Methods of purifying antibodies

Patent metadata

Field	Value
Publication number	US-10047144-B2
Application number	US-201213655955-A
Country	US
Kind code	B2
Filing date	Oct 19, 2012
Priority date	Oct 19, 2011
Publication date	Aug 14, 2018
Grant date	Aug 14, 2018

Abstract

The invention provides methods of purifying antibodies using various antibody-specific purification media to rapidly and efficiently separate mixtures of antibodies, antibody fragments and/or antibody components to isolate a desired antibody product from the mixture. The invention relates to the purification of bispecific monoclonal antibodies carrying a different specificity for each binding site of the immunoglobulin molecule, e.g., antibodies composed of a single heavy chain and two different light chains, one containing a Kappa constant domain and the other a Lambda constant domain, including antibodies of different specificities that share a common heavy chain. The invention also provides the methods of efficiently purifying intact antibodies by separating the intact antibody from non-intact antibodies including free light chains.

First claim

What is claimed is:

1. A method of purifying a bispecific antibody consisting of two copies of a single heavy chain polypeptide and a first light chain comprising a kappa constant region and a second light chain comprising a lambda constant region (κλ-bodies), the method consisting of the steps:
(a) providing a mixed antibody composition comprising κλ-bodies; one or more monospecific monoclonal antibodies having two lambda light chains (λ-MAb); and one or more monospecific monoclonal antibodies having two kappa light chains (κ-MAb);
(b) providing a separation means that has specific affinity for a kappa light chain constant region or a lambda light chain constant region;
(c) contacting the separation means with the mixed antibody composition under conditions that allow for the mixed antibody composition to specifically bind the separation means;
(d) washing the separation means to elute impurities;
(e) eluting the κλ-bodies under conditions that allow for preferential detachment of the κλ-bodies thereby purifying the κλ-bodies.

2. The method of claim 1 , wherein the separation means having specific affinity for kappa light chain constant region is
a resin, a membrane, a magnetic bead, a particle or a monolith coupled to a ligand having specific affinity for the kappa light chain constant region; and the separation means having specific affinity for lambda light chain constant region is
a resin, a membrane, a magnetic bead, a particle or a monolith coupled to a ligand having specific affinity for the lambda light chain constant region.

3. The method of claim 1 , wherein the elution conditions comprise a step variation in the pH level.

4. The method of claim 1 , wherein the elution conditions comprise a variation of the concentration of an amino acid in the composition.

5. The method of claim 4 , wherein the amino acid is arginine, histidine, proline, phenylalanine, tyrosine, tryptophan and/or glycine.

6. A single step affinity chromatography method of purifying a bispecific antibody consisting of two copies of a single heavy chain polypeptide and a first light chain comprising a kappa constant region and a second light chain comprising a lambda constant region (κλ-bodies), the method consisting of:
(a) providing a mixed antibody composition comprising κλ-bodies; one or more monospecific monoclonal antibodies having two lambda light chains (λ-MAb); and one or more monospecific monoclonal antibodies having two kappa light chains (κ-MAb);
(b) providing a separation means, wherein the separation means is an anti-kappa light chain antibody, or an anti-lambda light chain antibody coupled to a solid support;
(c) contacting the separation means with the mixed antibody composition under conditions that allow for the mixed antibody composition to specifically bind the separation means;
(d) washing the separation means to elute impurities with a wash solution having a pH of about 7.0 or higher; and
(e) eluting specifically the κλ-bodies with an elution solution having a pH of about 3.0, thereby purifying the κλ-bodies.

7. The method of claim 6 , wherein the solid support is a resin, a membrane, a magnetic bead, a particle or a monolith.

8. The method of claim 6 , wherein the solid support is a highly cross-linked agarose.

Assignees

• Novimmune Sa
• Novimmune Sa

Inventors

• Elson Greg
• Fouque Nicolas
• Depoisier Jean-Francois
• Fischer Nicolas
• Magistrelli Giovanni

Classifications

• C07K16/248 Primary

  IL-6 · CPC title

• C07K16/2866

  against receptors for cytokines, lymphokines, interferons · CPC title

• C07K16/468

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

• C07K2317/52

  Constant or Fc region; Isotype · CPC title

• C07K16/249

  Interferons · CPC title