# US 11,993,630 B2 · Method for purifying PEGylated erythropoietin > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,993,630 B2 - Document ID: US11993630B2 - Status: Granted - Kind: B2 - Application number: 17/145,653 - Filed: 2021-01-11 - Published: 2021-05-27 - Granted: 2024-05-28 - Earliest claimed: 2016-07-15 - Assignees: Hoffmann-La Roche Inc. - Applicants: Hoffmann-La Roche Inc. - Inventors: Roberto Falkenstein; Bernhard Spensberger - Web page: https://patentgrep.com/patent/US11993630B2 ## CPC - C07K 1/18: Ion-exchange chromatography - B01D 15/362: {Cation-exchange} - C07K 14/505: Erythropoietin [EPO] - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28) ## Abstract Herein is reported a method for the purification of a protein comprising erythropoietin and a single poly (ethylene glycol) residue from reaction by-products or not reacted starting material by a cation exchange chromatography method. It has been found that by employing a cation exchange Toyopearl® SP-650 chromatography material and employing a second wash step with an increased pH value compared to the first wash step a fusion protein of erythropoietin and a single poly (ethylene glycol) residue can be obtained in a single step with high purity and yield and suitability for large scale applications. ## Claims ### Claim 1 A method for purifying a protein, which comprises erythropoietin and a single poly (ethylene glycol) residue, comprising the following steps: a) applying a solution comprising a mixture of erythropoietin and conjugates of erythropoietin and poly (ethylene glycol) with one or more poly (ethylene glycol) residues per erythropoietin molecule to a column, comprising a chromatography material that has a matrix of methacrylate with a sulfopropyl as functional group, to which a first solution with a pH of about 2.4 to about 2.7 has been applied, b) applying a second solution with an increased pH value with respect to the first solution, c) applying a solution with increased or increasing conductivity to the column and thereby recovering the protein, which comprises erythropoietin and a single poly (ethylene glycol) residue, wherein the second solution with an increased pH value has a pH of about 2.7 to about 3.0 and has a conductivity value of about 17 mS/cm to about 19 mS/cm. ### Claim 2 (depends on 1) The method according to claim 1, characterized in that the method further comprises the step of re-applying the first solution with a pH of about 2.4 to about 2.7 after step b) and before step c). ### Claim 3 (depends on 1) The method according to claim 1, characterized in that the second solution with an increased pH value is a solution with a constant conductivity value. ### Claim 4 (depends on 1) The method according to claim 1, characterized in that the second solution with an increased pH value and the first solution with a pH of about 2.4 to about 2.7 have about the same constant conductivity value. ### Claim 5 (depends on 1) The method according to claim 1, characterized in that the second solution with an increased pH value and/or the first solution with a pH of about 2.4 to about 2.7 have a constant conductivity value of about 17 mS/cm to about 19 mS/cm. ### Claim 6 (depends on 1) The method according to claim 1, characterized in that the solution comprising a mixture of erythropoietin and conjugates of erythropoietin and poly (ethylene glycol) with one or more poly (ethylene glycol) residues per erythropoietin molecule is not adjusted to a conductivity value of about 19 mS/cm. ### Claim 7 (depends on 1) The method according to claim 1, characterized in that the solution with increasing conductivity is a solution with increasing sodium chloride concentration. ### Claim 8 (depends on 1) The method according to claim 1, characterized in that the solution with increasing conductivity has a conductivity that increases linearly or step-wise. ### Claim 9 (depends on 1) The method according to claim 1, characterized in that the method is used in large scale protein preparations wherein the chromatography column of step a) has a diameter of at least 30 cm. ### Claim 10 (depends on 1) The method according to claim 1, characterized in that the erythropoietin is human erythropoietin. ### Claim 11 (depends on 10) The method according to claim 10, characterized in that the human erythropoietin has the amino acid sequence of SEQ ID NO: 01 or SEQ ID NO: 02. ### Claim 12 (depends on 1) The method according to claim 1, characterized in that the single poly (ethylene glycol) residue has a molecular weight of from 20 kDa to 40 kDa.