Methods of producing immunoconjugates

Patent metadata

Field	Value
Publication number	US-9920124-B2
Application number	US-201314654384-A
Country	US
Kind code	B2
Filing date	Dec 19, 2013
Priority date	Dec 20, 2012
Publication date	Mar 20, 2018
Grant date	Mar 20, 2018

Abstract

The present invention provides methods of preparing active immunoconjugates, including anti-CD22 immunoconjugates. Suitably, the methods include a fed-batch refolding process and/or column stripping process that result in an increase in yield of the immunoconjugate over other processes that do not utilize the methods.

First claim

What is claimed is:

1. A method of preparing an active immunoconjugate, wherein said immunoconjugate is composed of two polypeptide chains joined by a disulfide bond, the method comprising refolding said immunoconjugate in a fed-batch refolding process in a refold buffer having a pH of 9.5 or less, and purifying the refolded immunoconjugate using a two cycle elution on an ion exchange column, wherein the column is stripped between a first elution and a second elution with a stripping buffer comprising arginine, urea and dithiothreitol (DTT), wherein the stripping buffer comprises 0.25M to about 0.75 M arginine, 7M to about 9 M urea and 9 mM to about 11 mM DTT.

2. The method of claim 1 , wherein an amount of the immunoconjugate recovered from the method of preparation is at least three-hundred % (300%) greater than an amount of the immunoconjugate recovered utilizing a method that does not comprise a fed-batch refolding process and/or purification on an ion exchange column that has been stripped using the stripping buffer.

3. The method of claim 2 , wherein the polypeptide or immunoconjugate comprises an antibody or antigen binding fragment thereof.

4. The method of claim 3 , 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.

5. The method of claim 2 , wherein the polypeptide or immunoconjugate comprises a toxin.

6. The method of claim 5 , wherein the toxin is a Pseudomonas exotoxin, or variant thereof.

7. The method of claim 6 , wherein said Pseudomonas exotoxin, or variant thereof has an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-22.

8. The method of claim 7 , wherein said Pseudomonas exotoxin, or variant thereof has the amino acid sequence of SEQ ID NO:22.

9. The method of claim 3 , wherein said antibody or antigen binding fragment thereof comprises a VH and a VL sequence.

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

11. The method of claim 9 , wherein said VL sequence is selected from the group consisting of SEQ ID NOs: 2, and 12-15.

12. The method of claim 2 , wherein the immunoconjugate comprises an anti-CD22 antibody or antigen binding fragment thereof and a PE or variant thereof.

13. The method of claim 12 , wherein the immunoconjugate is the Moxetumomab pasudotox immunotoxin comprising the VH-PE38 subunit of SEQ ID NO: 1 and the VL subunit of SEQ ID NO:2.

14. The method of claim 2 , wherein the refold buffer has a pH of 9.4.

15. The method of claim 2 , wherein the fed batch process uses an addition rate of about 52 mL of solubilized inclusion bodies per L of refold buffer per hour to about 13 mL solubilized inclusion bodies per L refold buffer per hour.

16. The method of claim 15 , wherein the fed batch process uses an addition rate of about 35 mL of solubilized inclusion bodies per L of refold buffer per hour to about 17 mL solubilized inclusion bodies per L refold buffer per hour.

17. The method of claim 16 , wherein the fed batch process uses an addition rate of about 30 mL of solubilized inclusion bodies per L of refold buffer per hour to about 18 mL solubilized inclusion bodies per L refold buffer per hour.

18. The method of claim 17 , wherein the fed batch process uses an addition rate of about 26 mL of solubilized inclusion bodies per L of refold buffer per hour.

19. The method of claim 15 , wherein the fed batch process occurs over a period of about 2 to about 8 hours.

Assignees

• Medimmune Llc

Inventors

• Hunter Alan
• Linke Thomas
• Pabst Timothy
• Wendeler Michaela
• Wang Xiangyang
• Thompson Christopher
• Xi Guoling
• Fulton Andrew

Classifications

• C07K14/21

  from Pseudomonadaceae (F) · CPC title

• C07K2317/14

  Specific host cells or culture conditions, e.g. components, pH or temperature · CPC title

• A61K39/39591 Primary

  Stabilisation, fragmentation · CPC title

• C07K16/2803

  against the immunoglobulin superfamily · CPC title

• C07K1/1136

  by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents · CPC title