STABILIZED PROTEIN CRYSTALS FORMULATIONS CONTAINING THEM AND METHODS OF MAKING THEM

• US 20020045582A1
• Filed: 08/10/1999
• Published: 04/18/2002
• Est. Priority Date: 12/31/1997
• Status: Active Grant

• Current Assignee: Ajinomoto Althea, Inc. (Ajinomoto Co., Inc.)
• Original Assignee: Altus Biologics, Inc.
• Inventors: MARGOLIN, ALEXEY L., SHENOY, BHAMI C., KHALAF, NAZAR K., RAKESTRAW, SCOTT L., CLAIR, NANCY L. ST.
• Granted Patent: US 6,541,606 B2
• Time in Patent Office: Days
• US Class Current: 514/21
• CPC Class Codes: A61K 38/27 Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/395 Antibodies agglutinins A61K38/36 ; as drug carriers A61K47/50; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• CPC Class Codes: A61K 47/10 Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• CPC Class Codes: A61K 47/26 Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• CPC Class Codes: A61K 47/40 Cyclodextrins; Derivatives thereof
• CPC Class Codes: A61K 47/42 Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein oligopeptides having up to five amino acids A61K47/183; polyamino acids A61K47/34
• CPC Class Codes: A61K 9/0014 Skin, i.e. galenical aspects of topical compositions non-active ingredients are additionally classified in A61K47/00; A61K9/0009, A61K9/0021, A61K9/7015, A61K9/7023 take precedence; cosmetic preparations A61K8/00, A61Q; preparations for wound dressings or bandages A61L26/00
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61K 9/1647 Polyesters, e.g. poly(lactide-co-glycolide)
• CPC Class Codes: A61K 9/50 Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals A61K9/2081 takes precedence; particles with a single coating comprising drug A61K9/167
• CPC Class Codes: A61P 15/08 for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/06 of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
• CPC Class Codes: A61P 7/12 Antidiuretics, e.g. drugs for diabetes insipidus ADH A61P5/10
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA
• CPC Class Codes: C12N 9/0004 Oxidoreductases (1.)
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/18 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: C12N 9/84 : Penicillin amidase (3.5.1.11)
• CPC Class Codes: C12Y 101/03004 : Glucose oxidase (1.1.3.4)
• CPC Class Codes: C12Y 301/01003 : Triacylglycerol lipase (3.1.1.3)
• CPC Class Codes: C12Y 305/01011 : Penicillin amidase (3.5.1.11), i.e. penicillin-amidohydrolase
• CPC Class Codes: Y10S 530/813 : Carrier is a saccharide
• CPC Class Codes: Y10S 530/815 : Carrier is a synthetic polymer

Abstract

This invention relates to methods for the stabilization, storage and delivery of biologically active macromolecules, such as proteins, peptides and nucleic acids. In particular, this invention relates to protein or nucleic acid crystals, formulations and compositions comprising them. Methods are provided for the crystallization of proteins and nucleic acids and for the preparation of stabilized protein or nucleic acid crystals for use in dry or slurry formulations. The present invention is further directed to encapsulating proteins, glycoproteins, enzymes, antibodies, hormones and peptide crystals or crystal formulations into compositions for biological delivery to humans and animals. According to this invention, protein crystals or crystal formulations are encapsulated within a matrix comprising a polymeric carrier to form a composition. The formulations and compositions enhance preservation of the native biologically active tertiary structure of the proteins and create a reservoir which can slowly release active protein where and when it is needed. Methods are provided preparing stabilized formulations using pharmaceutical ingredients or excipients and optionally encapsulating them in a polymeric carrier to produce compositions and using such protein crystal formulations and compositions for biomedical applications, including delivery of therapeutic proteins and vaccines. Additional uses for the protein crystal formulations and compositions of this invention involve protein delivery in human food, agricultural feeds, veterinary compositions, diagnostics, cosmetics and personal care compositions.

Claims

1. A formulation, said formulation comprising:

(a) a protein crystal, and (b) at least one ingredient, wherein said formulation is characterized by at least a 60 fold greater shelf life when stored at 50°

C. than the soluble form of said protein in solution at 50°

C., as measured by T_½

.

7. The formulation according to any one of claims 1, 2, 3, 4, 5 or 6, wherein said formulation is selected from the group of pharmaceutical formulations, food formulations, feed formulations, veterinary formulations, diagnostic formulations, cosmetic formulations and personal care formulations.

8. The formulation according to any one of claims 1, 2, 3, 4, 5 or 6, wherein said protein is an enzyme.

9. The formulation according to claim 8, wherein said enzyme is selected from the group consisting of hydrolases, isomerases, lyases, ligases, adenylate cyclases, transferases and oxidoreductases.

10. The formulation according to claim 9, wherein said enzyme is selected from the group consisting of lipase, glucose oxidase and penicillin acylase.

