METHODS OF PURIFYING CHARGE-SHIELDED FUSION PROTEINS

1 . A method of purifying a charge-shielded fusion protein from a cell lysate or periplasmic releasate, wherein the charge-shielded fusion protein comprises a biologically active domain and a charge-shielding domain, and wherein the method comprises hydrophobic interaction chromatography as a first chromatography step.

2 . A method for producing a charge-shielded fusion protein from a cell lysate or periplasmic releasate wherein the charge-shielded fusion protein comprises a biologically active domain and a charge-shielding domain, wherein the method comprises

i) culturing cells comprising a nucleic acid encoding the charge-shielded fusion protein; and

ii) purifying the charge-shielded fusion protein, wherein the charge-shielded protein is purified from the cell lysate or periplasmic releasate using hydrophobic interaction chromatography as a first chromatography step.

3 . The method of claim 1 , wherein the charge-shielded fusion protein is at least 45% pure after the first chromatography step.

4 . The method of claim 1 , wherein the method further comprises an anion exchange chromatography.

5 . The method of claim 1 , wherein the method further comprises a cation exchange chromatography.

6 . The method of claim 1 , wherein the method comprises a sequence of chromatography steps comprising in order:

i) hydrophobic interaction chromatography;

ii) anion exchange chromatography; and

iii) cation exchange chromatography.

7 . The method of claim 1 , wherein the biologically active domain is charged at pH of about 7.0, wherein the charge-shielding domain increases the hydrodynamic radius of the protein, and/or wherein the charge-shielding domain does not have a charge at pH of about 7.0.

8 . The method of claim 1 , wherein the molecular weight of the biologically active domain is less than the molecular weight of the charge-shielding domain.

9 . The method of claim 1 , wherein the molecular weight of the charge-shielding domain is between 10 kDa and 60 kDa.

10 - 13 . (canceled)

14 . The method of claim 1 , wherein the charge-shielding domain has a random coil or disordered structure.

15 . The method of claim 1 , wherein the charge-shielding domain is a polypeptide consisting of one or more of alanine, serine and proline residues.

16 . The method of claim 15 , wherein the charge-shielding domain is a polypeptide consisting of proline and alanine residues.

17 . A method for producing a PASylated biologically active fusion protein from a cell lysate or periplasmic releasate comprising

i) culturing cells comprising a nucleic acid encoding the PASylated biologically active protein; and

ii) purifying the PASylated biologically active protein,

wherein the PASylated biologically active protein is purified from the cell lysate or periplasmic releasate using hydrophobic interaction chromatography as a first chromatography step.

18 . A method for purifying a charge-shielded fusion protein comprising a biologically active domain and a charge-shielding domain from a cell lysate or periplasmic releasate, the method comprising the following steps in order

i) applying a load solution comprising the charge-shielded fusion protein to a hydrophobic interaction chromatography column;

ii) applying a wash solution to the hydrophobic interaction chromatography column;

iii) applying an elution solution to the hydrophobic interaction column to elute the charge-shielded protein;

iv) applying the eluted charge-shielded fusion protein in iii) as a load solution to an anion exchange chromatography column;

v) eluting the charge-shielded fusion protein from the anion exchange chromatography column;

vi) applying the eluted charge-shielded fusion protein in v) as a load solution to a cation exchange chromatography column;

vii) applying a wash solution to the cation exchange chromatography column;

viii) applying an elution solution to the cation exchange chromatography column to elute the charge-shielded fusion protein.

19 - 31 . (canceled)

32 . The method of claim 1 , wherein the biologically active domain is an asparaginase subunit.

33 . The method of claim 32 , wherein the asparaginase is selected from the group consisting of an E. coli asparaginase and an Erwinia asparaginase.

34 - 35 . (canceled)

36 . The method of claim 2 , wherein the cell is a bacterial cell.

37 . The method of claim 36 , wherein the cell is an E. coli cell or a Pseudomonas cell.

38 . A charge-shielded protein produced by the method of claim 1 .

39 . A pharmaceutical composition comprising the charge-shielded protein of claim 38 and a pharmaceutically acceptable carrier.

40 . A method of treatment comprising administering a composition comprising the charge-shielded protein of claim 38 to an individual in need thereof.

41 . A composition comprising a PASylated asparaginase, wherein the PASylated asparaginase is at least 45% pure.