Carbohydrate crosslinked glycoprotein crystals

Abstract

The present invention relates to the field of carbohydrate crosslinked glycoprotein crystals. Advantageously, such crosslinked glycoprotein crystals display stability to harsh environmental conditions, while maintaining the structural and functional integrity of the glycoprotein backbone. According to one embodiment, this invention relates to methods for concentrating proteins that have been modified by carbohydrates and for releasing their activity at controlled rates. This invention also provides methods for producing carbohydrate crosslinked glycoprotein crystals and methods for using them in pharmaceutical formulations, vaccines, immunotherapeutics, personal care compositions, including cosmetics, veterinary pharmaceutical compositions and vaccines, foods, feeds, diagnostics, cleaning agents, including detergents and decontamination formulations. The physical and chemical characteristics of carbohydrate crosslinked glycoprotein crystals render them particularly useful as sorbents for separations, such as chiral chromatography, or affinity chromatography—which are based on specific interactions between the active binding site of the glycoprotein component of the crystals and the substance or molecule of interest. Such characteristics also render carbohydrate crosslinked glycoprotein crystals useful as catalytic and binding components for the production of biosensing devices.

We claim:  
     
         1 . A crosslinked glycoprotein crystal, said crystal being crosslinked through one or more carbohydrate moieties in said glycoprotein.  
     
     
         2 . The crosslinked glycoprotein crystal according to  claim 1 , wherein said crystal is crosslinked with a multifunctional crosslinking agent.  
     
     
         3 . The crosslinked glycoprotein crystal according to  claim 1 , wherein said crystal is crosslinked with a diamine crosslinking agent.  
     
     
         4 . The crosslinked glycoprotein crystal according to  claim 3 , wherein said diamine crosslinking agent is selected from the group consisting of hexamethylenediamine, diaminooctane, and ethylenediamine.  
     
     
         5 . A crosslinked glycoprotein crystal, said crystal being crosslinked through one or more carbohydrate moieties in said glycoprotein and through one or more amino acid side chain functional groups in said glycoprotein.  
     
     
         6 . The crosslinked glycoprotein crystal according to  claim 5 , wherein said crystal is crosslinked through one or more carbohydrate moieties in said glycoprotein with a diamine crosslinking agent and said crystal is crosslinked through one or more amino acid side chain functional groups in said glycoprotein with a multifunctional crosslinking agent.  
     
     
         7 . The crosslinked glycoprotein crystal according to  claim 6 , wherein said diamine crosslinking agent is selected from the group consisting of hexamethylenediamine and diaminooctane and wherein said multifunctional crosslinking agent is selected from the group consisting of glutaraldehyde, succinic anhydride, phenylglutaric anhydride, salicylaldehyde, acetimidate, formalin, acrolein, succinic semialdehyde, butyraldehyde, dodecylaldehyde, glyceraldehyde or trans-oct-2-enal.  
     
     
         8 . The crosslinked glycoprotein crystal according to  claim 1  or  5 , said glycoprotein crystal having at least about 2 fold greater stability as measured by pronase digestion, thermal stability, or activity in 50% ethanol than the soluble form of the glycoprotein that is crystallized to form said glycoprotein crystal that is crosslinked.  
     
     
         9 . The crosslinked glycoprotein crystal according to  claim 1  or  5 , said glycoprotein crystal having greater stability at an alkaline pH or an acidic pH than the soluble form of the glycoprotein that is crystallized to form said glycoprotein crystal that is crosslinked.  
     
     
         10 . The crosslinked glycoprotein crystal according to  claim 1  or  5 , said glycoprotein crystal having greater stability at high temperature than the soluble form of the glycoprotein that is crystallized to form said glycoprotein crystal that is crosslinked.  
     
     
         11 . The crosslinked glycoprotein crystal according to  claim 1  or  5 , said glycoprotein crystal having activity in an organic solvent or aqueous/organic solvent mixture that is at least about 2 times greater than the activity of an equivalent amount of the soluble form of the glycoprotein that is crystallized to form said glycoprotein crystal that is crosslinked.  
     
     
         12 . The crosslinked glycoprotein crystal according to  claim 8 , wherein the stability of said glycoprotein crystal is at least about 5 to about 10 times that of the soluble form of the glycoprotein that is crystallized to form said glycoprotein crystal that is crosslinked.  
     
     
         13 . The crosslinked glycoprotein crystal according to  claim 1 , said glycoprotein crystal being substantially insoluble and stable in a composition under storage conditions and substantially soluble and active under conditions of use of said composition.  
     