11. The formulation according to any one of claims 1, 2, 3, 4, 5 or 6, wherein said protein is a therapeutic protein.

12. The formulation according to claim 11, wherein said therapeutic protein is selected from the group consisting of antibodies, human serum albumin, human growth hormones, nerve growth hormones, bone morphogenic hormones, fertility hormones, leukocyte markers, histocompatibility antigens, mucins, integrins, adhesion molecules, selectins, interleukins, interleukin receptors, chemokines, growth factors, growth factor receptors, interferon receptors, immunoglobulins, T-cell receptors, blood factors, leukocyte markers, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD11a, CD11b, CD11c, CD13, CD14, CD18, CD19, CE20, CD22, CD23, CD27, CD28, B7.1, B7.2, B7.3, CD29, CD30, CD40, gp39, CD44, CD45, Cdw52, CD56, CD58, CD69, CD72, CTLA-4, LFA-1, TCR, histocompatibility antigens, MHC class I, MHC class II, SLex, SLey, SLea, SLeb, VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, LFA-1, Mac-1, p150, p95, L-selectin, P-selectin, E-selectin, VCAM-1, ICAM-1, ICAM-2, LFA-3, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, PF4, RANTES, MIP1α

, MCP1, NAP-2, Groα

, Groβ

, and IL-8, TNFalpha, TGFbeta, TSH, VEGF/VPF, PTHrP, EGF family, EGF, PDGF family, endothelin, gastrin releasing peptide (GRP), TNFalphaR, RGFbetaR, TSHR, VEGFR/VPFR, FGFR, EGFR, PTHrPR, PDGFR family, EPO-R, GCSF-R, IFNα

R, IFNβ

R, IFNγ

R, IgE, FceRI, FceRII, complement C3b, complement C5a, complement C5b-9, Rh factor, fibrinogen, fibrin and myelin associated growth inhibitor.

13. The formulation according to any one of claims 1, 2, 3, 4, 5 or 6, wherein said therapeutic protein is a vaccine antigen.

14. The formulation according to claim 13, wherein said vaccine antigen is selected from the group consisting of viral surface proteins, HIV-1 envelope glycoproteins, RSV envelope glycoproteins, HSV envelope glycoproteins, EBV envelope glycoproteins, VZV envelope glycoproteins, HPV envelope glycoproteins, Influenza virus glycoproteins, Hepatitis family surface antigens;

viral structural proteins, viral enzymes, parasite proteins, parasite glycoproteins, parasite enzymes, bacterial proteins, tumor antigens, allergens and toxins.

15. The formulation according to any one of claims 1, 2, 3, 4, 5 or 6, wherein said ingredient is an excipient.

16. The formulation according to claim 15, wherein said excipient is selected from the group consisting of sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β

-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.

18. The formulation according to claim 17, wherein said nucleic acid is DNA.

19. The formulation according to claim 18, wherein said DNA codes for a ribozyme.

20. The formulation according to claim 17, wherein said nucleic acid is RNA.

21. The formulation according to claim 20, wherein said RNA is a ribozyme.

22. The formulation according to claim 17, wherein said nucleic acid codes for an enzyme.

23. The formulation according to claim 22, wherein said enzyme is selected from the group consisting of hydrolases, isomerases, lyases, ligases, adenylate cyclases, transferases and oxidoreductases.

24. The formulation according to claim 23, wherein said enzyme is selected from the group consisting of lipase, glucose oxidase and penicillin acylase.

25. The formulation according to claim 17, wherein said nucleic acid codes for a therapeutic protein.

26. The formulation according to claim 25, wherein said therapeutic protein is selected from the group consisting of antibodies, human serum albumin, human growth hormones, nerve growth hormones, bone morphogenic hormones, fertility hormones, leukocyte markers, histocompatibility antigens, mucins, integrins, adhesion molecules, selectins, interleukins, interleukin receptors, chemokines, growth factors, growth factor receptors, interferon receptors, immunoglobulins, T-cell receptors, blood factors, leukocyte markers, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD11a, CD11b, CD11c, CD13, CD14, CD18, CD19, CE20, CD22, CD23, CD27, CD28, B7.1, B7.2, B7.3, CD29, CD30, CD40, gp39, CD44, CD45, Cdw52, CD56, CD58, CD69, CD72, CTLA-4, LFA-1, TCR, histocompatibility antigens, MHC class I, MHC class II, SLex, SLey, SLea, SLeb, VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, LFA-1, Mac-1, p150, p95, L-selectin, P-selectin, E-selectin, VCAM-1, ICAM-1, ICAM-2, LFA-3, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-1R, IL-2R, IL-4R, IL-SR, IL-6R, IL-7R, IL-8R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, PF4, RANTES, MIP1α

, MCP1, NAP-2, Groα

, Groβ

, and IL-8, TNFalpha, TGFbeta, TSH, VEGF/VPF, PTHrP, EGF family, EGF, PDGF family, endothelin, gastrin releasing peptide (GRP), TNFalphaR, RGFbetaR, TSHR, VEGFR/VPFR, FGFR, EGFR, PTHrPR, PDGFR family, EPO-R, GCSF-R, IFNα

R, IFNβ

R, IFNγ

R, IgE, FceRI, FceRII, complement C3b, complement C5a, complement C5b-9, Rh factor, fibrinogen, fibrin and myelin associated growth inhibitor.

