Methods of purifying bispecific antibodies

Patent metadata

Field	Value
Publication number	US-10457749-B2
Application number	US-201615068916-A
Country	US
Kind code	B2
Filing date	Mar 14, 2016
Priority date	Mar 13, 2015
Publication date	Oct 29, 2019
Grant date	Oct 29, 2019

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) performing protein A chromatography on a biological sample to produce a mixed antibody composition comprising of (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) contacting the mixed antibody composition with a separation means 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, wherein the separation means comprises a combination of a hydrophobic interaction chromatography resin and a mix mode chromatography resin; and
(c) 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 3 , wherein the mixed mode chromatography resin comprises a methacrylic polymer with tryptophan as a ligand.

5. The method of claim 4 , wherein the hydrophobic interaction chromatography resin comprises a methacrylic polymer and a butyl ligand.

6. 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.

7. The method of claim 6 , wherein the mixed mode chromatography resin comprises a methacrylic polymer with tryptophan as a ligand.

8. The method of claim 6 , wherein the hydrophobic interaction chromatography resin comprises a methacrylic polymer and a butyl ligand.

9. The method of claim 1 , wherein the mixed mode chromatography resin comprises a methacrylic polymer with tryptophan as a ligand.

10. The method of claim 1 , wherein the hydrophobic interaction chromatography resin comprises a methacrylic polymer and a butyl ligand.

11. The method of claim 1 , wherein the biological sample is cell supernatant.

12. The method of claim 11 , 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.

13. The method of claim 1 , wherein the separation means comprises use of a hydrophobic interaction chromatography resin followed by use of a mixed mode chromatography resin.

14. The method of claim 13 , wherein the hydrophobic interaction chromatography resin comprises a methacrylic polymer and a butyl ligand, and wherein the mixed mode chromatography resin comprises a methacrylic polymer with tryptophan as a ligand.

15. The method of claim 1 , wherein the separation means comprises use of a mixed mode chromatography resin followed by use of a hydrophobic interaction chromatography resin.

16. The method of claim 15 , wherein the mixed mode chromatography resin comprises a methacrylic polymer with tryptophan as a ligand, and wherein the hydrophobic interaction chromatography resin comprises a methacrylic polymer and a butyl ligand.

Assignees

• Novimmune Sa

Inventors

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

Classifications

• C07K16/2866

  against receptors for cytokines, lymphokines, interferons · CPC title

• C07K16/468 Primary

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

• C07K16/249

  Interferons · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K1/22

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title