Enhanced capacity and purification of antibodies by mixed mode chromatography in the presence of aqueous-soluble nonionic organic polymers
This invention relates to the use of mixed mode chromatography for purification of at least one intact non-aggregated antibody from a mixture containing intact non-aggregated antibodies and undesirable materials, including fragmented or aggregated antibodies, host cell proteins, DNA, endotoxin, and/or virus. This invention further relates to the integration of such a method into a multi-step procedure with other fractionation methods for purification of antibodies suitable for in vivo applications.
1. A method for enhancing the antibody binding capacity of a mixed mode chromatography support, the method comprising
contacting said support with an antibody preparation in the presence of an aqueous-soluble nonionic organic polymer.
2. The method of claim 1 , wherein the contacting step occurs when the mixed mode chromatography support is operated in bind-elute mode.
3. The method of claim 1 , wherein the contacting step occurs when the mixed mode chromatography support is operated in flow-through mode such that non-aggregated antibodies flow through while aggregated antibodies bind to the mixed mode chromatography support.
4. The method of claim 1 , wherein the nonionic organic polymer is from the group consisting of dextran, starch, cellulose, polyvinylpyrrolidone, polypropylene glycol and polyethylene glycol (PEG).
5. The method of claim 1 , wherein the nonionic organic polymer comprises two or more nonionic inorganic polymers.
6. The method of claim 1 , wherein the nonionic organic polymer is at a concentration ranging from 0.01 to 50%.
7. The method of claim 1 , wherein the nonionic organic polymer has an average molecular weight of 100 to 10,000 daltons.
8. The method of claim 1 , wherein the antibody preparation comprises at least one of IgG, IgA, IgE, IgM or IgD.
9. The method of claim 1 , wherein the antibody preparation contains an antibody fusion protein.
10. The method of claim 1 , wherein the antibody preparation contains an antibody fragment.
11. The method of claim 1 , wherein the antibody preparation is unpurified.
12. The method of claim 1 , wherein the antibody preparation is partially purified.
13. The method of claim 12 , wherein the antibody preparation was previously purified with protein A.
14. The method of claim 1 , wherein the mixed mode support exploits a combination of two or more of the following functionalities to adsorb components of the antibody preparation: cation exchange, anion exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding, pi-pi bonding, metal affinity.
15. The method of claim 1 , wherein the mixed mode support comprises hydroxyapatite.
16. The method of claim 15 , wherein the mixed mode support is selected from the group consisting of hydroxypatite CHT Type I, 20 micron; hydroxypatite CHT Type I, 40 micron; hydroxypatite CHT Type I, 80 micron; hydroxypatite CHT Type II, 20 micron; hydroxypatite CHT Type II, 40 micron; and hydroxypatite CHT Type II, 80 micron.
17. The method of claim 1 , wherein the mixed mode support comprises fluoroapatite.
18. The method of claim 17 , wherein the mixed mode support comprises fluoroapatite CFT Type I, 40 micron or fluoroapatite CFT Type II, 40 micron.
19. The method of claim 1 , wherein the mixed mode support comprises a ligand selected from the group consisting of Capto-MMC, Capto-Adhere, Capto-S, Capto-Q, MEP Hypercel, and ABx.
20. The method of claim 1 , wherein the method comprises at least one other purification step.
21. The method of claim 1 , wherein antibody aggregate binding capacity of the support is preferentially enhanced in flow-through mode such that a larger volume of non-aggregated antibody is obtained before aggregated antibody begins to appear in the flow-through than would be obtained in the absence of the aqueous-soluble nonionic organic polymer.
22. The method of claim 1 , wherein separation of antibody aggregates from non-aggregated antibody is enhanced on the mixed mode chromatography support operated in bind-elute mode compared to the separation that would occur in the absence of the aqueous-soluble nonionic organic polymer.
23. The method of claim 21 , wherein virus binding capacity of the mixed mode support operated in flow-through mode is preferentially enhanced such that a larger volume of antibody is obtained before virus begins to appear in the flow-through compared to the volume of antibody that would be obtained in the absence of the aqueous-soluble nonionic organic polymer.
24. The method of claim 1 , wherein separation of virus from antibody on the mixed mode chromatography support operated in bind elute mode is enhanced compared to the separation that would occur in the absence of the aqueous-soluble nonionic organic polymer by conducting elution in the presence of an aqueous-soluble nonionic organic polymer.
25. The method of claim 1 , wherein antibody aggregates, virus, leached protein A, DNA, and/or endotoxin are separated from antibodies in the antibody preparation on the mixed mode support operated in bind-elute mode by conducting elution in the presence of an aqueous-soluble nonionic organic polymer.
26. The method of claim 21 , wherein virus, leached protein A, DNA, and/or endotoxin binding capacity of the mixed mode support operated in flow-through mode is enhanced such that a larger volume of non-aggregated antibody is obtained before virus, leached protein A, DNA, and endotoxin begin to appear in the flow-through than would be obtained in the absence of the aqueous-soluble nonionic organic polymer.
27. The method of claim 1 , wherein the separation of non-antibody proteins from antibody on a mixed mode chromatography support operated in bind elute mode is enhanced compared to the separation that would occur in the absence of the aqueous-soluble nonionic organic polymer.