Method of purifying polypeptides by simulated moving bed chromatography
View Patent ↗Provided are methods of separating an immunoreactive compound from at least one immaterial component, using a simulated moving bed (“SMB”) system and a SMB apparatus for use in these methods. Also provided are purified immunoreactive compounds prepared using the SMB methods and apparatus and methods of treatment with the purified immunoreactive compounds.
1. A method of separating an antibody or antibody fragment from at least one immaterial component in a fluid mixture, comprising:
(a) providing a simulated moving bed (SMB) chromatography apparatus that comprises a plurality of modules in fluid conducting communication with said apparatus, wherein said modules comprise at least one solid phase comprising a ligand for which the antibody or antibody fragment has selective affinity, and wherein said apparatus comprises a plurality of zones through which the modules pass;
(b) introducing the fluid mixture into a module in an association zone wherein the fluid mixture contacts the solid phase in a countercurrent mode and the antibody or antibody fragment associates with the solid phase;
(c) introducing a wash buffer into the module comprising the associated antibody or antibody fragment in at least one wash zone, wherein the wash buffer contacts the solid phase and substantially removes at least one immaterial component from said module;
(d) introducing an eluent into the module comprising the associated antibody or antibody fragment from step (c) in an elution zone, wherein the eluent contacts the solid phase and promotes disassociation of the antibody or antibody fragment from the solid phase;
(e) removing the antibody or antibody fragment that is substantially separated from at least one immaterial component in said fluid mixture;
(f) introducing an elution wash buffer into the module from step (e) in an elution wash zone;
(g) introducing a solution comprising a regenerant into the module from step (f) in a regeneration zone; and
(h) introducing an equilibration buffer into the module in an equilibration zone, wherein the equilibration buffer creates an environment in the module that permits binding of the antibody or antibody fragment to the solid phase comprising the ligand.
2. A method according to claim 1 , wherein step (g) comprises introducing a regeneration buffer comprising urea into the module.
3. A method according to claim 1 , wherein step (g) comprises introducing a clean in place (CIP) solution into the module.
4. A method according to claim 3 , wherein the CIP solution comprises phosphoric acid.
5. A method according to claim 1 , wherein the antibody or antibody fragment comprises a constant region of an immunoglobulin and the solid phase comprises Protein A or Protein G.
6. A method according to claim 5 , wherein the eluent comprises an acidic buffer.
7. A method according to claim 1 , wherein effluent of a module in at least one wash zone is fed back into a module in the association zone.
8. A method according to claim 1 , wherein step (c) of said method comprises introducing a high salt wash buffer into a module comprising the associated antibody or antibody fragment in a first wash zone.
9. A method according to claim 8 , wherein step (c) of said method further comprises introducing a low salt wash buffer into a module comprising the associated antibody or antibody fragment in a second wash zone.
10. A method according to claim 1 , further comprising a step following wash step (c) that comprises introducing solution containing purified antibody or antibody fragment into a module in an entrainment rejection zone prior to elution of antibody or antibody fragment from said module.
11. A method according to claim 1 , wherein the concentration of antibody or antibody fragment obtained in step (e) is greater than the concentration of antibody or antibody fragment in the fluid mixture that is introduced in step (a).