Spherical Protein Particles and Methods of Making and Using Them

Abstract

This invention relates to SPPs, spherical nanocrystalline composite particles or crystalline SPPs of biologically active proteins or compositions, including formulations, comprising such SPPs, spherical nanocrystalline composite particles or crystalline SPPs. More particularly, methods are provided for the production of SPPs, spherical nanocrystalline composite particles or crystalline SPPs of high concentrations of biologically active proteins, and for the preparation of stabilized SPPs, spherical nanocrystalline composite particles or crystalline SPPs for use alone, or in dry or slurry compositions. This invention also relates to methods for stabilization, storage and delivery of biologically active proteins using SPPs, spherical nanocrystalline composite particles or crystalline SPPs. The present invention further relates to methods using SPPs, spherical nanocrystalline composite particles or crystalline SPPs, or compositions or formulations comprising such SPPs, spherical nanocrystalline composite particles or crystalline SPPs, for biomedical applications, including biological delivery to humans and animals.

1 . A spherical protein particle. 
     
     
         2 . The spherical protein particle according to  claim 1 , wherein said spherical protein particle is a spherical nanocrystalline composite particle. 
     
     
         3 . The spherical protein particle according to  claim 1  wherein said protein is an antibody or a single-chain Fv fragment of said antibody. 
     
     
         4 . The spherical protein particle according to  claim 1 , wherein said spherical protein particle has a particle size range from about 0.04 microns to about 300 microns in diameter. 
     
     
         5 . The spherical protein particle according to  claim 1  wherein said spherical protein particle has a particle size range from about 0.04 microns to about 200 microns in diameter. 
     
     
         6 . The spherical protein particle according to  claim 1 , wherein said spherical protein particle has a particle size range from about 0.04 microns to about 100 microns in diameter. 
     
     
         7 .- 12 . (canceled) 
     
     
         13 . The spherical nanocrystalline composite particle according to  claim 2 , wherein the spherical nanocrystalline composite particle is from about 1 μm to about 300 μm in diameter, and comprises protein nanocrystals having a diameter of about 40 nm to about 999 nm. 
     
     
         14 . The spherical nanocrystalline composite particle according to  claim 2 , wherein the spherical nanocrystalline composite particle is from about 1 μm to about 300 μm in diameter, and comprises protein nanocrystals having a diameter of about 40 nm to about 499 nm. 
     
     
         15 . The spherical protein particle according to  claim 1 , wherein said protein has a conformation which is the same as that of the native, soluble counterpart of the protein, as indicated by an ELISA using a monoclonal antibody which specifically binds the native, soluble counterpart. 
     
     
         16 . The spherical protein particle according to  claim 1 , wherein said protein has a β-sheet structural content that is less than about 20% different from the β-sheet structural content of the native, soluble counterpart of the protein, and has lost less than about 20% of the α-helical structural content of its native, soluble counterpart, as indicated by FTIR or Circular Dichroism (CD) spectroscopy. 
     
     
         17 . The spherical protein particle according to  claim 16 , wherein said protein has a β-sheet structural content that is less than about 20% different from the β-sheet structural content of the native, soluble counterpart of the protein, as indicated by a correlation spectra as compared to its native, soluble counterpart, as indicated by FTIR. 
     
     
         18 . The spherical protein particle according to  claim 16 , wherein said protein loses less than about 20% of its α-helical structural content after storage for from about 4 days to about 180 days at from about 4° C. to about 50° C., as compared to its native, soluble counterpart after storage under identical conditions. 
     
     
         19 . The spherical protein particle according to  claim 16 , wherein said protein has a β-sheet structural content that is less than about 20% different from the β-sheet structural content of the native, soluble counterpart of the protein, and has lost less than about 20% of the α-helical structural content of the native, soluble counterpart of the protein, as indicated by Circular Dichroism (CD) spectroscopy. 
     
     
         20 . The spherical protein particle according to  claim 1 , wherein said protein has a biologically active conformation, as indicated by comparing the biological activity of the protein component of a dissolved spherical protein particle with the biological activity of its native, soluble counterpart. 
     
     
         21 .- 42 . (canceled) 
     
     
         43 . The spherical protein particle according to  claim 1 , wherein said protein is selected from the group consisting of: enzymes, protein hormones, viruses, viral proteins, antibodies, antibody fragments, receptors and peptides. 
     
     
         44 . The spherical protein particle according to  claim 3 , wherein said antibody is selected from the group consisting of: chimeric antibodies, humanized antibodies, non-glycosylated antibodies, bispecific antibodies, human antibodies and mouse antibodies. 
     
     
         45 .- 47 . (canceled) 
     
     
         48 . A composition, said composition comprising:
 (a) a spherical protein particle according to  claim 1 , and   (b) at least one ingredient.   
     
     
         49 .- 57 . (canceled) 
     
     
         58 . The composition according to  claim 48 , wherein said ingredient is a polymeric carrier. 
     
     
         59 . The composition according to  claim 58 , wherein said polymeric carrier is a biodegradable polymer or a biocompatible polymer. 
     
     
         60 . The composition according to  claim 58 , 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 (b-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. 
     
     
         61 .- 66 . (canceled) 
     
     
         67 . The composition according to  claim 48 , wherein said ingredient is selected from the group consisting of sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol. 
     
     
         68 .- 97 . (canceled)