27. The formulation according to claim 25, wherein said therapeutic protein is a vaccine antigen.

28. The formulation according to claim 27, wherein said vaccine antigen is selected from the group consisting of viral surface proteins, HIV-1 envelope glycoproteins, RSV envelope glycoproteins, HSV envelope glycoproteins, EBV envelope glycoproteins, VZV envelope glycoproteins, HPV envelope glycoproteins, Influenza virus glycoproteins, Hepatitis family surface antigens;

viral structural proteins, viral enzymes, parasite proteins, parasite glycoproteins, parasite enzymes, bacterial proteins, tumor antigens, allergens and toxins.

29. The formulation according to claim 17, wherein said ingredient is an excipient.

30. The formulation according to claim 29, wherein said excipient is selected from the group consisting of sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β

-cyclodextrin (HPCD), methoxypolyethylene glycol (MOPEG) and polyethylene glycol (PEG).

31. A method of treating a mammal comprising the step of administering to the mammal an effective amount of the formulation according to any one of claims 1, 2, 3, 4, 5, 6 or 16.

32. The method according to claim 31, wherein the formulation is administered by the route selected from a group consisting of oral, intramuscular, intravenous, pulmonary inhalation and parenteral.

2. A formulation, said formulation comprising:

(a) a protein crystal, and

(b) at least one ingredient, wherein said formulation is characterized by at least a 59 fold greater shelf life when stored at 40°

C. and 75% humidity than the nonformulated form of said protein crystal when stored at 40°

C. and 75% humidity, as measured by T_½

.

3. A formulation, said formulation comprising:

(a) a protein crystal, and

(b) at least one ingredient, wherein said formulation is characterized by at least a 60% greater shelf life when stored at 50°

C. than the nonformulated form of said protein crystal when stored at 50°

C., as measured by T_½

.

4. A formulation, said formulation comprising:

(a) a protein crystal, and

(b) at least one ingredient, wherein said formulation is characterized by the loss of less than 20% α

-helical structural content of the protein after storage for 4 days at 50°

C., wherein the soluble form of said protein loses more than 50% of its α

-helical structural content after storage for 6 hours at 50°

C., as measured by FTIR.

5. A formulation, said formulation comprising:

(a) a protein crystal, and

(b) at least one ingredient, wherein said formulation is characterized by the loss of less than 20% α

-helical structural content of the protein after storage for 4 days at 50°

C., wherein the soluble form of said protein loses more than 50% of its α

-helical structural content after storage for 6 hours at 50°

C., as measured by FTIR, and wherein said formulation is characterized by at least a 60 fold greater shelf life when stored at 50°

C. than the soluble form of said protein in solution at 50°

C., as measured by T_½

.

6. A formulation, said formulation comprising:

(a) a protein crystal, wherein said protein is greater than about 10,000 Daltons in molecular weight; and

(b) at least one ingredient.

17. A formulation, said formulation comprising:

(a) a nucleic acid crystal; and

(b) at least one ingredient.

33. A composition for the release of a protein, said composition comprising:

(a) a protein crystal formulation, wherein said formulation comprises a protein crystal and an ingredient; and

(b) at least one polymeric carrier, wherein said formulation is encapsulated within a matrix of said polymeric carrier, and wherein said formulation is characterized by at least a 60 fold greater shelf life when stored at 50°

C. than the soluble form of said protein in solution at 50°

C., as measured by T_½

.

39. The composition according to any one of claims 33, 34, 35, 36, 37 or 38, wherein said protein is an enzyme.

40. The composition according to claim 39, wherein said enzyme is selected from the group consisting of hydrolases, isomerases, lyases, ligases, adenylate cyclases, transferases and oxidoreductases.

41. The composition according to claim 40, wherein said enzyme is selected from the group consisting of lipase, glucose oxidase and penicillin acylase.

42. The composition according to any one of claims 33, 34, 35, 36, 37 or 38, wherein said protein is a therapeutic protein.

43. The composition according to claim 42, wherein said therapeutic protein is selected from the group consisting of antibodies, human serum albumin, human growth hormones, nerve growth hormones, bone morphogenic hormones, fertility hormones, leukocyte markers, histocompatibility antigens, mucins, integrins, adhesion molecules, selectins, interleukins, interleukin receptors, chemokines, growth factors, growth factor receptors, interferon receptors, immunoglobulins, T-cell receptors, blood factors, leukocyte markers, such as CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD11a, CD11b, CD11c, CD13, CD14, CD18, CD19, CE20, CD22, CD23, CD27, CD28, B7.1, B7.2, B7.3, CD29, CD30, CD40, gp39, CD44, CD45, Cdw52, CD56, CD58, CD69, CD72, CTLA-4, LFA-1, TCR, histocompatibility antigens, MHC class I, MHC class II, SLex, SLey, SLea, SLeb, VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, LFA-1, Mac-1, p150, p95, L-selectin, P-selectin, E-selectin, VCAM-1, ICAM-1, ICAM-2, LFA-3, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, PF4, RANTES, MIP1α

, MCP1, NAP-2, Groα

, Groβ

, IL-8, TNFalpha, TGFbeta, TSH, VEGF/VPF, PTHrP, EGF family, EGF, PDGF family, endothelin, gastrin releasing peptide (GRP), TNFalphaR, RGFbetaR, TSHR, VEGFR/VPFR, FGFR, EGFR, PTHrPR, PDGFR family, EPO-R, GCSF-R, IFNα

R, IFNβ

R, IFNγ

R, IgE, FceRI, FceRII, complement C3b, complement C5a, complement C5b-9, Rh factor, fibrinogen, fibrin and myelin associated growth inhibitor.

