Polypeptide Separation Methods
AssigneeZepteon, Incorporated
InventorsGlen Reed Bolton, Austin Wayne Boesch
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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.
Claims20
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.
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.
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.
Dependencies are inferred from the claim text.