Method for purifying active polypeptides or immunoconjugates

Patent metadata

Field	Value
Publication number	US-9580461-B2
Application number	US-201113813083-A
Country	US
Kind code	B2
Filing date	Jul 27, 2011
Priority date	Jul 30, 2010
Publication date	Feb 28, 2017
Grant date	Feb 28, 2017

Abstract

The present invention provides methods for isolating an active polypeptide or immunoconjugate by purification of a solution containing both the active polypeptide or immunoconjugate and an acidic variant thereof, such as a deamidated variant, using anion exchange chromatography.

First claim

What is claimed is:

1. A method of producing a purified polypeptide from a solution comprising the polypeptide and a deamidated species of the polypeptide, wherein said deamidated species of the polypeptide results in an inhibition of potency of said polypeptide, the method comprising: (a) contacting the polypeptide with an anion exchange (AIEX) chromatography matrix; and (b) eluting the bound polypeptide from the AIEX chromatography matrix with a linear or step salt gradient, thereby separating said polypeptide from deamidated species of said polypeptide and producing a purified polypeptide having less than between about 25% and about 1% deamidated species;
wherein the polypeptide comprises an antibody or antigen binding fragment thereof that binds the cell surface receptor CD22 and a Pseudomonas exotoxin, a cytotoxic fragment of Pseudomonas exotoxin, PE40, PE38, PE35, PE-LR, PE-LR-6X or PE-38.

2. The method of claim 1 , wherein the AIEX matrix contains quaternary amine and tertiary amine ion exchange groups.

3. The method of claim 2 , wherein the AIEX matrix contains a quaternary amino (Q) group.

4. The method of claim 3 , wherein the AIEX matrix is Q sepharose.

5. The method of claim 1 , wherein the polypeptide is eluted with a linear salt gradient that is from about 150 mM NaCl in Tris/HCl, pH 8.0 to about 300 mM NaCl in Tris/HCl, pH 8.0, from about 175 mM NaCl in Tris/HCl, pH 8.0 to about 275 mM NaCl in Tris/HCl, pH 8.0, or from about 192 mM NaCl in Tris/HCl, pH 8.0 to about 245 mM NaCl in Tris/HCl, pH 8.0.

6. The method of claim 1 , wherein the antibody or antigen binding fragment comprises a Fab, a Fab′, a F(ab′) 2 , a Fd, a single chain Fv or scFv, a disulfide linked Fv, a V-NAR domain, an IgNar, an intrabody, an IgGΔCH2, a minibody, a F(ab′) 3 , a tetrabody, a triabody, a diabody, a single-domain antibody, DVD-Ig, Fcab, mAb 2 , a (scFv) 2 , or a scFv-Fc.

7. The method of claim 1 , wherein said Pseudomonas exotoxin has an amino acid sequence selected from the group consisting of SEQ ID NOs:16-22.

8. The method of claim 1 , wherein said antibody or antigen binding fragment thereof comprises a VH and a VL sequence.

9. The method of claim 8 , wherein said VH sequence is selected from the group consisting of SEQ ID NOs: 6-11.

10. The method of claim 8 , wherein said VL sequence is selected from the group consisting of SEQ ID NOs: 2, and 12-15.

11. The method of claim 1 , wherein the polypeptide is the CAT-8015 immunotoxin comprising the V H -PE38 subunit of SEQ ID NO:1 and the V L subunit of SEQ ID NO:2.

12. A composition comprising a purified polypeptide having less than between about 25% and about 1% deamidated species, wherein said polypeptide is purified by the method of claim 1 .

13. A pharmaceutical composition comprising the purified polypeptide of claim 12 and a pharmaceutically acceptable carrier.

14. A formulation comprising the composition of claim 12 and at least one excipient selected from the group consisting of sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium hydroxide and water.

15. A method of modifying the bioactivity of a polypeptide solution comprising a polypeptide and a deamidated species of said polypeptide, the method comprising separating the polypeptide from the deamidated species by linear elution AIEX chromatography; and combining the purified polypeptide and deamidated species of said polypeptide in fixed quantities to obtain the desired bioactivity of the polypeptide solution.

16. The composition of claim 12 , wherein the polypeptide is the CAT-8015 immunotoxin comprising the V H -PE38 subunit of SEQ ID NO:1 and the V L subunit of SEQ ID NO:2.

Assignees

• Linke Thomas
• Wang William K
• Shah Ambarish
• Sathish Hasige
• Hunter Alan
• Thompson Christopher
• Medimmune Llc

Inventors

• Linke Thomas
• Wang William K
• Shah Ambarish
• Sathish Hasige
• Hunter Alan
• Thompson Christopher

Classifications

• A61P39/02

  Antidotes · CPC title

• B01D15/166 Primary

  Fluid composition conditioning, e.g. gradient · CPC title

• B01J41/20

  Anion exchangers for chromatographic processes · CPC title

• A61K39/3955

  against proteinaceous materials, e.g. enzymes, hormones, lymphokines · CPC title

• C07K2317/56

  variable (Fv) region, i.e. VH and/or VL · CPC title