Fabrication of protein-encapsulating microgels
The present invention provides methods for fabricating protein-encapsulating microgels using hydrocarbon-in-fluorocarbon emulsions. The non-aqueous emulsion-based microgel fabrication methods can be used for the encapsulation of a wide range of proteins and peptides, including antibodies and antibody-fusion proteins, for therapeutic use with ease of administration.
1. A method of producing microparticles comprising:
(a) combining a polyethylene glycol-amine polymer, a polyethylene glycol-N-hydroxysuccinimide polymer, a polyethylene glycol (PEG) polymer, and a powder including a vascular endothelial growth factor-Trap (VEGF-Trap) protein with dichloromethane to form a dispersed phase suspension;
(b) adding said dispersed phase suspension to a continuous phase solution, wherein said continuous phase solution includes perfluorotripentylamine and a triblock copolymer perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether (PFPE-PEG-PFPE), to form a combined dispersed phase suspension and continuous phase solution;
(c) applying blending forces to said combined dispersed phase suspension and continuous phase solution to form a non-aqueous emulsion having multiple dichloromethane droplets including said polyethylene glycol-amine polymer, said polyethylene glycol-N-hydroxysuccinimide polymer and said powder including VEGF-Trap in the perfluorotripentylamine; and
(d) removing the dichloromethane and the perfluorotripentylamine from said non-aqueous emulsion to form isolated microparticles.
2. The method of claim 1 , wherein the molar ratio of said amine to said N-hydroxysuccinimide is between 1:1 and 1:2.
3. The method of claim 1 , wherein said polyethylene glycol-amine polymer and/or said polyethylene glycol-N-hydroxysuccinimide polymer is a 4-armed or an 8-armed compound.
4. The method of claim 1 , wherein said VEGF-Trap protein is a truncated form of VEGF-Trap protein.
5. The method of claim 1 , wherein said VEGF-Trap protein is aflibercept.
6. The method of claim 1 , wherein said isolated microparticles have a diameter between 1·m and 200·m as measured by imaging analysis.
7. The method of claim 1 , wherein said powder including a VEGF-Trap protein is micronized by spray-drying, electrospray drying, reversible precipitation, spray freezing, microtemplating, or a combination thereof.
8. The method of claim 1 , wherein said blending forces comprise homogenization, vortexing, sonication, cavitation, agitation, or a combination thereof.
9. The method of claim 1 , wherein said microparticles are sustained release microparticles.
10. The method of claim 1 , wherein a concentration of said powder including a VEGF-Trap protein in said dispersed phase suspension is between 1.0% and 30% w/v.
11. The method of claim 1 , wherein a total concentration of said polyethylene glycol-amine polymer and said polyethylene glycol-N-hydroxysuccinimide polymer in said dispersed phase suspension is between 5.0% and 35% w/v.
12. The method of claim 1 , wherein a concentration of said PFPE-PEG-PFPE in said continuous phase solution is between 0.1% and 5.0% w/v.
13. The method of claim 1 , further comprising suspending said isolated microparticles in a pharmaceutically acceptable formulation.
14. The method of claim 13 , wherein said formulation comprises pH buffered saline, an aqueous solution, or a non-aqueous solution.
15. The method of claim 1 , wherein said powder including a VEGF-Trap protein further comprises at least one excipient.
16. A microparticle made by the method of claim 1 .
17. The method of claim 1 , wherein a concentration of said PEG polymer in said dispersed phase suspension is between 5.0% and 35% w/v.