44. The composition according to any one of claims 33, 34, 35, 36, 37 or 38, wherein said therapeutic protein is a vaccine antigen.

45. The composition according to claim 44, wherein said vaccine antigen is selected from the group consisting of viral surface proteins, HIV-1 envelope glycoproteins, RSV envelope glycoproteins, HSV envelope glycoproteins, EBV envelope glycoproteins, VZV envelope glycoproteins, HPV envelope glycoproteins, Influenza virus glycoproteins, Hepatitis family surface antigens;

viral structural proteins, viral enzymes, parasite proteins, parasite glycoproteins, parasite enzymes, bacterial proteins, tumor antigens, allergens and toxins.

46. The composition according to any one of claims 33, 34, 35, 36, 37 or 38, wherein said ingredient is an excipient.

47. The composition according to claim 46, wherein said excipient is selected from the group consisting of sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β

-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.

48. The composition according to any one of claims 33, 34, 35, 36, 37 or 38, wherein said polymeric carrier is a biodegradable polymer.

49. The composition according to any one of claims 33, 34, 35, 36, 37 or 38, wherein said polymeric carrier is a biocompatible polymer.

50. The composition according to any one of claims 33, 34, 35, 36, 37 or 38, wherein said polymeric carrier is a polymer selected from one or more of the group consisting of poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (β

-hydroxybutryate), poly (caprolactone), poly (dioxanone);

poly (eth ylene glycol), poly ((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof.

51. The composition according to any one of claims 33, 34, 35, 36, 37 or 38, wherein said polymeric carrier is poly(lactic-co-glycolic acid).

52. The composition according to any one of claims 33, 34, 35, 36, 37 or 38, wherein said polymeric carrier is emulsified with poly(vinyl alcohol).

53. The composition according to any one of claims 33, 34, 35, 36, 37 or 38, wherein said polymeric carrier is a co-polymer.

54. The composition according to any one of claims 33, 34, 35, 36, 37 or 38, wherein said polymeric carrier is albumin.

76. A method of treating a mammal comprising the step of administering to the mammal an effective amount of the composition according to any one of claims 33, 34, 35, 36, 37, 38 or 55.

34. A composition for the release of a protein, said composition comprising:

(a) a protein crystal formulation, and

(b) at least one polymeric carrier, wherein said formulation is encapsulated within a matrix of said polymeric carrier, and wherein said formulation is characterized by at least a 59 fold greater shelf life when stored at 40°

C. and 75% humidity than the nonformulated form of said protein crystal when stored at 40°

C. and 75% humidity, as measured by T_½

.

35. A composition for the release of a protein, said composition comprising:

(a) a protein crystal formulation, wherein said formulation comprises a protein crystal and a ingredient; and

(b) at least one polymeric carrier, wherein said formulation is encapsulated within a matrix of said polymeric carrier, and wherein said formulation is characterized by at least a 60% greater shelf life when stored at 50°

C. than the nonformulated form of said protein crystal when stored at 50°

C, as measured by T_½

.

36. A composition for the release of a protein, said composition comprising:

(a) a protein crystal formulation, wherein said formulation comprises a protein crystal and a ingredient; and

(b) at least one polymeric carrier, wherein said formulation is encapsulated within a matrix of said polymeric carrier, and wherein said formulation is characterized by the loss of less than 20% of α

-helical structural content of the protein after storage for 4 days at 50°

C., wherein the soluble form of said protein loses more than 50% of its α

-helical structural content after storage for 6 hours at 50°

C., as measured by FTIR.

37. A composition for the release of proteins, said composition comprising:

(a) a protein crystal formulation, wherein said formulation comprises a protein crystal and a ingredient; and

(b) at least one polymeric carrier, wherein said formulation is encapsulated within a matrix of said polymeric carrier, and wherein said formulation is characterized by the loss of less than 20% of α

-helical structural content of the protein after storage for 4 days at 50°

C., wherein the soluble form of said protein loses more than 50% of its α

-helical structural content after storage for 6 hours at 50°

C., as measured by FTIR, and wherein said formulation is characterized by at least a 60 fold greater shelf life when stored at 50°

C. than the soluble form of said protein in solution at 50°

C., as measured by T_½

.

38. A composition for the release of proteins, said composition comprising:

(a) a protein crystal formulation, wherein said protein is greater than about 10,000 Daltons in molecular weight; and

(b) at least one polymeric carrier, wherein said formulation is encapsulated within a matrix of said polymeric carrier.

55. A composition for the release of a nucleic acid, said composition comprising:

(a) a nucleic acid crystal formulation, wherein said formulation comprises a nucleic acid crystal and a ingredient; and

(b) at least one polymeric carrier, wherein said formulation is encapsulated within a matrix of said polymeric carrier.

