Hoffmann-la Roche Inc.

Roche Diagnostics Gmbh

F. Hoffmann-la Roche Ag

Josef Burg

Hartmut Ernst Schurig

Klaus Reichert

Axel Schroth

Axel Wessner

Bernd Hilger

Karl-Heinz Sellinger

Hans-Peter Josel

Wilhelm Tischer

Alfred Engel

Reinhard Franze

Hans Koll

Walter Schneider

Manfred Wozny

Anton Haselbeck

Claus Wallerius

Werner F&uuml;rst

Alexander Wrba

Hartmut Schurig

Thorsten Kaiser

Lars Stiens

Bernhard Hilger

The invention is a method for the purification of mono-PEGylated erythropoietin using two cation exchange chromatography steps wherein the same type of cation exchange material is used in both cation exchange chromatography steps and a method for producing a mono-PEGylated erythropoietin in substantially homogeneous form.

Purification of pegylated polypeptides

The current invention comprises a method for the regeneration of a cation exchange chromatography column.

Chromatographic methods

The invention concerns new EPO compositions with high specific activity which are characterized by a high proportion of N-acetyl-lactosamine units or/and tetraantennary branches in the carbohydrate structure. In addition the invention concerns a process for producing such EPO products.

Erythropoietin with high specific activity

The present invention refers to conjugates of erythropoietin with poly(ethylene glycol) comprising an erythropoietin glycoprotein having an N-terminal &#x3b1;-amino group and having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells and selected from the group consisting of human erythropoietin and analogs thereof which have the sequence of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site; said glycoprotein being covalently linked to one poly(ethylene glycol) group of the formula&#x2014;CO&#x2014;(CH)&#x2014;(OCHCH)&#x2014;ORwherein the &#x2014;CO of the poly(ethylene glycol) group forms an amide bond with said N-terminal &#x3b1;-amino group; and wherein R is lower alkyl; x is 2 or 3; and m is from about 450 to about 1350.

Erythropoietin conjugates

A process for producing erythropoietin which is free of foreign animal proteins except for the proteins of the host cell, wherein DNA coding for EPO is expressed in a eukaryotic host cell and the host cell is cultured in a medium free of natural mammalian proteins. The erythropoietin is chromatographically purified using dye affinity chromatography, chromatography on hydroxyapatite, reversed phase chromatography, and anion exchange chromatography. The resulting preparation contains less than 100 ppm of proteins derived from the host cell, and less than 10 pg of host cell DNA per 83 &mgr;g erythropoietin.

Process for producing erythropoietin containing no animal proteins

Erythropoietin glycoprotein products are disclosed which have the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells. The present conjugates have an increased circulating half-life and plasma residence time, decreased clearance, and increased clinical activity in vivo. In addition, compared with conventional PEG-EPO conjugates, the conjugates of this invention have superior clinical properties. The present invention also includes a method for the treatment of anemia in a human employing the novel erythropoietin glycoprotein products as well as a method for preparing the erythropoietin glycoprotein products.

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Weilheim, Germany

Josef Burg