Method of weak partitioning chromatography
View Patent ↗This invention relates to methods of using weak partitioning chromatography for the purification of a product from a load fluid containing one or more impurities. Further, the invention relates to methods of weak partitioning chromatography defined by operating conditions which cause a medium to bind least 1 mg of product per mL of medium, or alternatively, defined by a partition coefficient of at least 0.1.
1. A method of recovering a purified product from a load fluid, comprising the steps of:
passing the load fluid through a medium to obtain a column effluent, at operating conditions which cause the medium to bind at least about 2.8 mg of product per mL of medium, wherein the medium is selected from the group consisting of a charged ion exchange medium, a hydrophobic interaction chromatography resin, and an immobilized metal affinity chromatography resin; and
recovering the purified product in the column effluent.
2. A method of recovering a purified product from a load fluid, comprising the steps of:
passing the load fluid through a medium to obtain a column effluent, at operating conditions defined by a partition coefficient of at least about 0.1; and
recovering the purified product in the column effluent.
3. A method of recovering a purified product from a load fluid, comprising the steps of:
passing the load fluid through a medium to obtain a column effluent, at operating conditions which cause the medium to bind about 2.8 to about 13 mg of product per mL of medium; and
recovering the purified product in the column effluent.
4. A method of recovering a purified product from a load fluid, comprising the steps of:
passing the load fluid through a medium at operating conditions which cause the medium to bind at least about 1 mg of product per mL of medium, wherein the medium is selected from the group consisting of a hydrophobic interaction chromatography resin and an immobilized metal affinity chromatography resin; and
recovering the purified product from column effluent.
5. A method of recovering a purified antibody from a load fluid, comprising the steps of:
passing the load fluid through a charged anion exchange medium to obtain a column effluent, at operating conditions which cause the medium to bind at least about 2.8 mg of antibody per mL of medium, wherein the operating conditions comprise a pH of about 7.5 to about 8.2 and a chloride concentration of about 12 to about 55 mM, and wherein the load fluid is a product-containing fluid eluted from a Protein A column; and
recovering the purified antibody in the column effluent.
6. A method of recovering a purified antibody from a load fluid, comprising the steps of:
passing the load fluid through a charged anion exchange medium to obtain a column effluent, at operating conditions defined by a partition coefficient of at least about 1.0; wherein the operating conditions comprise a pH of about 7.5 to about 8.2 and a chloride concentration of about 12 to about 55 mM, and wherein the load fluid is a product-containing fluid eluted from a Protein A column; and
recovering the purified antibody in the column effluent during the load cycle and any essentially isocratic wash.
7. A method of recovering a purified product from a load fluid, comprising the steps of:
passing the load fluid through a medium to obtain a column effluent, at operating conditions which cause the medium to bind at least about 1 mg of product per mL of medium; and
recovering at least about 98% of the product in the column effluent.
8. A method of recovering a purified product from a load fluid, comprising the steps of:
passing the load fluid through a medium to obtain a column effluent, at operating conditions which cause the medium to bind at least about 3.0 mg of product per mL of medium; and
recovering at least about 93% of the product in the column effluent.
9. The method of claim 1 , wherein the operating conditions cause the medium to bind at least about 10 mg of product per mL of medium.
10. The method of claim 2 , wherein the value of the partition coefficient is in the range of about 0.2 to about 10.0.
11. The method of any one of claims 1 - 3 and 6 - 8 , further comprising the step of recovering the purified product from the medium using an essentially isocratic wash.
12. The method of any one of claims 1 - 3 and 5 - 8 , wherein the purified product is a protein selected from the group consisting of fusion proteins, Fc-containing proteins, immunoconjugates, cytokines, interleukins, hormones, and therapeutic enzymes.
13. The method of any one of claims 1 , 2 , 3 , 7 and 8 , wherein the operating conditions comprise a pH of about 7.5 to about 8.2 and a chloride concentration of about 12 to about 55 mM.
14. The method of any one of claims 1 - 3 and 5 - 8 , wherein the load fluid further comprises at least one of phosphate, calcium, arginine, glycine, HEPES, and counterligand.
15. The method of claim 14 , wherein the counterligand is imidazole.
16. The method of claim 1 , wherein the operating conditions cause the medium to bind at least about 8 mg of product per mL of medium.
