Process for providing PEGylated protein composition
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Publication Number
US-11518781-B2
Publication Date
2022-12-06
Expiration Date
Abstract
A process for providing a mono-PEGylated protein composition is provided. The process is particularly suitable for providing mono-PEGylated erythropoietin composition. The process comprises subjecting a mixture comprising non-PEGylated, mono-PEGylated and oligo-PEGylated to a hydrophobic interaction chromatography process.
Core Innovation
The invention provides a process for producing a mono-PEGylated protein composition comprising at least about 90% mono-PEGylated protein. A protein mixture comprising non-PEGylated protein, mono-PEGylated protein, and oligo-PEGylated protein is subjected to a two-stage hydrophobic interaction chromatography (HIC) step. The mixture is applied to a first HIC material to provide a first HIC flow-through solution, and then the first HIC flow-through solution is applied to a second HIC material to provide a second HIC flow-through solution.
In the two-stage HIC, the second HIC material is different from the first HIC material. The two-stage HIC conditions are suitable for binding oligo-PEGylated protein to the first HIC material and binding mono-PEGylated protein to the second HIC material. The process then elutes the mono-PEGylated protein from the second HIC material to provide a second HIC eluate, where the second HIC eluate provides the mono-PEGylated protein composition.
The disclosed workflow separates mono-PEGylated protein from the non-PEGylated protein and oligo-PEGylated protein using a first stage that selectively binds oligo-PEGylated protein and a second stage that selectively binds mono-PEGylated protein. The description emphasizes producing high-purity mono-PEGylated compositions, including mono-PEGylated erythropoietin, by using different phenyl-based HIC resins in series under suitable two-stage HIC conditions, followed by elution of the mono-PEGylated fraction from the second stage.
Claims Coverage
The independent claim covers a two-stage HIC process using different first and second HIC materials with two-stage HIC conditions tailored to bind oligo-PEGylated protein in the first stage and bind mono-PEGylated protein in the second stage, followed by elution of the mono-PEGylated fraction. Five inventive features are reflected across the claims.
Two-stage HIC separation of non-, mono-, and oligo-PEGylated proteins using different HIC materials
A process for producing a mono-PEGylated protein composition comprising at least about 90% mono-PEGylated protein, including providing a protein mixture comprising non-PEGylated protein, mono-PEGylated protein and oligo-PEGylated protein; subjecting the mixture to a two-stage HIC step with a first HIC material and then a second HIC material that is different from the first; wherein the two-stage HIC conditions are suitable for binding oligo-PEGylated protein to the first HIC material and binding mono-PEGylated protein to the second HIC material.
Elution of the mono-PEGylated protein from the second HIC material
Eluting the mono-PEGylated protein from the second HIC material to provide a second HIC eluate, wherein the second HIC eluate provides the mono-PEGylated protein composition.
Purity of mono-PEGylated protein composition at least about 99%
The mono-PEGylated protein composition contains at least about 99% mono-PEGylated protein.
Named phenyl HIC resin selection for first and/or second HIC materials
The process uses Phenyl Sepharose HP as the first HIC material and/or Toyopearl Phenyl 650M as the second HIC material.
Linear gradient elution of oligo-PEGylated protein from the first HIC material
Eluting an oligo-PEGylated protein from the first HIC material using a linear gradient elution.
The inventive coverage centers on a two-stage HIC workflow using different HIC materials with conditions that differentially bind oligo-PEGylated protein in the first stage and mono-PEGylated protein in the second stage, followed by elution of the mono-PEGylated protein from the second stage to yield a mono-PEGylated protein composition. Dependent claims refine the process by tightening purity requirements, specifying named phenyl HIC resins, and optionally defining linear gradient elution from the first HIC material.
Stated Advantages
Improved recovery of mono-PEGylated acidic EPO variants versus cation exchange chromatography.
Early recovery and recycling of non-PEGylated protein.
Faster and efficient operation without buffer switching.
Operation under physiological-to-near-physiological pH.
Production of a mono-PEGylated protein composition with high purity, reported at least about 90% and up to about 99% to 100% in the description.
Documented Applications
Producing a mono-PEGylated erythropoietin (EPO) composition, including mono-PEGylated acidic EPO variants.
Production of mono-PEGylated protein compositions associated with marketed erythropoiesis-stimulating agents (Mircera®, Peglntron®, Pegasys®) as referenced in the description.
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