Protein purification
View Patent ↗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.
1. 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 ion exchange material with wash buffer using a multi-slope gradient, wherein the multi-slope gradient comprises two or more segments of linear salt gradients with different slopes wherein the slope is greater in the first segment than in any additional segments, the increase in the salt concentration of the wash buffer is greater in the first segment of the multi-slope gradient wash than in subsequent segments, and each segment of the multi-slope wash ends when a predetermined polypeptide concentration is measured in the flowthrough, wherein each segment of the multi-slope gradient has a progressively shallower slope; and
(c) eluting the polypeptide from the ion exchange material.
2. The method of claim 1 , additionally comprising the step between steps (b) and (c) of washing the column with from 0.4 to 1 column volumes of wash buffer.
3. The method of claim 2 wherein the wash buffer has the composition of the wash buffer at the end of step (b).
4. The method of claim 1 , 2 , or 3 , further comprising subjecting the composition comprising the polypeptide to one or more further purification steps so as to obtain a homogenous preparation of the polypeptide.
5. The method of claim 1 , wherein the polypeptide is an antibody.
6. The method of claim 5 , wherein the antibody is an anti-HER2 antibody.
7. The method of claim 6 , wherein the anti-HER2 antibody comprises the light chain amino acid sequence of SEQ ID NO: 1 and the heavy chain amino acid sequence of SEQ ID NO: 2.
8. The method of claim 7 , wherein the antibody is rhuMAb HER2 comprising the light chain amino acid sequence of SEQ ID NO: 1, and the heavy chain amino acid sequence of SEQ ID NO: 2, and wherein the contaminants comprise a deamidated variant having Asn30 in CDR1 of either or both of the light chain variable regions (VL) converted to aspartate.
9. The method of claim 7 or 8 , wherein the multi-slope gradient comprises three segments.
10. The method of claim 7 or 8 , wherein the wash buffer comprises a mixture of equilibration buffer and elution buffer, and wherein the increase in the salt concentration of the wash buffer during step (b) is achieved by increasing the proportion of elution buffer in the wash buffer.
11. The method of claim 7 or 8 , wherein the amount of antibody in the composition loaded onto the ion exchange resin is from 15 mg to 45 mg per ml of ion exchange resin.