Methods of purifying antibodies

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.

1 - 8 . (canceled) 
     
     
         9 . A method of purifying at least one bispecific antibody from a mixture, the method comprising the steps of:
 (a) providing a mixed antibody composition that includes one or more bispecific antibodies and at least one non-intact antibody molecule, where the non-intact antibody molecule comprises one or more antibody components, one or more dimers of an antibody component, one or more antibody fragments, and combinations thereof;   (b) providing a separation means that has a differential affinity between a bispecific antibody and a non-intact antibody molecule;   (c) contacting the separation means with the mixed antibody composition under conditions that allow for differential binding to the separation means by the one or more bispecific antibodies as compared to the binding to the separation means by the non-intact antibody molecule; and   (d) separating the one or more bispecific antibodies from the separation means and retaining the one or more bispecific antibodies.   
     
     
         10 . The method of  claim 9 , wherein the non-intact antibody molecule is a free light chain. 
     
     
         11 . The method of  claim 9 , wherein the separation means is a resin, a membrane, a magnetic bead, a particle or a monolith. 
     
     
         12 . The method of  claim 9 , wherein the separation means is coupled to a ligand having high specificity and affinity for a bispecific antibody molecule. 
     
     
         13 . The method of  claim 12 , wherein the separation means is a mixed mode chromatography resin. 
     
     
         14 . The method of  claim 13 , wherein the separation means is a Mep HyperCel™ resin. 
     
     
         15 . The method of  claim 9 , wherein step (d) comprises eluting the bispecific antibody or combination of bispecific antibodies from the separation means under conditions that allow for preferential detachment of the at least one bispecific antibodies from the separation means as compared to the detachment of the non-intact antibody molecule. 
     
     
         16 . The method of  claim 9 , wherein step (c) comprises contacting the separation means under conditions that allow for the binding of the one or more bispecific antibodies to the separation means but do not allow for the binding of the non-intact antibody molecule to the separation means. 
     
     
         17 . The method of  claim 16 , wherein step (d) comprises eluting the bispecific antibody or combination of bispecific antibodies from the separation means under conditions that allow for detachment of the at least one bispecific antibody. 
     
     
         18 . The method of  claim 16 , wherein step (c) further comprises the step of removing any unbound, non-intact antibody molecules from the composition. 
     
     
         19 . A method of purifying at least one bispecific antibody from a mixture, the method comprising the steps of:
 (a) providing a mixed antibody composition that includes one or more bispecific antibodies and at least one non-intact antibody molecule, where the non-intact antibody molecule comprises one or more antibody components, one or more dimers of an antibody component, one or more antibody fragments, and combinations thereof;   (b) providing a separation means that has a differential affinity between an intact antibody and a non-intact antibody molecule;   (c) contacting the separation means with the mixed antibody composition under conditions that allow for binding to the separation means by the non-intact antibody molecule but do not allow for binding to the separation means by the one or more bispecific antibodies such that the one or more bispecific antibodies remain in an unbound fraction; and   (d) retaining the unbound fraction comprising one or more bispecific antibodies.   
     
     
         20 . The method of  claim 19 , wherein the non-intact antibody molecule is a free light chain. 
     
     
         21 . The method of  claim 19 , wherein the separation means is a resin, a membrane, a magnetic bead, a particle or a monolith. 
     
     
         22 . The method of  claim 19 , wherein the separation means is coupled to a ligand having high specificity and affinity for a non-intact antibody molecule. 
     
     
         23 . The method of  claim 22 , wherein the separation means is a mixed mode chromatography resin. 
     
     
         24 . The method of  claim 23 , wherein the separation means is a Mep HyperCel™ resin. 
     
     
         25 . The method of  claim 15 , wherein the step of eluting comprises an acetate elution buffer at pH 5.0.