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 an antibody from a composition comprising the antibody 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.
2. The method of claim 1 wherein the cation exchange resin comprises sulphopropyl immobilized on agarose.
3. The method of claim 1 wherein the conductivity of the wash buffer increases at a constant rate from the second conductivity to the third conductivity.
4. The method of claim 1 wherein the conductivity of the wash buffer increases at two or more different rates from the second conductivity to the third conductivity.
5. The method of claim 4 wherein the conductivity of the wash buffer increases at a first rate for a first segment of the washing, at a second rate for a second segment of the washing and at a third rate for a third segment of the washing.
6. The method of claim 5 wherein the wash buffer comprises a mixture of equilibration buffer and elution buffer.
7. The method of claim 6 wherein the conductivity of the wash buffer is increased by increasing the proportion of elution buffer in the wash buffer.
8. The method of claim 7 wherein the proportion of elution buffer in the wash buffer increases at a constant rate of about 6% during the first segment, at a constant rate of about 3.5% during the second segment and at a constant rate of about 2% during the third segment.
9. The method of claim 7 wherein the proportion of elution buffer in the wash buffer increases from about 26% to about 54% during the first segment, from about 54% to about 61% during the second segment and from about 61% to about 74% during the second segment.
10. The method of claim 5 wherein the cation exchange material is washed with about 5 column volumes of wash buffer in the first segment, about 2 column volumes of wash buffer in the second segment and about 6 column volumes of wash buffer in the third segment.
11. The method of claim 1 wherein the conductivity of the wash buffer is increased by increasing the proportion of elution buffer in the wash buffer.
12. The method of claim 1 wherein the conductivity of the wash buffer is increased by increasing the salt concentration therein.
13. The method of claim 1 wherein the fixed volume of wash buffer passed over the cation exchange material in step (c) is between about 0.4 column volumes and about 1.0 column volumes.
14. The method of claim 1 further comprising washing the ion exchange material with a regeneration buffer after step (d).