flocculant and a salt.
11. The process of claim 1 , wherein removing impurities in step (b) comprises use of one or more depth filters.
12. The process of claim 1 , wherein removing impurities in step (b) comprises use of centrifugation.
13. The process of claim 1 , wherein the bind and elute chromatography step in (c) employs continuous multi-column chromatography.
14. The process of claim 1 , wherein the bind and elute chromatography step in (c) is selected from the group consisting of affinity chromatography, cation exchange chromatography and mixed-mode chromatography.
15. The process of claim 1 , wherein the bind and elute chromatography step in (c) employs Protein A affinity chromatography.
16. The process of claim 15 , wherein Protein A affinity chromatography employs a Protein A ligand coupled to a matrix selected from the group consisting of rigid hydrophilic polyvinylether polymer, controlled pore glass and agarose.
17. The process of claim 1 , wherein the sample in step (a) is a cell culture.
18. The process of claim 17 , wherein the cell culture is provided in a bioreactor.
19. The process of claim 18 , wherein the bioreactor is a single use bioreactor.
20. The process of claim 17 , wherein the cell culture is provided in a vessel other than a bioreactor.
21. The process of claim 1 , wherein the precipitant in step (b) is added to a bioreactor comprising a cell culture.
22. The process of claim 21 , wherein the precipitant is added using a static mixer.
23. The process of claim 1 , wherein the precipitant in step (b) is added to a vessel other than a bioreactor which comprises the sample comprising the target molecule.
24. The process of claim 1 , wherein the flow-through purification in step (d) further comprises use of a virus filtration membrane.
25. The process of claim 1 , wherein the cation exchange chromatography media is in the form of a membrane, a bead or a fiber.
26. The process of claim 1 , wherein the process comprises use of one or more surge tanks and does not employ any pool tanks between process steps.
27. The process of claim 1 , further comprising a formulation step.
28. The process of claim 27 , wherein formulation comprises diafiltration, concentration and sterile filtration.
29. The process of claim 28 , further concentration comprises tangential flow filtration.