Polypeptide Separation Methods

Abstract

The present disclosure provides methods and compositions for separating polypeptide glycoforms using a medium that includes an Fc receptor. In certain embodiments, a medium includes an Fc receptor which comprises an extracellular portion of an Fc gamma RIII receptor.

We claim: 
     
         1 . A method of enriching for afucosylated immunoglobulin C (IgG), comprising:
 (a) providing a medium comprising an immunoglobulin Fc receptor, wherein the Fc receptor comprises an extracellular domain of an Fc gamma RIII receptor or Fc gamma RIV receptor, wherein the Fc gamma RIII receptor comprises an amino acid sequence having at least 85% identity to an amino acid sequence as set forth in residues 21-208 of SEQ ID NO: 1, wherein F176 is changed to V176, or an amino acid sequence as set forth in residues 20-208 of SEQ ID NO: 2;   (b) contacting the medium with a load fluid, wherein the load fluid comprises IgG, under conditions in which the IgG binds to the immunoglobulin Fc receptor, wherein the IgG comprises an immunoglobulin Fc receptor binding moiety that is monomeric, wherein the load fluid comprises a plurality of glycoforms of the IgG, and wherein the Fc receptor preferentially binds to monomeric afucosylated glycoforms of the IgG with an affinity that is at least 2 fold greater than the affinity with which the Fc receptor binds to fucosylated glycoforms of the IgG;   (c) contacting the medium with an elution solution under conditions in which the bound IgG elutes from the medium; and   (d) recovering the bound IgG that elutes from the medium, thereby producing an eluate, wherein the percentage of afucosylated glycoforms of the IgG present in the eluate is at least 2-fold greater than the percentage of afucosylated glycoforms of the IgG present in the load fluid prior to being contacted with the medium, thereby enriching for afucosylated IgG.   
     
     
         2 . The method of  claim 1 , wherein the load fluid comprises serum selected from the group consisting of: serum of an immunized subject, serum of a subject who has been exposed to an infectious agent, serum of a subject who has developed immunity to an infectious agent, and serum of a naive subject. 
     
     
         3 . The method of  claim 2 , wherein the serum is from a subject who has been exposed to an infectious agent or from a subject who has developed immunity to an infectious agent. 
     
     
         4 . The method of  claim 3 , wherein a biological activity of the IgG present in the eluate is altered relative to the activity of the IgG in the load fluid. 
     
     
         5 . The method of  claim 4 , wherein the biological activity comprises therapeutic efficacy of the IgG against the infectious agent, and wherein the therapeutic efficacy of the IgG against the infectious agent is increased. 
     
     
         6 . The method of  claim 5 , wherein the infectious agent is a virus. 
     
     
         7 . The method of  claim 6 , wherein the virus is an RNA virus. 
     
     
         8 . The method of  claim 4 , wherein the biological activity is antibody-dependent cell-mediated cytotoxicity (ADCC), and wherein ADCC is increased. 
     
     
         9 . The method of  claim 4 , wherein the biological activity is antibody-dependent cellular phagocytosis (ADCP), and wherein ADCP is increased. 
     
     
         10 . The method of  claim 1 , wherein the load fluid comprises IgG1 IgG2, IgG3, and IgG4. 
     
     
         11 . The method of  claim 10 , wherein the bound IgG that elutes from the medium is IgG1 and/or IgG3. 
     
     
         12 . The method of  claim 1 , wherein the Fc receptor comprises an extracellular domain of an Fc gamma RIII receptor comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino add sequence as set forth in residues 21-208 of SEQ ID NO: 1, wherein F176 is changed to V176, or an amino add sequence as set forth in residues 20-208 of SEQ ID NO: 2. 
     
     
         13 . The method of  claim 1 , wherein the Fc receptor is a full-length Fc gamma RIII receptor or full-length Fc gamma RIV receptor. 
     
     
         14 . The method of  claim 5 , further comprising producing a pharmaceutical composition from the IgG in the eluate. 
     
     
         15 . A method of treating a viral infection, comprising administering to a subject in need thereof the pharmaceutical composition of  claim 14 . 
     
     
         16 . A method of enriching for one or more immunoglobulin G (IgG) glycoforms, comprising:
 (a) providing a medium comprising an immunoglobulin Fc receptor, wherein the Fc receptor comprises an extracellular domain of an Fc gamma RIII receptor or Fc gamma RIV receptor, wherein the Fc gamma RIII receptor comprises an amino add sequence having at feast 85% identity to an amino add sequence as set forth in residues 21-208 of SEQ ID NO: 1, wherein F176 is changed to V178, or an amino add sequence as set forth in residues 20-208 of SEQ ID NO: 2;   (b) contacting the medium with a load fluid, wherein the load fluid comprises IgG, under conditions in which the IgG binds to the immunoglobulin Fc receptor, wherein the IgG comprises an immunoglobulin Fc receptor binding moiety, wherein the load fluid comprises a plurality of glycoforms of the IgG, and wherein the Fc receptor preferentially binds one or more of the glycoforms of the IgG;   (c) contacting the medium with an elution solution under conditions in which the bound IgG elutes from the medium; and   (d) recovering the bound IgG that elutes from the medium, thereby producing an eluate comprising one or more IgG glycoforms.   
     
     
         17 . The method of  claim 16 , wherein the IgG is IgG1 and/or IgG3. 
     
     
         18 . A medium comprising an Fc receptor coupled to a solid support, wherein the Fc receptor comprises an extracellular binding domain of an Fc gamma RIII receptor or Fc gamma RIV receptor, and wherein the medium maximizes antibody binding capacity of the Fc receptor. 
     
     
         19 . The medium of  claim 18 , wherein the medium is equilibrated for about 1-2 hours with a solution that has a pH between about 24. 
     
     
         20 . The medium of  claim 18 , wherein the Fc receptor comprises an extracellular binding domain of an Fc gamma RIII receptor comprising an amino add sequence as set forth in residues 21-192 of SEQ ID NO: 1, wherein F176 is changed to V176, or an amino add sequence as set forth in residues 20-208 SEQ ID NO: 2.