Abstract
Processes for producing biocompatible and biodegradable protein microspheres with controlled porosity and microsphere sizes are disclosed. Generally, the processes are accomplished as follows: (a) dissolving a source of protein in a mildly acidic or basic aqueous medium, in the presence or absence of a biomodifying agent, (b) adding a cross-linking reagent to the dissolved protein molecules in an amount effective subsequently to cross-link the protein molecules without gelation; (c) adding a water soluble surfactant to modify the surface of the partially cross-linked protein molecules, (d) adding a water soluble organic desolubilizer to desolubilize the protein molecules to allow cross-linkage of nearby protein molecules into water-insoluble microspheres. Alternatively, the cross-linking step of (b) is employed after desolvation of the microspheres. Incorporation of the biomodifying agent, if any, can be achieved by prior bonding to the protein molecules, addition during the...
Filing date: Aug 7, 1989
Issue date: Dec 3, 1991
Inventor: Richard C. K. Yen
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What is claimed is:
1. A process for manufacturing protein microspheres, comprising:
- a) dissolving protein molecules in an aqueous buffer solution to form a protein solution;
- b) adding a surfactant;
- c) adding a desolvating agent to the admixture of protein solution and surfactant to produce a turbid mixture comprising substantially monodispersed protein microspheres; and
- d) adding a cross-linking agent to the turbid mixture formed in step c).
2. The process described in claim 1 wherein said protein molecules have at least one biological element attached thereto.
3. The process described in claim 1 additionally comprising adding at least one biological element to said protein solution prior to adding said desolvating agent.
4. The process described in claim 1 additionally comprising adding at least one biological element to said turbid mixture prior to adding said cross-linking agent and resulting in attachment of biological element to said protein microspheres.
5. The process described in claim 1 additionally comprising adding at least one biological element subsequent to the addition of said cross-linking agent and resulting in attachment of biological element to said protein microspheres.
6. The process described in claims 2, 3, 4 or 5 wherein the biological element is chosen from the group consisting of the following: alkaloids, amino acids, antibiotics, carbohydrates, carcinogens, immunoglobulins or globulins, halogenated compounds, hormones, lipids, nucleotides, polypeptides, porphyrins, steroids, vitamins, lecithins, metal sulfides, metal halides, metal oxides, antifungal compounds, enzymes, and chemotherapeutic agents.
7. The process described in claim 1 wherein said protein molecules are chosen from any one or more of the group consisting of albumin, collagen, hemoglobin, and immunoglobulin.
8. The process described in claim 7 wherein the concentration of protein molecules just prior to the addition of desolvating agent is within the range of approximately 0.225-62.50 mg/ml.
9. The process described in claim 7 wherein said protein molecules comprise albumin.
10. The process described in claim 9 wherein the concentration of albumin just prior to the addition of desolvating agent is within the range of approximately 9.00-20.00 mg/ml.
11. The process described in claim 10 wherein the concentration of albumin just prior to the addition of desolvating agent is 15 mg/ml within an accuracy of plus or minus 10%.
12. The process described in claim 7 wherein said protein molecules are hemoglobin.
13. The process described in claim 12 wherein the concentration of hemoglobin just prior to the addition of desolvating agent is 3.7 mg/ml within an accuracy of plus or minus 20%.
14. The process described in claim 1 wherein said aqueous buffer solution is maintained between 0 and 42 degrees Centigrade.
15. The process described in claim 1 wherein the pH of said aqueous buffer solution is maintained between 5 and 9.
16. The process described in claim 1 wherein the concentration of said surfactant is approximately 0.1 to 50 mg/ml of said aqueous buffer solution.
17. The process described in claim 11 wherein said surfactant is a detergent.
18. The process described in claim 1 wherein said surfactant is sodium lauryl sulfate.
19. The process described in claim 1 wherein said desolvating agent is an alcohol.
20. The process described in claim 19 wherein said alcohol is chosen from the group consisting of methanol, ethanol, propanol, and butanol.
21. The process described in claim 11 wherein said cross-linking agent is a polyaldehyde which is added in an amount in the range of 0.002 to 10.0 volume per 100 volume of said aqueous buffer solution.
22. The process described in claim 21 wherein polyaldehyde is selected from the group consisting of glutaraldehyde, polyglutaraldehyde, and polyacrotein.
23. The process described in claim 1 additionally comprising the step of removing said desolvating agent and other unreacted or partially-reacted soluble material from said protein microspheres.
24. The process described in claim 23 wherein said step of removing desolvating agent is chosen from one or more of the following methods: dialysis, centrifugation and washing, gradient centrifugation, gel-filtration, electrophoresis, column chromatography, gradient centrifugation, thin-layer chromatography, hollow-fiber ultrafiltration, and tangential filtration.