Purification of recombinantly produced interferon

A method of separating the oxidized monomeric isofonn of an interferon (IFN) from one or more undesired isoforms of that IFN in a mixture of recombinantly produced isoforms of the IFN, the method comprising:

• (a) providing the mixture of 1FN isoforms in a first buffer solution;
• (b) providing a chromatography column that is greater than 15 cm in length and packed with an anion exchange resin that is equilibrated with the first or a second buffer solution;
• (c) loading the buffered IFN solution onto the anion exchange column;
• (d) washing the loaded column with a wash solution;
• (e) applying to the washed column a strong elution solution in an amount that is 1 to 10 bed volumes of the column, wherein the strong elution solution comprises a first phosphate concentration of 10 to 30 mM and has a pH of 5.4 to 6.6;
• (f) applying to the column from step (e) a weak elution solution in an amount that is 2 to 20 bed volumes of the column, wherein the weak elution solution comprises a second phosphate concentration that is less than the first phosphate concentration and has a pH of 5.4 to 6.6;
• and
• (g) collecting a plurality of eluate fractions that contain the oxidized IFN monomeric isoform.

A method of separating isoform 1 of interferon alpha-2b (IFN-α2b) from isoform 4 of IFN-α2b in a mixture of recombinantly produced isoforms of IFN-α2b, the method comprising:

• (a) providing a diethylaminoethyl (DEAE) anion exchange chromatography column that is at least about 20 cm in length and equilibrated with a buffer solution which consists essentially of 10 mM Tris and a pH of 8.0;
• (b) loading the IFN-α2b mixture onto the DEAE column in a loading buffer that consists essentially of 10 mM Tris, 40 mM NaCI, and has a pH of from 7.5 to 8.0, wherein the IFN-α2b mixture is loaded at a flow rate of 2 cm per minute;
• (c) washing the loaded column with 3 bed volumes of a wash solution at a flow rate of 2 cm per minute, wherein the wash solution consists essentially of 10 mM Tris HCI and 13 mM NaCI, and has a pH of 8.0;
• (d) applying to the washed column 6 bed volumes of a strong elution solution at a flow rate of 1 cm per minute, wherein the strong elution solution consists essentially of 17.5 mM sodium phosphate and has a pH of 5.85;
• (e) applying to the column from step (d) 15 bed volumes of a weak elution solution at a flow rate of 1 cm per minute, wherein the weak elution solution consists essentially of 5 mM sodium phosphate and has a pH of 5.85;
• and
• (f) collecting a plurality of eluate fractions that contain isoform 1.

A method of separating a desired isoform of an interferon (IFN) from one or more undesired isoforms of that IFN in a mixture of recombinantly produced isoforms of the IFN, the method comprising:

• (a) providing a chromatography column that is at least about 20 cm in length and packed with a diethylaminoethyl anion exchange resin that is equilibrated with a buffer solution, wherein the buffer solution consists essentially of about 10 mM Tris and a pH of about 8.0;
• (b) applying the IFN isoform mixture to the anion exchange column in a loading solution of about 10 mM Tris and about 40 mM NaCl and which has a pH of about 8.0;
• (c) washing the column from step (b) with about 3 bed volumes of a wash solution, wherein the wash solution consists essentially of about 10 mM Tris HC1 and about 13 mM NaCl, and has a pH of about 8.0;
• (d) applying to the washed column about 6 bed volumes of a strong elution solution at a flow rate of 0.5 to 2.5 cm/min, wherein the strong elution solution has a first phosphate concentration of 15 to 25 mM and a pH of between 5.7 and 6.1;
• (e) applying to the column from step (d) about 15 bed volumes of a weak elution solution at a flow rate of 0.5 to 2.5 cm/min, wherein the weak elution solution has a second phosphate concentration that is less than the first phosphate concentration and has a pH of between 5.7 and 6.1;
• and
• (f) collecting a plurality of eluate fractions that contain the desired IFN isoform;
• and
• (g) combining the collected eluate fractions in which the amount of the undesired IFN isoforms is less than a desired purity criteria.