Methods of purifying bispecific antibodies

Patent metadata

Field	Value
Publication number	US-2016264685-A1
Application number	US-201615068916-A
Country	US
Kind code	A1
Filing date	Mar 14, 2016
Priority date	Mar 13, 2015
Publication date	Sep 15, 2016
Grant date	—

Abstract

The invention relates to the purification of bispecific antibodies carrying a different specificity for each binding site of the immunoglobulin molecule from a mixture of monospecific antibodies. The bispecific antibodies are composed of a single heavy chain and two different light chains, one containing a Kappa constant domain and the other a Lambda constant domain. This invention in particular relates to the isolation of these bispecific antibodies from mixtures that contain monospecific antibodies having two Kappa light chains or portions thereof and monospecific antibodies having two Lambda light chains or portions thereof. The invention also provides the methods of efficiently purifying these bispecific antibodies.

First claim

What is claimed is:

1 . A method of purifying a bispecific antibody from a mixture of antibodies, the method comprising the steps of:
(a) providing a mixed antibody composition that comprises (i) at least one bispecific antibody with a different specificity in each combining site and two copies of a single heavy chain polypeptide, a first light chain with a kappa constant region, and a second light chain with a lambda constant region (κλ-body); (ii) at least one monospecific antibody having two lambda light chains or portions thereof (λ mono-Ab); and (iii) at least one monospecific antibody having two kappa light chains or portions thereof (κ mono-Ab); (b) providing a separation means; (c) contacting the separation means with the mixed antibody composition under conditions that allow for differential binding to the separation means by the κλ-body as compared to the binding to the separation means by the κ mono-Ab and the λ mono-Ab; and (d) eluting the κλ-body, the κ mono-Ab, and the λ mono-Ab from the separation means under conditions that allow for preferential detachment of the κλ-body from the separation means as compared to detachment of κ mono-Ab and of the λ mono-Ab from the separation means.

2 . The method of claim 1 , wherein the separation means is a resin, a membrane, a magnetic bead, a particle or a monolith.

3 . The method of claim 1 , wherein the binding conditions comprise a variation in pH level, salt level, or both pH level and salt level.

4 . The method of claim 1 , wherein the elution conditions comprise a step variation in pH level, salt level, both pH level and salt level, Hofmeister ion level, both pH and Hofmeister ion level, buffer concentration, buffer composition, both buffer concentration and composition, and combinations thereof.

5 . The method of claim 1 , wherein the separation means is a mixed mode chromatography resin.

6 . The method of claim 5 , wherein the separation means is a TOYOPEARL MX-Trp 650M resin.

7 . The method of claim 4 , wherein the separation means is a mixed mode chromatography resin.

8 . The method of claim 7 , wherein the separation means is a TOYOPEARL MX-Trp 650M resin.

9 . The method of claim 1 , wherein the separation means is a hydrophobic interaction chromatography resin.

10 . The method of claim 9 , wherein the separation means is a TOYOPEARL Butyl 600M resin.

11 . The method of claim 4 , wherein the separation means is a hydrophobic interaction chromatography resin.

12 . The method of claim 11 , wherein the separation means is a TOYOPEARL Butyl 600M resin.

13 . The method of claim 1 , wherein step (a) comprises performing affinity chromatography on a biological sample to provide the mixed antibody composition.

14 . The method of claim 13 , wherein the biological sample is cell supernatant.

15 . The method of claim 14 , wherein the cell is transfected with a κλ bispecific expression vector comprising one γ1 heavy chain cDNA sequence, one κ light chain cDNA sequence, and one λ cDNA sequence.

16 . The method of claim 13 , wherein the affinity chromatography is Protein A chromatography.

17 . The method of claim 13 , wherein the affinity chromatography is not Protein A chromatography.

18 . The method of claim 13 , wherein the separation means comprises a combination of a hydrophobic interaction chromatography resin and a mixed mode chromatography resin.

19 . The method of claim 18 , wherein the separation means comprises use of a hydrophobic interaction chromatography resin followed by use of a mixed mode chromatography resin.

20 . The method of claim 19 , wherein the hydrophobic interaction chromatography resin comprises a TOYOPEARL Butyl 600M resin, and wherein the mixed mode chromatography resin comprises a TOYOPEARL MX-Trp 650M resin.

21 . The method of claim 18 , wherein the separation means comprises use of a mixed mode chromatography resin followed by use of a hydrophobic interaction chromatography resin.

22 . The method of claim 21 , wherein the mixed mode chromatography resin comprises a TOYOPEARL MX-Trp 650M resin, and wherein the hydrophobic interaction chromatography resin comprises a TOYOPEARL Butyl 600M resin.

Assignees

• Novimmune Sa

Inventors

• Fouque Nicolas
• Depoisier Jean François
• Wilson Keith
• Vajda Judith
• Müller Egbert
• Dabre Romain

Classifications

• C07K2317/32

  specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor · CPC title

• C07K16/468 Primary

  Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies · CPC title

• C07K1/36

  by a combination of two or more processes of different types · CPC title

• C07K16/2866

  against receptors for cytokines, lymphokines, interferons · CPC title

• C07K1/20

  Partition-, reverse-phase or hydrophobic interaction chromatography · CPC title