17. The method of claim 16 , wherein the operating conditions cause the medium to bind at least about 14 mg of product per mL of medium.
18. The method of claim 1 , wherein the operating conditions cause the medium to bind about 2.8 to about 12 mg of product per mL of medium.
19. The method of claim 18 , wherein the operating conditions cause the medium to bind about 2.8 to about 5 mg of product per mL of medium.
20. The method of claim 18 , wherein the operating conditions cause the medium to bind about 8 to about 12 mg of product per mL of medium.
21. The method of claim 2 , wherein the value of the partition coefficient is in the range of about 0.5 to about 15.
22. The method of claim 21 , wherein the value of the partition coefficient is in the range of about 0.5 to about 2.5.
23. The method of claim 12 , wherein the Fc-containing protein is an antibody.
24. The method of claim 2 , wherein the medium comprises a charged ion exchange medium.
25. The method of claim 1 or 24 , wherein the charged ion exchange medium comprises an anion exchange resin.
26. The method of claim 1 or 24 , wherein the charged ion exchange medium comprises a cation exchange resin.
27. The method of claim 2 , wherein the medium comprises a hydrophobic interaction chromatography resin.
28. The method of claim 2 , wherein the medium comprises a hydroxyapatite resin.
29. The method of claim 2 , wherein the medium comprises an immobilized metal affinity chromatography resin.
30. The method of any one of claims 1 - 3 and 5 - 8 , wherein the load fluid comprises one or more impurities selected from the group consisting of host cell proteins, nucleic acids, product variants, endotoxins, Protein A, and viruses, or any combinations thereof.
31. The method of any one of claims 1 - 3 and 5 - 8 , wherein the load fluid is a product-containing fluid eluted from a Protein A column using an elution buffer and the pH and conductivity of the product-containing fluid is adjusted using a neutralization buffer which results in no more than about 20 mM of the ionic strength of the product-containing fluid.
32. The method of claim 31 , wherein the elution buffer comprises molecules with a charged anionic group with a pKa of about 2 to about 5.
33. The method of claim 31 , wherein the elution buffer further comprises molecules with a charged cationic group with a pKa of about 6.5 to about 10.
34. The method of claim 31 , wherein the elution buffer comprises a molecule that is a zwitterion at a pH of about 4 to about 9.
35. The method of claim 34 , wherein the zwitterion is selected from the group consisting of glycine; 1,4-piperazinebis-(ethanesulfonic acid); glycylglycine; cyclopentanetetra-1,2,3,4-carboxylic acid; N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonicacid; 2-(N-morpholino)propane-sulfonic acid; N-tris (hydroxymethyl)methyl-2-aminoethane sulfonic acid; N-2-hydroxyethylpiperazine-N′-2-ethanesulfonicacid; 4-(2-hydroxyethyl)-l-piperazinepropane sulfonic acid; N-tris (hydroxymethyl)methylglycine; glycinamide; N,N-bis(2-hydroxyethyl)glycine; N-tris (hydroxymethyl)methyl-2-aminopropane sulfonic acid; and N-glycylglycine.
36. The method of claim 35 , wherein the zwitterion is glycine.
37. The method of any one of claims 1 - 3 and 5 - 8 , wherein the product is loaded onto the medium at a concentration of least about 100 mg of product per mL of medium.
38. The method of claim 37 , wherein the product is loaded onto the medium at a concentration of least about 500 mg of product per mL of medium.
39. The method of claim 38 , wherein the product is loaded onto the medium at a concentration of least about 1000 mg of product per mL of medium.
40. The method of any one of claims 1 - 3 and 5 - 8 , wherein the concentration of product in the load fluid is at least about 1 mg of product per mL of load fluid.
41. The method of claim 40 , wherein the concentration of product in the load fluid is at least about 10 mg of product per mL of load fluid.
42. The method of claim 41 , wherein the concentration of product in the load fluid is at least about 100 mg of product per mL of load fluid.
43. The method of any one of claims 1 - 3 and 5 - 6 , wherein at least about 89% of the product is recovered in the column effluent.
44. The method of claim 43 , wherein at least about 95% of the product is recovered in the column effluent.