Methods for quantitating individual antibodies from a mixture

Abstract

The present disclosure relates to, inter alia, a method of quantitating an amount of an antibody molecule from a mixture comprising two or more antibody molecules, comprising separating each of the two or more antibody molecules from the mixture by hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC) and quantitating an amount of each antibody molecule, wherein the molecular weight of each antibody molecule is within 15 kDa of any other antibody molecule in the mixture and either each antibody molecule is different from another antibody molecule in the mixture by more than about 0.25 unit on the Kyte & Doolittle hydropathy scale or each of the antibody molecules when nm alone on HIC-HPLC elutes at distinct run time with little overlap from the other antibody molecules in the mixture, or both.

1 - 22 . (canceled) 
     
     
         23 . A method of quantitating contents of a mixture including a first antibody and a second antibody, the method comprising:
 generating a first standard curve for the first antibody;   separating the first and second antibodies within the mixture using hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC); and   using the first standard curve, quantitating an amount of the first antibody in the mixture.   
     
     
         24 . The method of  claim 23 , wherein the first and second antibodies are monoclonal antibodies. 
     
     
         25 . The method of  claim 24 , wherein one or more of the monoclonal antibodies are human monoclonal antibodies. 
     
     
         26 . The method of  claim 23 , wherein the first antibody and the second antibody have protein sequences that are at least 90% homologous; and/or
 the first antibody and the second antibody have protein structures that are at least 90% homologous, as determined by their protein sequences.   
     
     
         27 . The method of  claim 23 , wherein the first antibody elutes at a first run time during the HIC-HPLC separation, the second antibody elutes at a second run time during the HIC-HPLC separation, and the first and second run times do not overlap. 
     
     
         28 . The method of  claim 23 , further comprising:
 generating a second standard curve for the second antibody; and   using the second standard curve, quantitating an amount of the second antibody in the mixture.   
     
     
         29 . The method of  claim 23 , wherein the mixture further comprises a third antibody, separating the first and second antibodies within using HIC-HPLC includes separating the first, second, and third antibodies using HIC-HPLC, and the method further comprises:
 generating a third standard curve for the third antibody; and   using the third standard curve, quantitating an amount of the third antibody in the mixture.   
     
     
         30 . A method of quantitating contents of a mixture including a first antibody and a second antibody, the method comprising:
 separating the first and second antibodies within the mixture using hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC); and   quantitating an amount of the first antibody in the mixture.   
     
     
         31 . The method of  claim 30 , further comprising quantitating an amount of the second antibody in the mixture. 
     
     
         32 . The method of  claim 30 , wherein the first and second antibodies are variants of each other. 
     
     
         33 . The method of  claim 32 , wherein the second antibody is a deletion variant, an insertion variant, and/or a substitution variant of the first antibody. 
     
     
         34 . The method of  claim 30 , wherein the first and second antibodies bind to the same antigen. 
     
     
         35 . The method of  claim 30 , wherein a difference between an isoelectric point of the first antibody and an isoelectric point of the second antibody is less than or equal to about 0.6. 
     
     
         36 . The method of  claim 30 , further comprising determining a first surface hydrophobicity of the first antibody and determining a second surface hydrophobicity of the second antibody. 
     
     
         37 . The method of  claim 36 , wherein a difference of the first surface hydrophobicity and the second surface hydrophobicity is about 0.25 to about 1.0 unites on the Kyte & Doolittle hydropathy scale. 
     
     
         38 . The method of  claim 36 , wherein the first surface hydrophobicity is calculated based on a protein structure or sequence of the first antibody. 
     
     
         39 . A method of quantitating contents of a mixture including a first antibody and a second antibody, the method comprising:
 separating the first and second antibodies within the mixture using hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC); and   quantitating an amount of the first antibody in the mixture, wherein the mixture is a co-formulated composition.   
     
     
         40 . The method of  claim 39 , wherein the co-formulated composition is configured to treat macular degeneration in a human patient. 
     
     
         41 . The method of  claim 39 , wherein the first and second antibodies bind to the same antigen, and the antigen is a viral protein. 
     
     
         42 . The method of  claim 39 , wherein the first and second antibodies are bispecific antibodies. 
     
     
         43 . The method of  claim 42 , wherein the first antibody binds to two or more proteins selected from the group consisting of: EGFR, CD28, PSMA, PD-1, CD3, CD20, and LAG3.