Controlled dissolution crosslinked protein crystals

Abstract

The present invention relates to crosslinked protein crystals characterized by the ability to change from insoluble and stable form to soluble and active form upon a change in the environment of said crystals, said change being selected from the group consisting of change in temperature, change in pH, change in chemical composition, change from concentrate to dilute form, change in shear force acting upon the crystals and combinations thereof. According to one embodiment of this invention, such crosslinked protein crystals are capable of releasing their protein activity at a controlled rate. This invention also provides methods for producing such crosslinked protein crystals, methods using them for protein delivery and methods using them in cleaning agents, including detergents, pharmaceutical compositions, vaccines, personal care compositions, including cosmetics, veterinary compositions, foods, feeds, diagnostics and formulations for decontamination.

1 . A crosslinked protein crystal, said protein crystal being capable of change from insoluble and stable form to soluble and active form upon a change in the environment surrounding said crystal, said change being selected from the group consisting of: change in temperature, change in pH, change in chemical composition, change from concentrate to dilute form, change in shear force acting upon the crystal and combinations thereof.  
     
     
         2 . The crosslinked protein crystal according to  claim 1 , wherein said change from concentrate to dilute form comprises a change in solute concentration.  
     
     
         3 . The crosslinked protein crystal according to  claim 2 , wherein said change in solute concentration comprises an increase or decrease in salt concentration.  
     
     
         4 . The crosslinked protein crystal according to  claim 3 , wherein said change in solute concentration comprises a decrease in salt concentration.  
     
     
         5 . The crosslinked protein crystal according to  claim 2 , wherein said change in solute concentration comprises an increase or decrease in water concentration.  
     
     
         6 . The crosslinked protein crystal according to  claim 5 , wherein said change in solute concentration comprises an increase in water concentration.  
     
     
         7 . The crosslinked protein crystal according to  claim 2 , wherein said change in solute concentration comprises an increase or decrease in organic solvent concentration.  
     
     
         8 . The crosslinked protein crystal according to  claim 2 , wherein said change in solute concentration comprises a decrease in detergent concentration.  
     
     
         9 . The crosslinked protein crystal according to  claim 2 , wherein said change in solute concentration comprises a decrease in protein concentration.  
     
     
         10 . The crosslinked protein crystal according to  claim 1 , wherein said change from concentrate to dilute form comprises a change in concentration of all solutes from about 2-fold to about 10,000-fold.  
     
     
         11 . The crosslinked protein crystal according to  claim 10 , wherein said change from concentrate to dilute form comprises a change in concentration of all solutes from about 2-fold to about 700-fold.  
     
     
         12 . The crosslinked protein crystal according to  claim 1 , wherein said change in pH comprises a change from acidic pH to basic pH.  
     
     
         13 . The crosslinked protein crystal according to  claim 1 , wherein said change in pH comprises a change from basic pH to acidic pH.  
     
     
         14 . The crosslinked protein crystal according to  claim 1 , wherein said change in temperature comprises an increase or decrease in temperature.  
     
     
         15 . The crosslinked protein crystal according to  claim 14 , wherein said change in temperature is an increase in temperature from a low temperature between about 0° C. and about 20° C. to a high temperature between about 25° C. and about 70° C.  
     
     
         16 . The crosslinked protein crystal according to  claim 1 , wherein said active form of said protein is a form which is active against macromolecular substrates.  
     
     
         17 . A crosslinked protein crystal, said protein crystal having a half-life of activity under storage conditions which is greater than at least 2 times that of the soluble form of the protein that is crystallized to form said crystal that is crosslinked and activity similar to that of the soluble form of the protein under conditions of use.  
     
     
         18 . A crosslinked protein crystal, said protein crystal being capable of releasing its protein activity at a controlled rate upon exposure to a change in the environment surrounding said crystal, said change being selected from the group consisting of change in pH, change in solute concentration, change in temperature, change in chemical composition, change in shear force acting upon the crystals and combinations thereof.  
     
     
         19 . The crosslinked protein crystal according to  claim 18 , wherein said controlled rate of releasing protein activity is determined by a factor selected from the group consisting of: the degree of crosslinking of said crosslinked protein crystal, the length of time of exposure of protein crystal to the crosslinker, the rate of addition of the crosslinking agent to said protein crystal, the nature of the crosslinker, the chain length of the crosslinker, the surface area of said crosslinked protein crystal, the size of said crosslinked protein crystal, the shape of said crosslinked protein crystal and combinations thereof.  
     
