Methods for purifying protein
AssigneeImmunex Corporation
InventorsGanesh Vedantham, Clayton A. Brooks III, Joanne M. Reeder, Andrew M. Goetze
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Abstract
A method is provided for separating a protein from one or more other proteins using hydroxyapatite chromatography in which the protein does not bind to hydroxyapatite but the other protein(s) does. In some embodiments, a second protein affixed to a solid support has been used previously to purify the protein by affinity chromatography, and small amounts of the second protein are introduced in the sample during this process. The protein being purified can comprise at least one constant antibody immunoglobulin domain. The second protein can bind to proteins comprising such a domain.
Claims30
A method for purifying a protein from a sample comprising the protein and at least one protein contaminant comprising subjecting the sample to hydroxyapatite chromatography,
- wherein the protein is separated from at least one protein contaminant by hydroxyapatite chromatography in a solution in which hydroxyapatite chromatography is performed,
- wherein the majority of molecules of the protein are recovered in the flow through and wash,
- wherein the protein has been previously purified by affinity chromatography using a second protein affixed to a solid support as an absorbent, and
- wherein the second protein binds to a constant antibody immunoglobulin domain.
A method for purifying a recombinant fusion protein from sample comprising the recombinant fusion protein and at least one protein contaminant comprising subjecting the sample to hydroxyapatite chromatography,
- wherein the recombinant fusion protein comprises an FC portion of an antibody and part or all of a non-antibody protein, and
- wherein the recombinant fusion protein has been previously purified by affinity chromatography using a second protein affixed to a solid support as an absorbent.
A method for purifying TNFR:FC comprising subjecting a sample comprising TNFR:FC and at least one protein contaminant to hydroxyapatite chromatography under conditions wherein the majority of molecules of TNFR:FC are recovered in the flow through and wash, wherein at least one protein contaminant is separated from TNFR:FC.
A method for separating a protein from host cell proteins comprising subjecting a sample comprising the protein and at least one host cell protein to hydroxyapatite chromatography,
- whereby at least one host cell protein is separated from the protein,
- wherein the protein comprises a constant antibody immunoglobulin domain,
- wherein the majority of molecules of the protein are recovered in the flow through and wash, and
- wherein both the protein and the host cell protein have been secreted into a culture medium by cultured animal cells.
A purified preparation of TNFR:FC, wherein the preparation comprises less than 3 parts per million Protein A.
Dependencies are inferred from the claim text.