Method for purifying PEGylated erythropoietin
Inventors
Falkenstein, Roberto • Spensberger, Bernhard
Assignees
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Publication Number
US-11993630-B2
Publication Date
2024-05-28
Expiration Date
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.
Core Innovation
The invention relates to a method for purifying a protein comprising erythropoietin and a single poly (ethylene glycol) residue. The method includes 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 chromatography column having a matrix of methacrylate with a sulfopropyl as functional group, with a first solution at a pH of about 2.4 to about 2.7.
After the first low-pH application, the method applies a second solution with an increased pH value with respect to the first solution. The second solution has a pH of about 2.7 to about 3.0 and a conductivity value of about 17 mS/cm to about 19 mS/cm, and is used in combination with applying a solution with increased or increasing conductivity to recover the protein comprising erythropoietin and a single poly (ethylene glycol) residue.
The method uses conductivity behavior and pH conditions to improve purification outcomes for mono-PEGylated erythropoietin. Dependent refinements include constant conductivity and increasing conductivity that is linear or step-wise.
Claims Coverage
The independent claim covers a purification method for erythropoietin conjugated to a single poly(ethylene glycol) residue using a methacrylate/sulfopropyl chromatography material, with a defined initial low-pH solution and a second increased-pH solution combined with specified conductivity behavior. The independent inventive features are further refined by dependent claims that add sequence specificity, PEG residue molecular-weight constraints, conductivity behavior, and scale constraints.
Methacrylate/sulfopropyl chromatography with initial low pH
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.
Increased pH solution with defined conductivity window
Applying a second solution with an increased pH value with respect to the first solution, wherein the second solution has a pH of about 2.7 to about 3.0 and a conductivity value of about 17 mS/cm to about 19 mS/cm.
Conductivity increase for recovery of mono-PEGylated erythropoietin
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.
Overall, the claim coverage centers on purifying erythropoietin with a single poly(ethylene glycol) residue by combining methacrylate/sulfopropyl chromatography under an initial pH of about 2.4 to about 2.7, an intermediate increased-pH solution at about 2.7 to about 3.0 with conductivity about 17 mS/cm to about 19 mS/cm, and recovery by applying increased or increasing conductivity.
Stated Advantages
Improves mono-PEG purity/yield by adding an increased-pH wash prior to conductivity-based elution.
Documented Applications
Not explicitly described in patent.
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