     
         20 . The crosslinked protein crystal according to  claim 18 , wherein said crystal has a protein activity release rate of between about 0.1% per day and about 100% per day.  
     
     
         21 . The crosslinked protein crystal according to  claim 18 , wherein said crystal has a protein activity release rate between about 0.01% per hour and about 100% per hour.  
     
     
         22 . The crosslinked protein crystal according to  claim 18 , wherein said crystal has a protein activity release rate between about 1% per minute and about 50% per minute.  
     
     
         23 . The crosslinked protein crystal according to any one of claims  1 ,  17  or  18 , said protein crystal being substantially insoluble and stable in a composition under storage conditions and substantially soluble and active under conditions of use of said composition.  
     
     
         24 . The crosslinked protein crystal according to  claim 23 , wherein said composition is selected from the group consisting of cleaning agents, detergents, personal care compositions, cosmetics, pharmaceuticals, veterinary compounds, vaccines, foods, feeds, diagnostics and formulations for decontamination.  
     
     
         25 . The crosslinked protein crystal according to  claim 24 , wherein said detergent is selected from the group consisting of powdered detergents, liquid detergents, bleaches, household cleaners, hard surface cleaners, industrial cleaners, carpet shampoos and upholstery shampoos.  
     
     
         26 . The crosslinked protein crystal according to  claim 24 , wherein said cosmetic is selected from the group consisting of creams, emulsions, lotions, foams, washes, gels, compacts, mousses, sunscreens, slurries, powders, sprays, foams, pastes, ointments, salves, balms, shampoos, sunscreens and drops.  
     
     
         27 . The crosslinked protein crystal according to any one of claims  1 ,  17  or  18 , wherein said protein is an enzyme.  
     
     
         28 . The crosslinked protein crystal according to  claim 27 , wherein said enzyme is selected from the group consisting of hydrolases, isomerases, lyases, ligases, transferases and oxidoreductases.  
     
     
         29 . The crosslinked protein crystal according to  claim 28 , wherein said enzyme is selected from the group consisting of proteases, amylases, cellulases, lipases and oxidases.  
     
     
         30 . The crosslinked protein crystal according to any one of claims  1 ,  17  or  18 , wherein said protein is selected from the group consisting of therapeutic proteins, cleaning agent proteins, personal care proteins, veterinary proteins, food proteins, feed proteins, diagnostic proteins and decontamination proteins.  
     
     
         31 . The crosslinked protein crystal according to any one of claims  1 ,  17  or  18 , wherein said protein is selected from the group consisting of hormones, antibodies, inhibitors, growth factors, trophic factors, cytokines, lymphokines, toxoids, growth hormones, nerve growth hormones, bone morphogenic proteins, and nutrients.  
     
     
         32 . The crosslinked protein crystal according to any one of claims  1 ,  17  or  18 , wherein said protein is selected from the group consisting of insulin, amylin, erythropoietin, Factor VIII, TPA, dornase-α, α-1-antitrypsin, urease, fertility hormones, FSH, LSH, postridical hormones, tetanus toxoid and diptheria toxoid.  
     
     
         33 . The crosslinked protein crystal according to any one of claims  1 ,  17  or  18 , said crystal having a longest dimension of between about 0.01 μm and about 500 μm.  
     
     
         34 . The crosslinked protein crystal according to any one of claims  1 ,  20  or  21 , said crystal having a longest dimension of between about 0.1 μm and about 50 μm.  
     
     
         35 . The crosslinked protein crystal according to any one of claims  1 ,  17  or  18 , said crystal having a shape selected from the group consisting of: spheres, needles, rods, plates, rhomboids, cubes, bipyramids and prisms.  
     
     
         36 . A composition comprising a crosslinked protein crystal according to any one of claims  1 ,  17  or  18 , said composition being selected from the group consisting of cleaning agents, detergents, personal care compositions, cosmetics, pharmaceuticals, veterinary compounds, vaccines, foods, feeds, diagnostics and formulations for decontamination.  
     
     
         37 . The composition according to  claim 36 , wherein said detergent is selected from the group consisting of powdered detergents, liquid detergents, bleaches, household cleaners, hard surface cleaners, industrial cleaners, carpet shampoos and upholstery shampoos.  
     
     
         38 . The composition according to  claim 36 , wherein said cosmetic is selected from the group consisting of creams, emulsions, lotions, foams, washes, gels, compacts, sunscreens, slurries, powders, sprays, foams, pastes, ointments, salves, balms, shampoos, sunscreens and drops.  
     
