Methods of Engineering Surface Charge for Bispecific Antibody Production

Abstract

The present disclosure relates to methods of modifying the isoelectric point of an antibody. The method includes providing an antibody comprising a first polypeptide comprising a heavy chain variable region and a second polypeptide comprising heavy chain variable region and substituting, in at least one of the first and second polypeptides of the antibody, one or more amino acid residues of the heavy chain variable region (VH) at positions 7, 9, 11, 14, 41, 70, 74, 82a, 84, and 113, according to the Kabat numbering system, wherein the substituting increases or decreases the isoelectric point of the antibody.

What is claimed: 
     
         1 . A method of enhancing separation of a bispecific antibody from its two parental antibodies, said method comprising:
 providing a first and second parental antibody, each parental antibody comprising a heavy chain variable region;   substituting, in at least one of the first and second parental antibodies, one or more amino acid residues in the heavy chain variable region (V H ) at positions 7, 9, 11, 14, 41, 70, 74, 82a, 84, and 113, according to the Kabat numbering system, wherein said substituting increases or decreases the isoelectric point of the first parental antibody relative to the second parental antibody;   producing the bispecific antibody from the two parental antibodies after said substituting;   separating the produced bispecific antibody from its two parental antibodies, wherein said separating is enhanced as a result of said substituting.   
     
     
         2 . The method of  claim 1 , wherein when the one or more amino acid residues of the V H  at positions 7, 9, 11, 14, 41, 74, 84, and 113 (Kabat numbering) of the first parental antibody are neutrally charged amino acid residues, said substituting comprises:
 exchanging said one or more neutrally charged amino acid residues with one or more positively charged amino acid residues to increase the isoelectric point of the first parental antibody.   
     
     
         3 . The method of  claim 1 , wherein when the one or more amino acid residues of the V H  at positions 7, 9, 11, 14, 41, 74, 84, and 113 (Kabat numbering) of the first parental antibody are negatively charged amino acid residues, said substituting comprises:
 exchanging said one or more negatively charged amino acid residues with one or more neutral or positively charged amino acid residues to increase the isoelectric point of the first parental antibody.   
     
     
         4 . The method of  claim 1 , wherein when the one or more amino acid residues of the V H  at positions 9, 70, 74, 82a, and 84 (Kabat numbering) of the first parental antibody are neutrally charged amino acid residues, said substituting comprises:
 exchanging said one or more neutrally charged amino acid residues with one or more negatively charged amino acid residues to decrease the isoelectric point of the first parental antibody.   
     
     
         5 . The method of  claim 1 , wherein when the one or more amino acid residues of the V H  at positions 9, 70, 74, 82a, and 84 (Kabat numbering) of the first parental antibody are positively charged amino acid residues, said substituting comprises:
 exchanging said one or more positively charged amino acid residues with one or more neutral or negatively charged amino acid residues to decrease the isoelectric point of the first parental antibody.   
     
     
         6 . The method of  claim 1 , wherein said substituting comprises:
 exchanging the V H  amino acid residue at position 74 (Kabat numbering) in the first parental antibody with a negatively charged amino acid residue to decrease the isoelectric point of the first parental antibody.   
     
     
         7 . The method of  claim 1 , wherein said substituting comprises:
 exchanging the V H  amino acid residue at position 82a (Kabat numbering) in the first parental antibody with a negatively charged amino acid residue to decrease the isoelectric point of the first parental antibody.   
     
     
         8 . The method of  claim 1 , wherein said substituting comprises:
 exchanging the V H  amino acid residue at position 84 (Kabat numbering) in the first parental antibody with a negatively charged amino acid residue to decrease the isoelectric point of the first parental antibody.   
     
     
         9 . The method of  claim 1 , wherein said substituting comprises:
 exchanging the V H  amino acid residue at at least two of the positions selected from positions 74, 82a, and 84 (Kabat numbering) in the first parental antibody with a negatively charged amino acid residue to decrease the isoelectric point of the first parental antibody.   
     
     
         10 . The method of  claim 1 , wherein said substituting comprises:
 exchanging the V H  amino acid residue at all three positions 74, 82a, and 84 (Kabat numbering) in the first parental antibody with a negatively charged amino acid residue to decrease the isoelectric point of the first parental antibody.   
     
     
         11 . A multi-specific antibody comprising:
 a first polypeptide comprising a heavy chain variable region and   a second polypeptide comprising a heavy chain variable region, wherein the isoelectric point of the first polypeptide is less than the isoelectric point of the second polypeptide and one or more amino acid residues at positions 9, 70, 74, 82a, and 84 (Kabat numbering) of the heavy chain variable region of the first polypeptide comprise a neutral or negatively charged amino acid residue and one or more amino acid residues at the corresponding positions of the heavy chain variable region of the second polypeptide comprise a differentially charged amino acid residue when compared to the first polypeptide.   
     
     
         12 . The multi-specific antibody of  claim 11 , wherein one or more amino acid residues at positions 7, 9, 11, 14, 41, 74, 84, and 113 of the heavy chain variable region of the second polypeptide comprise a neutral or positively charged amino acid residue, and one or more amino acid residues at the corresponding positions of the heavy chain variable region of the first polypeptide comprise a differentially charged amino acid residue when compared to the second polypeptide. 
     
     
         13 . The multi-specific antibody of  claim 11 , wherein the neutral or negatively charged amino acid residues at one or more positions 9, 70, 74, 82a, and 84 (Kabat numbering) are amino acid substitutions. 
     
     
         14 . A multi-specific antibody comprising:
 a first polypeptide comprising a heavy chain variable region and   a second polypeptide comprising a heavy chain variable region, wherein the isoelectric point of the first polypeptide is higher than the isoelectric point of the second polypeptide and one or more amino acid residues at positions 7, 9, 11, 14, 41, 74, 84, and 113 (Kabat numbering) of the heavy chain variable region of the first polypeptide comprise a neutral or positively charged amino acid residue and one or more amino acid residues at the corresponding positions of the heavy chain variable region of the second polypeptide comprise a differentially charged amino acid residue when compared to the first polypeptide.   
     
     
         15 . The multi-specific antibody of  claim 14 , wherein one or more amino acid residues at positions 9, 70, 74, 82a, and 84 (Kabat numbering) of the heavy chain variable region of the second polypeptide comprise a neutral or negatively charged amino acid residue and one or more amino acid residues at the corresponding positions of the heavy chain variable region of the first polypeptide comprise a differentially charged amino acid residue when compared to the second polypeptide. 
     
     
         16 . The multi-specific antibody of  claim 14 , wherein the neutral or positively charged amino acid residues at one or more positions 7, 9, 11, 14, 41, 74, 84, and 113 (Kabat numbering) are amino acid substitutions. 
     
     
         17 . The multi-specific antibody of any one of  claims 11 - 16 , wherein said multi-specific antibody is a bi-specific antibody.