56. The composition according to claim 55, wherein said nucleic acid is DNA.

57. The composition according to claim 56, wherein said DNA codes for a ribozyme.

58. The composition according to claim 55, wherein said nucleic acid is RNA.

59. The composition according to claim 58, wherein said RNA is a ribozyme.

60. The composition according to claim 55, wherein said nucleic acid codes for an enzyme.

61. The composition according to claim 60, wherein said enzyme is selected from the group consisting of hydrolases, isomerases, lyases, ligases, adenylate cyclases, transferases and oxidoreductases.

62. The composition according to claim 61, wherein said enzyme is selected from the group consisting of lipase, glucose oxidase and penicillin acylase.

63. The composition according to claim 55, wherein said nucleic acid codes for a therapeutic protein.

64. The composition according to claim 63, wherein said therapeutic protein is selected from the group consisting of antibodies, human serum albumin, human growth hormones, nerve growth hormones, bone morphogenic hormones, fertility hormones, leukocyte markers, histocompatibility antigens, mucins, integrins, adhesion molecules, selectins, interleukins, interleukin receptors, chemokines, growth factors, growth factor receptors, interferon receptors, immunoglobulins, T-cell receptors, blood factors, leukocyte markers, such as CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD11a, CD11b, CD11c, CD13, CD14, CD18, CD19, CE20, CD22, CD23, CD27, CD28, B7.1, B7.2, B7.3, CD29, CD30, CD40, gp39, CD44, CD45, Cdw52, CD56, CD58, CD69, CD72, CTLA-4, LFA-1, TCR, histocompatibility antigens, MHC class I, MHC class II, SLex, SLey, SLea, SLeb, VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, LFA-1, Mac-1, p150, p95, L-selectin, P-selectin, E-selectin, VCAM-1, ICAM-1, ICAM-2, LFA-3, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, PF4, RANTES, MIP1α

, MCP1, NAP-2, Groα

, Groβ and

IL-8, TNFalpha, TGFbeta, TSH, VEGF/VPF, PTHrP, EGF family, EGF, PDGF family, endothelin, gastrin releasing peptide (GRP), TNFalphaR, RGFbetaR, TSHR, VEGFR/VPFR, FGFR, EGFR, PTHrPR, PDGFR family, EPO-R, GCSF-R, IFNα

R, IFNβ

R, IFNγ

R, IgE, FceRI, FceRII, complement C3b, complement C5a, complement C5b-9, Rh factor, fibrinogen, fibrin and myelin associated growth inhibitor.

65. The composition according to claim 63, wherein said therapeutic protein is a vaccine antigen.

66. The composition according to claim 65, wherein said vaccine antigen is selected from the group consisting of viral surface proteins, HIV-1 envelope glycoproteins, RSV envelope glycoproteins, HSV envelope glycoproteins, EBV envelope glycoproteins, VZV envelope glycoproteins, HPV envelope glycoproteins, Influenza virus glycoproteins, Hepatitis family surface antigens;

viral structural proteins, viral enzymes, parasite proteins, parasite glycoproteins, parasite enzymes, bacterial proteins, tumor antigens, allergens and toxins.

67. The composition according to claim 55, wherein said ingredient is an excipient.

68. The composition according to claim 67, wherein said excipient is selected from the group consisting of sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β

-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.

69. The composition according to claim 55, wherein said polymeric carrier is a biodegradable polymer.

70. The composition according to claim 55, wherein said polymeric carrier is a biocompatible polymer.

71. The composition according to claim 55, wherein said polymeric carrier is a polymer selected from one or more of the group consisting of poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (β

-hydroxybutryate), poly (caprolactone) and poly (dioxanone);

poly (ethylene glycol), poly ((hydroxypropyl)methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof.

72. The composition according to claim 55, wherein said polymeric carrier is poly(lactic-co-glycolic acid).

73. The composition according to claim 55, wherein said polymeric carrier is emulsified with poly(vinyl alcohol).

74. The composition according to claim 55, wherein said polymeric carrier is a co-polymer.

75. The composition according to claim 55, wherein said polymeric carrier is albumin.

77. The method according to claim 72, wherein the composition is administered by a route selected from the group consisting of oral, intramuscular, intravenous, pulmonary inhalation and parenteral.

79. The protein crystal composition according to claim 78, wherein said protein is selected from the group consisting of glycoproteins, enzymes, hormones, antibodies, cytokines peptides.

80. The protein crystal composition according to claim 78, wherein said polymeric carrier is a biodegradable polymer.

81. The protein crystal composition according to claim 78, wherein said polymeric carrier is a biocompatible polymer.

82. The protein crystal composition according to claim 78, wherein said polymeric carrier is a polymer selected from one or more of the group consisting of poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (β

-hydroxybutryate), poly (caprolactone) and poly (dioxanone);

poly (ethylene glycol), poly ((hydroxypropyl)methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof.

83. The protein crystal composition according to claim 82, wherein said polymeric carrier is poly(lactic-co-glycolic acid).

84. The protein crystal composition according to claim 82, wherein said polymeric carrier is emulsified with polyvinyl alcohol).

