Methods of analyzing heterodimeric ¿proteins
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsPallavi PRASAD, Jiann-Kae Luo, Svetlana Schussler, Michelle Mazzeo, Michael Perrone
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Abstract
The present disclosure provides methods for analyzing a mixture of proteins using affinity chromatography. In some embodiments, the proteins are heterodimeric bispecific antibodies or antigen-binding fragments thereof. In some embodiments, guanidinium salts are used in the mobile phase of the affinity chromatography. This disclosure provides protein analysis methods that provide superior resolution and lower machine maintenance than other methods.
Claims25
A method of analyzing a heterodimeric protein in a mixture comprising the heterodimeric protein and impurities, the method comprising:
- (a) loading an affinity matrix containing a protein-binding ligand with a sample of the mixture, wherein the heterodimeric protein comprises first and second polypeptides with differing affinities for the protein-binding ligand, and wherein at least one impurity binds the protein-binding ligand with greater affinity than the heterodimeric protein and at least one impurity does not bind the protein-binding ligand or binds with lower affinity than the heterodimeric protein;
- (b) washing the mixture on the affinity matrix with a first mobile phase comprising a guanidinium salt at a concentration of from 50 mM to 300 mM;
- (c) eluting the heterodimeric protein in an eluate from the affinity matrix with a second mobile phase comprising an elution buffer;
- and
- (d) quantitating the heterodimeric protein in the eluate.
(canceled)
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-32. (canceled)
A method of analyzing a heterodimeric protein in a mixture comprising the heterodimeric protein and impurities, the method comprising:
- (a) loading an affinity matrix containing Protein A with a sample of the mixture, wherein the heterodimeric protein comprises first and second polypeptides with differing affinities for Protein A, and wherein at least one impurity binds the Protein A with greater affinity than the heterodimeric protein and at least one impurity does not bind the Protein A or binds the Protein A with lower affinity than the heterodimeric protein;
- (b) washing the mixture on the affinity matrix with a first mobile phase comprising guanidinium chloride and a buffer, wherein the guanidinium chloride is at a concentration of from 50 mM to 250 mM, and wherein the buffer is sodium acetate at a concentration of from 150 mM to 250 mM;
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- (c) eluting the heterodimeric protein from the affinity matrix in an eluate with a second mobile phase comprising an elution buffer, wherein the elution buffer is acetic acid at a concentration of from 450 mM to 550 mM, and wherein the second mobile phase has a pH of 4.0 to 4.5;
- and
- (d) quantitating the heterodimeric protein in the eluate.
(canceled)
A method of analyzing a heterodimeric protein and two types of homodimeric proteins in a mixture comprising the heterodimeric protein and the two types of homodimeric proteins, the method comprising:
- (a) loading a first affinity matrix containing a first protein-binding ligand with a sample of the mixture, wherein the heterodimeric protein comprises first and second polypeptides with differing affinities for the first protein-binding ligand, wherein a first type of homodimeric protein binds the protein-binding ligand with greater affinity than the heterodimeric protein and a second type of homodimeric protein does not bind the protein-binding ligand or binds with lower affinity than the heterodimeric protein, and wherein the first protein-binding ligand is Protein A;
- (b) loading a second affinity matrix containing a second protein-binding ligand with a sample of the mixture, wherein the second type of homodimeric protein binds the second protein-binding ligand with greater affinity than the heterodimeric protein, and wherein the second protein-binding ligand is Protein G;
- (c) washing the mixture on the affinity matrix with a first mobile phase comprising a guanidinium salt at a concentration of from 50 mM to 300 mM and a buffer, wherein the buffer is sodium acetate at a concentration of from 100 to 300 mM sodium acetate;
- (d) eluting the heterodimeric protein and the first type of homodimeric protein from the first affinity matrix with a second mobile phase comprising an elution buffer, wherein the elution buffer comprises acetic acid at a concentration of from 300 mM to 700 mM mixed with the first mobile phase in increasing volumes to decrease pH of the first mobile phase over time;
- (e) eluting the second type of homodimeric protein from the second affinity matrix;
- and
- (f) quantitating the first type of homodimeric protein, the heterodimeric protein, and the second type of homodimeric protein.
-41. (canceled)
Dependencies are inferred from the claim text.