PROTEIN PURIFICATION USING HCIC AND ION EXCHANGE CHROMATOGRAPHY
The present invention provides methods for purifying proteins. In particular, the methods employ a two-step non-affinity chromatography process without the use of an in-process tangential flow filtration step.
1 . A method for purifying a target protein from a mixture, which comprises the target protein and one or more contaminants, comprising:
(a) subjecting the mixture to a cation exchange chromatography step and a hydrophobic charge induction chromatography step, in either order, wherein there is no in-process tangential flow filtration step; and
(b) isolating the target protein.
2 - 3 . (canceled)
4 . The method claim 1 , wherein the target protein is isolated to a purity of about 100 parts per million (ppm) or less of host cell protein and about 10 pg/mg or less of nucleic acids.
5 . The method of claim 1 , which further includes a viral inactivation step.
6 . The method of claim 5 , wherein the viral inactivation step is an in-process step.
7 . The method of claim 1 , wherein the mixture is prepared from a cell culture supernatant.
8 . The method of claim 1 , wherein the cation exchange chromatography is performed at a pH range from 3 to 10.
9 . The method of claim 1 , wherein the cation exchange chromatography is performed at a pH range from 4.0 to 8.0.
10 . The method of claim 1 , wherein the hydrophobic charge induction chromatography step is performed at a pH range from 3 to 10.
11 . The method of claim 1 , wherein the hydrophobic charge induction chromatography is performed using at a pH range from 4.0 to 9.0.
12 . The method of claim 1 , wherein the target protein binds to a cation exchange chromatography column at pH 3 to 9 and at a conductivity range from 0.1 to 40 mS/cm.
13 . The method of claim 1 , wherein the target protein binds to a cation exchange chromatography column at pH 4.0 to 8.0 and at a conductivity from 0.5 to 10 mS/cm.
14 . The method of claim 1 , wherein the target protein binds to a hydrophobic charge induction chromatography column at pH 5 to 10 and at a conductivity range from 0 to 90 mS/cm.
15 . The method of claim 1 , wherein the target protein binds to a hydrophobic charge induction chromatography column at pH 6 to 9 and conductivity range from 2 to 9 mS/cm.
16 . The method of claim 1 , wherein the target protein is bound to the cation exchange chromatography column and the column is washed using a wash buffer having a pH range from 3 to 9 and a conductivity range from 0.1 to 40 mS/cm.
17 . The method of claim 1 , wherein the target protein is bound to a cation exchange chromatography column and the column is washed using a wash buffer having a pH range from 4.0 to 8.0 and a conductivity range from 0.5 to 10 mS/cm.
18 . The method of claim 1 , wherein the target protein is bound to a hydrophobic charge induction chromatography column and the column is washed using a wash buffer having a pH range from 5 to 10 and a conductivity range from 0 to 90 mS/cm.
19 . The method of claim 1 , wherein the target protein is bound to a hydrophobic charge induction chromatography column and the column is washed using a wash buffer having a pH range from 6 to 9 and a conductivity range from 0.1 to 9 mS/cm.
20 . The method of claim 1 , wherein the target protein elutes from the cation exchange chromatography column at a pH range from 3 to 10 and a conductivity range from 0.1 to 40 mS/cm.
21 . The method of claim 1 , wherein the target protein elutes from the cation exchange chromatography column at a pH range from 4.0 to 8.0 and a conductivity range from 5 to 15 mS/cm.
22 . The method of claim 1 , wherein the target protein elutes from the hydrophobic charge induction exchange chromatography column at a pH range from 3.0 to 7.0 and a conductivity range from 0 to 20 mS/cm.
23 . The method of claim 1 , wherein the target protein elutes from the hydrophobic charge induction exchange chromatography column at a pH range from 4.0 to 6.0 and a conductivity range from 0.1 to 2.0 mS/cm.
24 - 29 . (canceled)
30 . The method of claim 1 , wherein the target protein is a monoclonal antibody or antibody fragment.
31 . (canceled)
32 . The method of claim 30 wherein the antibody is a fully human antibody.
33 . The method of claim 30 wherein the antibody is selected from anti-CTLA4 antibody and anti-CD30 antibody.
34 - 36 . (canceled)
37 . The method of claim 1 , further comprising the step of formulating the isolated protein into a pharmaceutical composition.