85. The protein crystal composition according to claim 82, wherein said polymeric carrier is a co-polymer.

86. The protein crystal composition according to claim 85, wherein said polymeric carrier is albumin.

87. The protein crystal composition according to claim 78, wherein said protein crystals are between about 0.01 μ

m and about 500 μ

m in their largest diameter.

88. The protein crystal composition according to claim 78, wherein said protein crystals are between about 50 μ

m and 100 μ

m in their largest diameter.

89. The protein crystal composition according to claim 78, wherein said protein crystals are microcrystals.

90. The protein crystal composition according to claim 89, wherein said protein microcrystals are nicroencapsulated within a matrix of said polymeric carrier.

91. The protein crystal composition according to claim 78, wherein said protein crystals are crosslinked.

92. The protein crystal composition according to claim 91, wherein said protein crystals are crosslinked with a multifunctional crosslinking agent.

93. The protein crystal composition according to claim 92, wherein said multifunctional crosslinking agent is a bifunctional crosslinking agent.

94. The protein crystal composition according to claim 92, wherein said multifunctional crosslinking agent is selected from the group consisting of glutaraldehyde, succinaldehyde, octanedialdehyde and glyoxal.

95. The protein crystal composition according to claim 78, wherein said protein is selected from the group consisting of phosphoproteins, sulphoproteins, iodoproteins, methylated proteins and fusion proteins.

96. The protein crystal composition according to claim 78, wherein said protein is selected from the group consisting of therapeutic proteins, prophylactic proteins, vaccine proteins, veterinary proteins, cleaning agent proteins, detergent proteins, personal care proteins, cosmetic proteins, food proteins, feed proteins, diagnostic proteins and decontamination proteins.

97. The protein crystal composition according to claim 78, wherein said protein is selected from the group consisting of insulin, amylin, erythropoietin, glucagon-like insulinotropic peptide, Factor VIII, dornase-α

α

-1-antitripsin, fertility hormones, FSH, LSH, postridical hormones, tetanus toxoid and diptheria toxoid.

98. The protein crystal composition according to claim 79, wherein said enzyme is selected from the group consisting of hydrolases, isomerases, lyases, ligases, transferases and oxidoreductases.

99. The protein crystal composition according to claim 98, wherein said enzyme is a hydrolase.

100. The protein crystal composition according to claim 99, wherein said hydrolase is selected from the group consisting of thermolysin, elastase, esterase, lipase, nitrilase, hydantoinase, protease, asparaginase, urease and lysozyme.

101. The protein crystal composition according to claim 100, wherein said hydrolase is a lipase.

102. The protein crystal composition according to claim 101, wherein said lipase is Candida rugosa lipase or Pseudomonas cepacia lipase.

103. The composition according to claim 78, said composition being selected from the group consisting of pharmaceuticals, vaccines, veterinary compounds, foods and feeds.

104. The composition according to claim 78, said composition being selected from the group consisting of diagnostics, cleaning agents, personal care compositions, cosmetics and formulations for decontamination.

105. The composition according to claim 103, wherein said pharmaceutical is a topical composition selected from the group consisting of creams, emulsions, lotions, foams, washes, drops, gels, compacts, slurries, powders, sprays, foams, pastes, ointments, salves, balms, shampoos and sunscreens.

106. The composition according to claim 104, wherein said cosmetic is selected from the group consisting of creams, emulsions, lotions, foams, washes, drops, gels, compacts, slurries, powders, sprays, foams, pastes, ointments, salves, balms, shampoos and sunscreens.

107. The protein crystal composition according to claim 78, wherein said composition is a pharmaceutical controlled release composition.

108. The pharmaceutical controlled release composition according to claim 107, wherein said composition is formulated for administration by an oral, pulmonary, nasal, aural, anal, dermal, ocular, intravenous, intramuscular, intraarterial, intraperitoneal, mucosal, sublingual, subcutaneous or intracranial route.

109. The composition according to claim 103, wherein said composition is a vaccine.

127. A protein delivery system, said system comprising the protein crystal composition according to claim 79

128. A method for delivering a protein to a subject, said method comprising the step of administering to said subject a protein crystal composition according to claim 79.

78. A protein crystal composition comprising:

(a) at least one polymeric carrier; and

(b) protein crystals, wherein said protein crystals are encapsulated within a matrix of said polymeric carrier.

110. A process for producing microspheres by encapsulating protein crystals while maintaining their crystallinity, comprising the steps of:

(a) suspending said protein crystals in a polymeric carrier dissolved in an organic solvent to produce coated crystals in the suspension;

(b) transferring said suspension of coated crystals, polymeric carrier and organic solvent to an aqueous solution containing an emulsifier; and

(c) hardening said polymeric carrier by evaporating the organic solvent in the presence of said emulsifier to produce said microspheres.

111. The process according to claim 110, wherein said protein is selected from the group consisting of glycoproteins, enzymes, hormones, a ntibodies and peptides.

112. The process according to claim 110, wherein said polymeric carrier is a biodegradable polymer.

113. The process according to claim 110, wherein said polymeric carrier is a biocompatible polymer.

114. The process according to claim 110, wherein said emulsifier is poly(vinyl alcohol).

115. The process according to claim 110, wherein said emulsifier is a surfactant.

116. The process according to claim 110, wherein said polymeric carrier is a polymer selected from one or more of the group consisting of poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (β

-hydroxybutryate), poly (caprolactone), poly (dioxanone), poly (ethylene glycol), poly ((hydroxypropyl)methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof.

