Process for providing PEGylated protein composition

1. A process for producing a mono-PEGylated protein composition comprising at least about 90% mono-PEGylated protein, comprising the steps of:

a) providing a protein mixture comprising non-PEGylated protein, mono-PEGylated protein and oligo-PEGylated protein;

b) subjecting the protein mixture to a two-stage hydrophobic interaction chromatography (HIC) step, comprising:

applying the protein mixture to a first HIC material to provide a first HIC flow-through solution; and

applying the first HIC flow-through solution to a second HIC material to provide a second HIC flow-through solution,

wherein the second HIC material is different from the first HIC material; and

wherein the two-stage HIC step is performed under two-stage HIC conditions, which 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; and

c) 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.

2. The process according to claim 1 , wherein the protein is a hormone, a cytokine, an enzyme or an antibody.

3. The process according to claim 1 , wherein the protein is erythropoietin.

4. The process according to claim 1 , wherein the first HIC material and the second HIC material are directly connected in series.

5. The process according to claim 1 , wherein under the two-stage HIC conditions the first HIC material does not bind non-PEGylated protein or mono-PEGylated protein, and wherein the second HIC material does not bind non-PEGylated protein.

6. The process according to claim 1 , wherein in comparative HIC elution chromatograms the first HIC material and the second HIC material have well-resolved peaks for mono-PEGylated protein.

7. The process according to claim 1 , wherein

a) the first HIC material has substantially the same selectivity as Phenyl Sepharose HP under the two-stage HIC conditions; and

b) the second HIC material has substantially the same selectivity as Toyopearl Phenyl 650M under the two-stage HIC conditions.

8. The process according to claim 1 , wherein

a) the first HIC material is Phenyl Sepharose HP; and/or

b) the second HIC material is Toyopearl Phenyl 650M.

9. The process according to claim 1 , comprising the further step of:

d) eluting the oligo-PEGylated protein from the first HIC material to provide a first HIC eluate.

10. The process according to claim 9 , wherein eluting the oligo-PEGylated protein from the first HIC material comprises a linear gradient elution.

11. The process according to claim 1 , wherein

i. the two-stage HIC conditions are at a conductivity of about 54-55 mS/cm; and/or

ii. eluting the mono-PEGylated protein from the second HIC material is at a conductivity of about 40-41 mS/cm; and/or

iii. eluting the oligo-PEGylated protein from the first HIC material is carried out by linear gradient from about 54-55 mS/cm to about 1-5 mS.cm

and wherein optionally the protein is EPO.

12. The process according to claim 1 , wherein the process is carried out using a mixture of buffer A and buffer B, wherein buffer A comprises 25 mM HEPES, pH 7.5, 500 mM Na 2 SO 4 , and wherein buffer B comprises 25 mM HEPES, pH 7.5, and wherein

i. the two stage HIC conditions are at about 13.5% buffer B; and/or

ii. eluting the mono-PEGylated protein from the second HIC material is at about 40% buffer B; and/or

iii. eluting the oligo-PEGylated protein from the first HIC material is carried out by linear gradient from about 13.5% to about 80% buffer B

and wherein optionally the protein is EPO.

13. The process according to claim 1 , wherein elution of the mono-PEGylated protein from the second HIC material comprises a step elution, optionally followed by a gradient elution.

14. The process according to claim 1 , wherein the mono-PEGylated protein composition comprises at least about 99% mono-PEGylated protein.

15. The process according to claim 1 , wherein the protein mixture comprises at least 10% oligo-PEGylated protein.

16. The process according to claim 1 , wherein mono-PEGylated protein comprises a PEG residue having a molecular weight of at least about 20 kDa.

17. The process according to claim 1 , wherein the mono-PEGylated protein composition is a pharmaceutical composition, the process further comprising formulating the second HIC eluate with a pharmaceutical excipient.