SINGLE UNIT CHROMATOGRAPHY ANTIBODY PURIFICATION

1 . Method for the purification of antibodies from a protein mixture produced in a bioreactor, at least comprising the steps of intermediate purification and polishing, wherein the intermediate purification and polishing steps comprise serial in-line anion exchange chromatography (AEX) and mixed-mode chromatography (MiMo) both in flow-through mode, wherein the AEX step yields as a flow-through fraction a separation mixture containing antibodies, wherein the separation mixture is subjected to a step without intermediate storage, yielding as a flow through fraction a purified antibody preparation, and wherein the purified antibody preparation is subjected to at least one further purification step, wherein the separation mixture prior to the MiMo step is supplemented with an adequate amount of a suitable adjusting solution in order to adjust the pH and/or conductivity and/or concentration or type of specific ionic components for removal of impurities from the antibodies in the step.

2 . Method according to claim 1 wherein anion exchange chromatography and mixed mode chromatography take place in two separate devices which are serially connected.

3 . Method according to claim 1 wherein the serial in-line AEX and MiMo are performed as a single unit operation.

4 . Method according to claim 1 wherein the separation mixture prior to MiMo is supplemented with an adequate amount salt or a combination of salts.

5 . Method according to claim 4 wherein the separation mixture prior to MiMo is supplemented with an adequate amount of ammonium sulfate, sodium sulfate, potassium sulfate, ammonium phosphate, sodium phosphate, potassium phosphate, potassium chloride and sodium chloride.

6 . Method according to claim 1 wherein the separation mixture prior to MiMo chromatography is supplemented with an adequate amount of an acidic solution.

7 . Method according to claim 6 wherein the separation mixture prior to MiMo chromatography is supplemented with an adequate amount of a solution containing citric acid (or its monobasic or dibasic sodium or potassium salts), phosphoric acid (or its monobasic or dibasic sodium or potassium salts), acetic acid, hydrochloric acid or sulfuric acid.

8 . Method according to claim 1 wherein the separation mixture prior to mixed mode chromatography is supplemented with an adequate amount of an alkaline solution.

9 . Method according to claim 8 wherein the separation mixture prior to MiMo chromatography is supplemented with an adequate amount of a solution containing sodium or potassium hydroxide, (or its mono or di basic sodium or potassium salts) or tris(hydroxymethyl)aminomethane

10 . A single operational unit which can be used in a method according to claim 1 comprising both an anion exchange chromatography part and a mixed mode chromatography part, which are serially connected, wherein the outlet of the anion exchange chromatography part is connected to the inlet of the mixed mode chromatography part, wherein the unit comprises an inlet at the upstream end of the anion exchange chromatography part and an outlet at the downstream end of the mixed mode chromatography part and wherein the unit also comprises an inlet between the anion exchange chromatography part and the mixed mode chromatography part.