Use of hydrophobic organic acids to increase hydrophobicity of proteins and protein conjugates
View Patent ↗Examples may include a microsphere. The microsphere may include a biodegradable polymer. Furthermore, the microsphere may include a protein mixture selected from the group consisting of a protein-polyethylene glycol conjugate, the protein-polyethylene glycol conjugate and the hydrophobic anion of the organic acid, a protein and the hydrophobic anion of the organic acid, and combinations thereof. Examples may also include a method of making the microspheres. The method may further include reacting a polyethylene glycol with a protein to form a protein-PEG conjugate. In addition, the method may include protonating an amino group on the protein-PEG conjugate with a hydrophobic organic acid to form the protein-PEG conjugate salt. Furthermore, the method may include mixing the protein-PEG conjugate salt in an organic solvent with a biodegradable polymer. The method may also include emulsifying the mixture. Additionally, the method may include hardening the emulsified mixture into the controlled-release microspheres.
1. A microsphere comprising:
a biodegradable polymer selected from the group consisting of a polylactide; a polyglycolide; a poly(d,l-lactide-co-glycolide); a polycaprolactone; a polyorthoester; a copolymer of a polyester and a polyether; and a copolymer of polylactide and polyethylene glycol; and
a protein mixture selected from the group consisting of a protein-polyethylene glycol conjugate and a hydrophobic anion of an organic acid, a protein and the hydrophobic anion of the organic acid, and combinations thereof, wherein the organic acid comprises pamoic acid, docusate hydrogen, furoic acid, or mixtures thereof;
and wherein the microsphere is formed by a process comprising:
adding the organic acid to the protein-polyethylene glycol conjugate or the protein in a water-immiscible solvent in an organic phase to form a water-immiscible solution, wherein the addition occurs without the presence of water.
2. The microsphere of claim 1 , wherein the protein of the protein-polyethylene glycol conjugate or the protein comprises human growth hormone.
3. The microsphere of claim 1 , wherein the protein of the protein-polyethylene glycol conjugate or the protein comprises glucagon-like peptide-1.
4. The microsphere of claim 3 , further comprising an insulin-polyethylene glycol conjugate.
5. The microsphere of claim 1 , wherein the protein of the protein-polyethylene glycol conjugate or the protein is selected from the group consisting of insulin, parathyroid hormone, a fragment of parathyroid hormone, enfuvirtide, and octreotide.
6. The microsphere of claim 1 , wherein:
the protein mixture comprises the protein-polyethylene glycol conjugate, and
the polyethylene glycol of the protein-polyethylene glycol conjugate comprises methoxy polyethylene glycol.
7. The microsphere of claim 1 , wherein:
the protein mixture comprises the hydrophobic anion of the organic acid, and
the organic acid comprises pamoic acid, and the hydrophobic anion comprises a pamoate anion.
8. The microsphere of claim 1 , wherein:
the protein mixture comprises the protein-polyethylene glycol conjugate and the hydrophobic anion of the organic acid, and
a molar ratio of the hydrophobic anion of the organic acid to the protein-polyethylene glycol conjugate ranges from 1:1 to 11:1.
9. The microsphere of claim 1 , wherein:
the protein mixture comprises the protein-polyethylene glycol conjugate, and
the protein-polyethylene glycol conjugate comprises a monoPEGylated conjugate.
10. The microsphere of claim 1 , wherein the biodegradable polymer comprises poly(d, l-lactide-co-glycolide).
11. The microsphere of claim 1 , wherein the biodegradable polymer has a molecular weight from 7,000 Da to 17,000 Da.
12. A method of making controlled-release microspheres containing a protein-PEG conjugate salt; 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;
protonating an amino group on the protein-PEG conjugate with a hydrophobic organic acid in a water-immiscible solvent in an organic phase without the presence of water to form a water-immiscible solution, wherein the protonation forms the protein-PEG conjugate salt having a hydrophobic anion;
mixing the protein-PEG conjugate salt in an organic solvent with a biodegradable polymer to form a mixture, wherein:
the hydrophobic anion of the protein-PEG conjugate salt increases the solubility of the protein-PEG conjugate salt in the organic solvent,
the biodegradable polymer is selected from the group consisting of a polylactide; a polyglycolide; a poly(d,l-lactide-co-glycolide); a polycaprolactone; a polyorthoester; a copolymer of a polyester and a polyether; and a copolymer of polylactide and polyethylene glycol, and the organic acid comprises pamoic acid, docusate hydrogen, furoic acid, or mixtures thereof;
emulsifying the mixture of the protein-PEG conjugate salt and the biodegradable polymer in an aqueous solution to form an emulsified mixture; and
hardening the emulsified mixture of the protein-PEG conjugate salt and the biodegradable polymer into the controlled-release microspheres.
13. The method of claim 12 , wherein the hydrophobic organic acid comprises pamoic acid.
14. The microsphere of claim 1 , wherein:
the protein mixture comprises the protein-polyethylene glycol conjugate and the hydrophobic anion of the organic acid, and
a molar ratio of the hydrophobic anion of the organic acid to the protein-polyethylene glycol conjugate ranges from 3:1 to 8:1.
15. The microsphere of claim 1 , wherein the hydrophobic anion has a water-octanol partition coefficient log P greater than 1.
16. The microsphere of claim 1 , wherein the process further comprises protonating at least one amino group on the protein or the protein-polyethylene glycol conjugate.