Protein purification
AssigneeGenentech, Inc.
InventorsJefferson C. Emery, Paul J. McDonald, Rhona O'Leary
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Abstract
A method for purifying a polypeptide by ion exchange chromatography is described in which a gradient wash is used to resolve a polypeptide of interest from one or more contaminants.
Claims46
A method for purifying a polypeptide from a composition comprising the polypeptide and contaminants, which method comprises the sequential steps of:
- (a) loading the composition onto an ion exchange resin with an equilibration buffer having a first salt concentration;
- (b) washing the ion exchange resin with a wash buffer until a predetermined protein concentration is measured in the flowthrough, wherein the salt concentration of the wash buffer increases from an initial, second salt concentration that is greater than the salt concentration of the equilibration buffer, to a final, third salt concentration;
- (c) passing a fixed volume of wash buffer at the final, third salt concentration over the cation exchange resin;
- and
- (d) eluting the polypeptide from the ion exchange resin with elution buffer that has a salt concentration that is greater than the final salt concentration of the wash buffer.
The method of claim 23 wherein the pH of the equilibration buffer, wash buffer and elution buffer is approximately 5.5.
A method for purifying an antibody from a composition comprising the polypeptide and a contaminant, which method comprises the following steps performed sequentially:
- (a) binding the antibody to a cation exchange material with an equilibration buffer at a first conductivity;
- (b) washing the cation exchange material with a wash buffer, wherein the conductivity of the wash buffer increases from a second conductivity that is higher than the first conductivity to a third conductivity during the washing;
- (c) passing a fixed volume of wash buffer at the third conductivity over the cation exchange material;
- and
- (d) eluting the antibody from the cation exchange material with an elution buffer at a fourth conductivity that is higher than the third conductivity.
A method for purifying an antibody from a composition comprising the antibody and a contaminant, which method comprises the following steps performed sequentially:
- (a) loading the composition onto a cation exchange material;
- (b) washing the cation exchange material with a wash buffer with a conductivity that increases at a first rate from a first conductivity to a second conductivity, at a second rate from the second conductivity to a third conductivity and at a third rate from the third conductivity to a fourth conductivity;
- and
- (c) eluting the antibody from the ion exchange material, wherein the amount of antibody in the composition loaded onto the cation exchange material is from about 15 mg to about 45 mg of the antibody per ml of cation exchange material.
A method for purifying a polypeptide from a composition comprising the polypeptide and a contaminant, which method comprises the following steps performed sequentially:
- (a) loading the composition onto an ion exchange material;
- (b) washing the cation exchange material with wash buffer using a multi-slope gradient until a predetermined protein concentration is measured in the flowthrough;
- and
- (c) eluting the polypeptide from the ion exchange material.
Dependencies are inferred from the claim text.