     
         39 . A protein delivery system, said system comprising crosslinked protein crystals according to any one of claims  1 ,  17  or  18 .  
     
     
         40 . The protein delivery system according to  claim 39 , wherein said protein is selected from the group consisting of: detergent enzymes, cosmetic proteins, pharmaceutical proteins, agricultural proteins, vaccine proteins and decontamination proteins.  
     
     
         41 . The protein delivery system according to  claim 40 , said protein delivery system being a microparticulate protein delivery system.  
     
     
         42 . The protein delivery system according to  claim 41 , wherein said microparticulate protein delivery system comprises crosslinked protein crystals having a longest dimension between about 0.01 μm and about 500 μm.  
     
     
         43 . The protein delivery system according to  claim 42 , wherein said microparticulate protein delivery system comprises crosslinked protein crystals having a longest dimension of between about 0.1 μm and about 50 μm.  
     
     
         44 . The protein delivery system according to  claim 41 , wherein said microparticulate protein delivery system comprises crosslinked protein crystals having a shape selected from the group consisting of: spheres, needles, rods, plates, rhomboids, cubes, bipyramids and prisms.  
     
     
         45 . A detergent formulation comprising a crosslinked protein crystal according to any one of claims  1 ,  17  or  18 .  
     
     
         46 . A controlled release formulation comprising a crosslinked protein crystal according to any one of claims  1 ,  17  or  18 .  
     
     
         47 . A pharmaceutical controlled release formulation comprising a crosslinked protein crystal according to any one of claims  1 ,  17  or  18 .  
     
     
         48 . A pharmaceutical controlled release formulation comprising a crosslinked protein crystal, said crystal being substantially insoluble under storage conditions and capable of releasing its protein activity in vivo at a controlled rate.  
     
     
         49 . The pharmaceutical controlled release formulation according to  claim 47 , said pharmaceutical being capable of administration by parenteral or non-parenteral routes.  
     
     
         50 . The pharmaceutical controlled release formulation according to  claim 49 , said pharmaceutical being capable of administration by oral, pulmonary, nasal, aural, anal, dermal, ocular, intravenous, intramuscular, intraarterial, intraperitoneal, mucosal, sublingual, subcutaneous or intracranial route.  
     
     
         51 . The pharmaceutical controlled release formulation according to  claim 47 , wherein said pharmaceutical is capable of administration by oral route and said crosslinked protein crystal is substantially insoluble under gastric pH conditions and substantially soluble under small intestine pH conditions.  
     
     
         52 . A vaccine comprising a crosslinked protein crystal according to any one of claims  1 ,  17  or  18 .  
     
     
         53 . A formulation comprising a crosslinked protein crystal according to any one of claims  1 ,  17  or  18 , said formulation being selected from the group consisting of tablets, liposomes, granules, spheres, microspheres, microparticles and capsules.  
     
     
         54 . A method for producing crosslinked protein crystals comprising the step of reacting protein crystals with a first crosslinking agent, or a first crosslinking agent and at least a second crosslinking agent, under conditions sufficient to induce crosslinking of said crystals to the extent that the resulting crosslinked crystals are characterized by the ability to change from insoluble and stable form to soluble and active form upon a change in their environment, said change being selected from the group consisting of change in temperature, change in pH, change in chemical composition, change from concentrate to dilute form, change in shear force acting upon the crystals and combinations thereof.  
     
     
         55 . A method for producing crosslinked protein crystals comprising the step of reacting protein crystals with a first crosslinking agent, or a first crosslinking agent and at least a second crosslinking agent, under conditions sufficient to induce crosslinking of said crystals to the extent that the resulting crosslinked crystals are characterized by a half-life of activity under storage conditions which is greater than at least 2 times that of the soluble form of the protein that is crystallized to form said crystals that are crosslinked and activity similar to that of the soluble form of the protein under conditions of use.  
     
     
         56 . A method for producing crosslinked protein crystals comprising the step of reacting protein crystals with a first crosslinking agent, or a first crosslinking agent and at least a second crosslinking agent, under conditions sufficient to induce crosslinking of said crystals to the extent that the resulting crosslinked crystals are characterized by being capable of releasing their protein activity at a controlled rate upon exposure to a change in their environment, said change being selected from the group consisting of change in pH, change in soluble concentration, change in temperature, change in chemical composition, change in shear force acting upon the crystals and combinations thereof.  
     
     
         57 - 85  (canceled)