Controlled dissolution crosslinked protein in 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 oxidation-reduction potential of the solution, change in the incident radiation, change in transition metal concentration, change in flouride concentration, change in free radical concentration, change in metal chelater concentration, 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 - 97 . (canceled)  
     
     
         98 . A controlled dissolution crosslinked lipase crystal produced by a process comprising the step of crosslinking lipase crystals with at least one crosslinking agent in a crosslinking solution, said crosslinking agent being present in said solution at a concentration between about 0.76 mM and 172.3 mM, wherein said controlled dissolution crosslinked lipase crystal is 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 and wherein said crosslinking agent is dimethyl 3,3′-dithiobispropionimidate.HCl (DTBP); dithiobis (succinimidylpropionate) (DSP); bismaleimidohexane (BMH); bis[sulfosuccinimidyl]suberate (BS); 1,5-difluoro-2,4-dinitrobenzene (DFDNB); dimethylsuberimidate-2HCl (DMS); disuccinimidyl glutarate (DSG); disulfosuccinimidyl tartarate (Sulfo-DST); 1-ethyl-3-[3-dimethylaminoproplyl]carbodiimide hydrochloride (EDC); ethylene glycolbis[sulfo-succinimidylsuccinate] (Sulfo-EGS); N-[γ-maleimido-butyryloxy]succinimide ester (GMBS); N-hydroxysulfo-succinimidyl-4-azidobenzoate (Sulfo-HSAB); sulfosuccinimidyl-6-(α-methyl-α-(2-pyridylthio)toluamido)hexanoate (Sulfo-LC-SMPT); Bis-(β-[4-azidosalicylamido]ethyl)disulfide (BASED); NHS-PEG-Vinylsulfone (NHS-PEG-VS); (Bis[2-(sulfosuccinimidooxycarbonyloxy)-ethyl]sulfone (Sulfo-BSOCOES); glutaraldehyde; succinaldehyde; octanedialdehyde; glyoxal; or epichlorohydrin.  
     
     
         99 . The controlled dissolution crosslinked lipase crystal according to  claim 98 , wherein said change from concentrate to dilute form comprises a change in solute concentration.  
     
     
         100 . The controlled dissolution crosslinked lipase crystal according to  claim 99 , wherein said change in solute concentration comprises an increase or decrease in salt concentration.  
     
     
         101 . The controlled dissolution crosslinked lipase crystal according to  claim 100 , wherein said change in solute concentration comprises a decrease in salt concentration.  
     
     
         102 . The controlled dissolution crosslinked lipase crystal according to  claim 99 , wherein said change in solute concentration comprises an increase in water concentration.  
     
     
         103 . The controlled dissolution crosslinked lipase crystal according to  claim 99 , wherein said change in solute concentration comprises an increase or decrease in organic solvent concentration.  
     
     
         104 . The controlled dissolution crosslinked lipase crystal according to  claim 99 , wherein said change in solute concentration comprises a decrease in detergent concentration.  
     
     
         105 . The controlled dissolution crosslinked lipase crystal according to  claim 99 , wherein said change in solute concentration comprises a decrease in lipase concentration.  
     
     
         106 . The controlled dissolution crosslinked lipase crystal according to  claim 98 , 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.  
     
     
         107 . The controlled dissolution crosslinked lipase crystal according to  claim 106 , wherein said change from concentrate to dilute form comprises a change in concentration of all solutes from about 2-fold to about 700-fold.  
     
     
         108 . The controlled dissolution crosslinked lipase crystal according to  claim 98 , wherein said change in pH comprises a change from acidic pH to basic pH.  
     
     
         109 . The controlled dissolution crosslinked lipase crystal according to  claim 98 , wherein said change in pH comprises a change from basic pH to acidic pH.  
     
     
         110 . The controlled dissolution crosslinked lipase crystal according to  claim 98 , wherein said change in temperature comprises an increase or decrease in temperature.  
     
     
         111 . The controlled dissolution crosslinked lipase crystal according to  claim 110 , wherein said change in temperature comprises 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.  
     
     
         112 . The controlled dissolution crosslinked lipase crystal according to  claim 98 , said lipase 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 lipase that is crystallized to form said crystal that is crosslinked and activity similar to that of said soluble form of the lipase under conditions of use.  
     
     
         113 . The controlled dissolution crosslinked lipase crystal according to  claim 98 , said lipase crystal being characterized by a lipase activity release rate of between about 0.1% and about 100% per day.  
     
     
         114 . The controlled dissolution crosslinked lipase crystal according to  claim 113 , wherein said lipase activity release rate is determined by a factor selected from the group consisting of: the degree of crosslinking of said crosslinked lipase crystal, the length of time of exposure of said lipase crystals to the crosslinker, the amino acids residues involved in the crosslinks, whether the crosslinker is homobifunctional or heterobifunctional, the rate of addition of the crosslinking agent to said lipase crystals, the nature of the crosslinker, the chain length of the crosslinker, the surface area of said crosslinked lipase crystal, the size of said crosslinked lipase crystal, the shape of said crosslinked lipase crystal and combinations thereof.  
     
     
         115 . The controlled dissolution crosslinked lipase crystal according to  claim 98 , said lipase crystal being substantially insoluble and stable in a composition under storage conditions and substantially soluble and active under conditions of use of said composition.  
     
     
         116 . The controlled dissolution crosslinked lipase crystal according to  claim 115 , wherein said composition is selected from the group consisting of cleaning agents, detergents, pharmaceuticals, therapeutics, veterinary compounds, personal care compositions, cosmetics, foods, feeds, vaccines, pulp, paper, textile processing, diagnostics and formulations for decontamination.  
     
     
         117 . The controlled dissolution crosslinked lipase crystal according to  claim 116 , 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.  
     
     
         118 . The controlled dissolution crosslinked lipase crystal according to  claim 116 , 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.  
     
     
         119 . The controlled dissolution crosslinked lipase crystal according to  claim 98 , said crystal having a longest dimension of between about 0.01 μm and about 500 μm.  
     
     
         120 . The controlled dissolution crosslinked lipase crystal according to  claim 119 , said crystal having a longest dimension of between about 0.1 μm and about 50 μm.  
     
     
         121 . The controlled dissolution crosslinked lipase crystal according to  claim 98 , said crystal having a shape selected from the group consisting of: spheres, needles, rods, plates, rhomboids, cubes, bipryamids and prisms.  
     
     
         122 . A controlled release formulation comprising a controlled dissolution crosslinked lipase crystal according to  claim 98 , said lipase crystal being substantially insoluble under storage conditions and capable of releasing its lipase activity in vivo at a controlled rate.  
     
     
         123 . The controlled release formulation according to  claim 122 , said formulation being capable of administration by parenteral or non-parenteral route.  
     
     
         124 . The controlled release formulation according to  claim 123 , said formulation being capable of administration by oral, pulmonary, nasal, aural, anal, dermal, ocular, intravenous, intramuscular, intraarterial, intraperitoneal, mucosal, sublingual, subcutaneous or intracranial route.  
     
     
         125 . The controlled release formulation according to  claim 122 , said formulation being capable of administration by oral route and said crosslinked lipase crystal being substantially insoluble under gastric pH conditions and substantially soluble under small intestine pH conditions.  
     
     
         126 . The controlled release formulation according to  claim 122 , said formulation being selected from the group consisting of tablets, liposomes, granules, spheres, microspheres, microparticles and capsules.  
     
     
         127 . The controlled dissolution crosslinked lipase crystal according to  claim 98 , wherein said crosslinking agent is bis[sulfosuccinimidyl]suberate (BS).