METHODS OF PURIFYING PROTEINS FROM EGG WHITE
The invention provides methods of purifying proteins, e.g., cytokines, from egg whites.
1 . A method of purifying a heterologous cytokine that is expressed in egg whites, the method comprising:
acidifying an egg white preparation comprising the heterologous cytokine;
performing a first and a second ion exchange chromatography; and
performing chromatography based on hydrophobic interaction wherein the cytokine is eluted in an IPA containing solution, thereby purifying the heterologous cytokine.
2 . The method of claim 1 wherein the heterologous cytokine is granulocyte colony-stimulating factor.
3 . The method of claim 1 wherein the heterologous cytokine is interferon-α.
4 . The method of claim 1 wherein the IPA containing solution comprises TFA.
5 . The method of claim 1 wherein the first and the second ion exchange chromatographies are cation exchange chromatography.
6 . The method of claim 5 wherein the second cation exchange chromatography is at a different pH than the first cation exchange chromatography.
7 . The method of claim 1 wherein the chromatography based on a hydrophobic interaction is a reversed phase chromatography.
8 . The method of claim 1 wherein the chromatography based on a hydrophobic interaction is a hydrophobic interaction chromatography.
9 . The method of claim 1 wherein the egg white preparation is acidified to a pH in the range of about 2.0 to about 6.5.
10 . The method of claim 9 wherein the egg white preparation is acidified to a pH in the range of about 4.0 to about 6.0.
11 . The method of claim 10 wherein the egg white preparation is acidified to a pH of about 5.0.
12 . A method of purifying a heterologous protein from an egg white preparation comprising the heterologous protein, the method comprising:
acidifying the egg white preparation to a pH of about 4.0 to about 6.0;
performing a first cation exchange chromatography;
performing a second cation exchange chromatography, wherein the second cation exchange chromatography is at a lower pH than the first cation exchange chromatography; and
performing chromatography based on hydrophobic interaction wherein the heterologous protein is eluted in an IPA containing solution; thereby purifying the heterologous protein from the egg white preparation.
13 . The method of claim 12 wherein the heterologous protein is a cytokine.
14 . The method of claim 12 wherein the heterologous protein is selected from the group consisting of granulocyte colony-stimulating factor (GCSF), GM-CSF, interferon-α, interferon-β, and erythropoietin.
15 . The method of claim 12 wherein the chromatography based on hydrophobic interaction is a reversed phase chromatography.
16 . The method of claim 12 wherein the chromatography based on hydrophobic interaction is a hydrophobic interaction chromatography.
17 . The method of claim 12 wherein the first cation exchange chromatography is at a pH of about 5.0 and the second cation exchange chromatography is at a pH of about 4.0.
18 . The method of claim 17 further wherein the hydrophobic interaction chromatography is a reversed phase chromatography.
19 . The method of claim 18 further wherein the heterologous protein is granulocyte colony-stimulating factor (GCSF).
20 . The method of claim 18 wherein the IPA containing solution comprises TFA.