Use of hydrophobic organic acids to increase hydrophobicity of proteins and protein conjugates

1. A method of making a protein-PEG conjugate salt with increased hydrophobicity, the method comprising:

providing an aqueous protein solution comprising a protein and a pH buffer;

reacting a polyethylene glycol with the protein to form a protein-PEG conjugate; and

protonating an amino group on the protein-PEG conjugate with an organic acid, wherein:

the protonation forms the protein-PEG conjugate salt having a hydrophobic anion that increases the hydrophobicity of the protein-PEG conjugate salt,

protonating the amino group on the protein-PEG conjugate with the organic acid is in a water-immiscible solvent in an organic phase without the presence of water, and

the organic acid comprises pamoic acid, docusate hydrogen, furoic acid, or mixtures thereof.

2. The method of claim 1 , wherein reacting the polyethylene glycol with the protein comprises forming an amine bond, an amide bond, an ester bond, or a disulfide bond between the polyethylene glycol and the protein.

3. The method of claim 1 , wherein reacting the polyethylene glycol with the protein comprises attaching a thiol-reactive polyethylene glycol to a cysteine residue of the protein.

4. The method of claim 1 , wherein the protein is selected from the group consisting of glucagon-like peptide-1, human growth hormone, insulin, parathyroid hormone, a fragment of parathyroid hormone, enfuvirtide, and octreotide.

5. The method of claim 4 , wherein the hydrophobic anion comprises a fatty acid anion, a phospholipid anion, a polystyrene sulfonate anion, or mixtures thereof.

6. The method of claim 5 , wherein the hydrophobic anion comprises the phospholipid anion, and the phospholipid is phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphocholine, or mixtures thereof.

7. The method of claim 1 , wherein the polyethylene glycol comprises methoxy polyethylene glycol aldehyde.

8. The method of claim 1 , wherein the organic acid comprises pamoic acid, and the hydrophobic anion comprises a pamoate anion.

9. The method of claim 1 , wherein the protein-PEG conjugate comprises a monoPEGylated conjugate.

10. The method of claim 1 , wherein a molar ratio of the organic acid to the protein-PEG conjugate is in a range from 3:1 to 8:1.

11. The method of claim 1 , wherein the hydrophobic anion has a water-octanol partition coefficient log P greater than 1.

12. The method of claim 1 , wherein the polyethylene glycol has a molecular weight of 5000 Daltons or less.

13. A method of making a protein salt with increased hydrophobicity, the method comprising:

providing an aqueous protein solution comprising a protein and a pH buffer; and

protonating an amino group on the protein with an organic acid, wherein:

the protonation forms the protein salt having a hydrophobic anion that increases the hydrophobicity of the protein salt,

protonating the amino group on the protein with the organic acid is in a water-immiscible solvent in an organic phase without the presence of water, and

the organic acid comprises pamoic acid, docusate hydrogen, furoic acid, or mixtures thereof.

14. The method of claim 13 , wherein the method does not comprise introducing a polyethylene glycol to the aqueous protein solution.

15. The method of claim 13 , wherein the organic acid comprises pamoic acid, and the hydrophobic anion comprises a pamoate anion.

16. The method of claim 13 , wherein the hydrophobic anion is comprises a fatty acid anion, a phospholipid anion, a polystyrene sulfonate anion, and mixtures thereof.

17. The method of claim 13 , wherein a molar ratio of the organic acid to the protein ranges from 1:1 to 11:1.