Methods of increasing protein purity using protein A based chromatography
AssigneeEMD Millipore Corporation
InventorsNanying Bian, Melissa Holstein
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Abstract
The present invention provides methods for increasing purity of an Fc-containing protein by removing protein aggregates during the Protein A chromatography step used during the purification of the Fc-containing protein.
Claims21
A method of reducing the level of protein aggregates in an elution pool containing an Fc-containing protein, the method comprising the steps of:
- (a) providing a sample comprising an Fc-containing protein and protein aggregates;
- (b) contacting the sample with a protein A ligand immobilized onto a solid support, wherein the protein A ligand consists of one or more C-based domains of Protein A, such that the Fc-region containing protein binds to the protein A ligand;
- (c) obtaining an elution pool containing the Fc-containing protein using a pH gradient method employing a high pH buffer and a low pH buffer;
- wherein at least 30% of the protein aggregates are removed prior to the elution of the Fc-containing protein in addition to protein aggregates that are removed after the elution of the Fc-containing protein, thereby reducing the level of protein aggregates in the elution pool.
A method of reducing the level of protein aggregates in an elution pool containing an Fc-containing protein, the method comprising the steps of:
- (d) providing a sample comprising an Fc-containing protein and protein aggregates;
- (e) contacting the sample with a protein A ligand immobilized onto a solid support, wherein the protein A ligand consists of one or more C-based domains of Protein A, such that the Fc-region containing protein binds to the protein A ligand;
- (f) obtaining an elution pool containing the Fc-containing protein using a pH step method employing two or more buffers used sequentially in the order of descending pH values,
- wherein at least 30% of the protein aggregates are removed prior to the elution of the Fe-containing protein in addition to protein aggregates that are removed after the elution of the Fc-containing protein, thereby reducing the level of protein aggregates in the elution pool.
A method of eluting Fc-containing protein aggregates before elution of Fc-containing monomer protein from an affinity ligand, the method comprising:
- (a) contacting a sample comprising a mixture of Fc-containing protein aggregates and an Fc-containing protein with an affinity ligand immobilized on a solid support, the affinity ligand consisting of one or more C-based domains of Protein A and which binds Fc-containing protein and Fc-containing protein aggregates;
- (b) applying a pH gradient elution buffer from a high pH to a low pH, thereby eluting at least 20% of aggregates from the mixture of aggregates and monomer bound to the affinity ligand on the solid support before elution of Fc-containing monomer protein from the affinity ligand.
A method of eluting Fc-containing protein aggregates before elution of Fc-containing monomer protein from an affinity ligand, the method comprising:
- (a) contacting a sample comprising a mixture of Fc-containing protein aggregates and an Fc-containing protein with an affinity ligand immobilized on a solid support, the affinity ligand consisting of one or more C-based domains of Protein A and which binds Fc-containing protein and Fc-containing protein aggregates;
- (b) applying a pH step elution using two or more buffers with sequentially descending pH values from a high pH to a low pH, thereby eluting at least 20% of aggregates from the mixture of aggregates and monomer bound to the affinity ligand on the solid support before elution of Fc-containing monomer protein from the affinity ligand.
Dependencies are inferred from the claim text.