117. The process according to claim 110, wherein said polymeric carrier is poly(lactic-co-glycolic acid).

118. The process according to claim 110, wherein said organic solvent is methylene chloride.

119. The process according to claim 110, wherein said polymeric carrier is a co-polymer.

120. The process according to claim 110, wherein said polymeric carrier is albumin.

121. The process according to claim 110, wherein said protein crystals are microcrystals.

122. The process according to claim 121, wherein said protein microcrystals are microencapsulated in said polymeric carrier.

123. The process according to claim 110, wherein said protein crystals are crosslinked.

124. The process according to claim 110, wherein said protein crystals are crosslinked with a multifunctional crosslinking agent.

125. The process according to claim 124, wherein said multifunctional crosslinking agent is a bifunctional crosslinking agent.

126. The process according to claim 124, wherein said multifunctional crosslinking agent is selected from the group consisting of glutaraldehyde, succinaldehyde, octanedialdehyde and glyoxal.

131. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 129 or 130, wherein after step (b) and before step (c), the method further comprises the step of washing the protein or nucleic acid crystals in a different organic solvent than the one used in step (b).

132. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 129, wherein after step (d), the method further comprises the step of dissolving the protein or nucleic acid crystals in an appropriate buffer or formulation with pharmaceutical ingredients.

133. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 129 or 130, wherein said crystals are washed with an organic solvent selected from the group consisting of:

acetone, methyl alcohol, methyl isobutyl ketone, ethanol, chloroform, isopropanol, 2-propanol, acetonitrile, butanol, cyclohexane, dioxane, ethyl acetate, dimethylformamide, dichloroethane, hexane, isooctane, methylene chloride, tert-butyl alchohol, toluene, carbon tetrachloride and combinations thereof.

134. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 129 or 130, wherein said crystals are dried by passing therethrough a stream of a gas selected from the group consisting of:

nitrogen, argon, helium, carbon dioxide, air and combinations thereof.

135. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 129 or 130, wherein said crystals are crystals of a protein.

136. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 135, wherein said protein is an enzyme.

137. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 135, wherein said protein is a hormone.

138. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 135, wherein said protein is a therapeutic protein.

139. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 135, wherein said protein is an antibody.

140. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 135, wherein said dried, non-crosslinked protein or nucleic acid crystals are selected from the group consisting of:

hydrolases, isomerases, lyases, ligases, multidrug resistance proteins, adenylate cyclases, transferases, oxidoreductases, human growth hormones, nerve growth hormones, bone morphogenic hormones, fertility hormones, leukocyte markers, histocompatibility antigens, mucins, integrins, adhesion molecules, selectins, interleukins, interleukin receptors, chemokines, growth factors, growth factor receptors, interferon receptors, immunoglobulins, T-cell receptors, blood factors, viral surface proteins, HIV-1 envelope glycoproteins, RSV envelope glycoproteins, HSV envelope glycoproteins, EBV envelope glycoproteins, VZV envelope glycoproteins, HPV envelope glycoproteins, Hepatitis family surface antigens;

viral structural proteins, viral enzymes, parasite proteins, parasite glycoproteins, parasite enzymes, bacterial proteins, tumor antigens, allergens and toxins.

141. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 135, wherein said protein is a viral protein or a protein comprising a viral protein sequence.

142. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 135, wherein said protein is a bacterial protein or a protein comprising a bacterial protein sequence.

143. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 135, wherein said protein is a tumor cell antigen or a protein comprising a tumor cell antigen protein sequence.

144. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 129 or 130, wherein said crystals are crystals of DNA.

145. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 144, wherein said crystals are crystals of DNA coding for an enzyme.

146. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 144, wherein said crystals are crystals of DNA coding for a hormone.

147. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 144, wherein said crystals are crystals of DNA coding for a therapeutic protein.

148. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 144, wherein said crystals are crystals of DNA coding for an antibody.

149. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 144, wherein said crystals are crystals of DNA coding for a viral protein or DNA coding for a protein comprising a viral protein sequence.

150. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 144, wherein said crystals are crystals of DNA coding for a bacterial protein or DNA coding for a protein comprising a bacterial protein sequence.

151. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 144, wherein said crystals are crystals of DNA coding for a tumor cell protein antigen or DNA coding for a protein comprising a tumor cell protein antigen sequence.

152. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 144, wherein said crystals comprise DNA operably linked to a promoter.

153. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 129 or 130, wherein said crystals comprise RNA.

154. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 153, wherein said crystals comprise RNA coding for an enzyme.

155. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 153, wherein said crystals comprise RNA coding for a hormone.

156. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 153, wherein said crystals comprise RNA coding for a therapeutic protein.

157. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 153, wherein said crystals comprise RNA coding for an antibody.

158. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 153, wherein said crystals comprise RNA coding for a viral protein or RNA coding for a protein comprising a viral protein sequence.

159. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 153, wherein said crystals comprise RNA coding for a bacterial protein or RNA coding for a protein comprising a bacterial protein sequence.

160. The method for producing dried, non-crosslinked protein or nucleic acid crystals according to claim 153, wherein said crystals comprise RNA coding for a tumor cell antigen protein or RNA coding for a protein comprising a tumor cell antigen protein sequence.

162. Dried, non-crosslinked protein or nucleic acid crystals produced by the method according to claim 129 or 130.

163. The dried, non-crosslinked protein or nucleic acid crystals according to claim 161 or 162, wherein the crystals are protein crystals.

164. The dried, non-crosslinked protein or nucleic acid crystals according to claim 161 or 162, wherein the protein is an enzyme.

165. The dried, non-crosslinked protein or nucleic acid crystals according to claim 161 or 162, wherein the protein is a hormone.

166. The dried, non-crosslinked protein or nucleic acid crystals according to claim 161 or 162, wherein the protein is a therapeutic protein.

167. The dried, non-crosslinked protein or nucleic acid crystals according to claim 161 or 162, wherein the protein is an antibody.

168. The dried, non-crosslinked protein or nucleic acid crystals according to claim 161 or 162, wherein the protein is a viral protein or a protein comprising a viral protein sequence.

169. The dried, non-crosslinked protein or nucleic acid crystals according to claim 161 or 162, wherein the protein is a bacterial protein or a protein comprising a bacterial protein sequence.

170. The dried, non-crosslinked protein or nucleic acid crystals according to claim 161 or 162, wherein the protein is a tumor cell protein antigen or a protein comprising a tumor cell antigen protein sequence.

171. The dried, non-crosslinked protein or nucleic acid crystals produced according to claim 161 or 162, wherein said crystals are crystals of DNA.

172. The dried, non-crosslinked protein or nucleic acid crystals according to claim 171, wherein said crystals are crystals of DNA coding for an enzyme.

173. The dried, non-crosslinked protein or nucleic acid crystals according to claim 171, wherein said crystals are crystals of DNA coding for a hormone.

174. The dried, non-crosslinked protein or nucleic acid crystals according to claim 171, wherein said crystals are crystals of DNA coding for a therapeutic protein.

175. The dried, non-crosslinked protein or nucleic acid crystals according to claim 171, wherein said crystals are crystals of DNA coding for an antibody.

176. The dried, non-crosslinked protein or nucleic acid crystals according to claim 171, wherein said crystals are crystals of DNA coding for a viral protein or DNA coding for a protein comprising a viral protein sequence.

177. The dried, non-crosslinked protein or nucleic acid crystals according to claim 171, wherein said crystals are crystals of DNA coding for a bacterial protein or DNA coding for a protein comprising a bacterial protein sequence.

178. The dried, non-crosslinked protein or nucleic acid crystals according to claim 171, wherein said crystals are crystals of DNA coding for a tumor cell protein antigen or crystals of DNA coding for a protein comprising a tumor cell protein antigen sequence.

179. The dried, non-crosslinked protein or nucleic acid crystals according to claim 171, wherein the crystals are crystals of DNA operably linked to a promoter.

180. The dried, non-crosslinked protein or nucleic acid crystals according to claim 161 or 162, wherein the crystals are crystals of RNA.

181. The dried, non-crosslinked protein or nucleic acid crystals according to claim 180, wherein said crystals are crystals of RNA coding for an enzyme.

182. The dried, non-crosslinked protein or nucleic acid crystals according to claim 180, wherein said crystals are crystals of RNA coding for a hormone.

183. The dried, non-crosslinked protein or nucleic acid crystals according to claim 180, wherein said crystals are crystals of RNA coding for a therapeutic protein.

184. The dried, non-crosslinked protein or nucleic acid crystals according to claim 180, wherein said crystals are crystals of RNA coding for an antibody.

185. The dried, non-crosslinked protein or nucleic acid crystals according to claim 180, wherein said crystals are crystals of RNA coding for a viral protein or RNA coding for a protein comprising a viral protein sequence.

186. The dried, non-crosslinked protein or nucleic acid crystals according to claim 180, wherein said crystals are crystals of RNA coding for a bacterial protein or RNA coding for a protein comprising a bacterial protein sequence.

187. The dried, non-crosslinked protein or nucleic acid crystals according to claim 180, wherein said crystals are crystals of RNA coding for a tumor cell antigen protein or RNA coding for a protein comprising a tumor cell antigen protein sequence.

129. A method for producing dried, non-crosslinked protein or nucleic acid crystals comprising the steps of:

(a) crystallizing protein or nucleic acid molecules to form protein or nucleic acid crystals;

(b) washing said protein or nucleic acid crystals with an organic solvent or a liquid polymer;

(c) removing said organic solvent from said protein or nucleic acid crystals; and

(d) drying said protein or nucleic acid crystals.

130. A method for producing dried, non-crosslinked protein or nucleic acid crystals comprising the steps of:

(a) crystallizing protein or nucleic acid molecules to form protein or nucleic acid crystals; and

(b) washing and suspending said protein or nucleic acid crystals in an organic solvent or a liquid polymer.

161. Dried, non-crosslinked protein or nucleic acid crystals.