     
         14 . The crosslinked glycoprotein crystal according to  claim 13 , wherein said composition is selected from the group consisting of pharmaceuticals, vaccines, immunotherapeutics, immune modifiers, adjuvants, veterinary pharmaceuticals and vaccines, agricultural compounds, foods, feeds, cosmetics, detergents, and decontamination agents.  
     
     
         15 . The crosslinked glycoprotein crystal according to  claim 1 , said glycoprotein 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.  
     
     
         16 . The crosslinked glycoprotein crystal according to  claim 1  or  5 , wherein said glycoprotein is selected from the group consisting of hormones, enzymes, antibodies, viral receptors, viral surface glycoproteins, parasite glycoproteins, parasite receptors, T-cell receptors, MHC molecules, immune modifiers, tumor antigens, mucins, inhibitors, growth factors, trophic factors, cytokines, lymphokines, cytokines, toxoids, nerve growth hormones, blood clotting factors, adhesion molecules, multidrug resistance proteins, therapeutic proteins, adenylate cyclases, bone morphogenic proteins and lectins.  
     
     
         17 . The crosslinked glycoprotein crystal according to  claim 16 , wherein said enzyme is selected from the group consisting of hydrolases, isomerases, lyases, ligases, transferases and oxidoreductases.  
     
     
         18 . The crosslinked glycoprotein crystal according to  claim 17 , wherein said enzyme is selected from the group consisting of lipases and oxidases.  
     
     
         19 . The crosslinked glycoprotein crystal according to  claim 17 , wherein said enzyme is glucose oxidase.  
     
     
         20 . The crosslinked glycoprotein crystal according to  claim 17 , wherein said enzyme is  Candida rugosa  lipase.  
     
     
         21 . The crosslinked glycoprotein crystal according to  claim 1  or  5 , said crystal having a longest dimension of between about 0.01 μm and about 500 μm.  
     
     
         22 . The crosslinked glycoprotein crystal according to  claim 21 , said crystal having a longest dimension of between about 0.1 μm and about 50 μm.  
     
     
         23 . The crosslinked glycoprotein crystal according to  claim 1  or  5 , said crystal having a shape selected from the group consisting of spheres, needles, rods, plates, rhomboids, cubes, squares, bipryamids and prisms.  
     
     
         24 . A method for producing a crosslinked glycoprotein crystal comprising the steps of: 
 (a) reacting a glycoprotein crystal with an oxidizing agent to oxidize the carbohydrate moieties of said glycoprotein; and    (b) reacting said glycoprotein crystal with a crosslinking agent, under conditions sufficient to induce crosslinking of said crystal through the carbohydrate moieties of said glycoprotein.    
     
     
         25 . The method for producing a crosslinked glycoprotein crystal according to  claim 24 , wherein said crosslinking agent is a multifunctional crosslinking agent.  
     
     
         26 . The method for producing a crosslinked glycoprotein crystal according to  claim 24 , wherein said crosslinking agent is a diamine crosslinking agent.  
     
     
         27 . The method for producing a crosslinked glycoprotein crystal according to  claim 26 , wherein said diamine crosslinking agent is selected from the group consisting of hexamethylenediamine, diaminooctane,and ethylenediamine.  
     
     
         28 . The method for producing a crosslinked glycoprotein crystal according to  claim 24 , wherein said oxidizing agent is sodium periodate (NaIO 4 ).  
     
     
         29 . A method for producing a crosslinked glycoprotein crystal comprising the step of reacting a glycoprotein crystal with a first crosslinking agent, said first crosslinking agent being capable of crosslinking said crystal through the amino acid side chain functional groups of said glycoprotein and at least a second crosslinking agent, said second crosslinking agent being capable of crosslinking said glycoprotein through carbohydrate moieties of said glycoprotein.  
     
     
         30 . The method according to  claim 29 , wherein said glycoprotein crystal is crosslinked with said first and said second crosslinking agent at the same time or in sequence.  
     
     
         31 . A method for producing a crosslinked glycoprotein crystal comprising the steps of: 
 (a) reacting a glycoprotein crystal with a first crosslinking agent, under conditions sufficient to induce crosslinking through one or more carbohydrate moieties of said glycoprotein; and    (b) reacting said crosslinked glycoprotein crystal with at least a second crosslinking agent, under conditions sufficient to induce crosslinking of said crystal through one or more carbohydrate moieties of said glycoprotein, said second crosslinking agent being different from said first crosslinking agent.    
     
     
         32 . A method for producing a crosslinked glycoprotein crystal comprising the steps of: 
 (a) reacting a glycoprotein crystal with a first crosslinking agent, under conditions sufficient to induce crosslinking through one or more amino acid side chain functional groups of said glycoprotein; and    (b) reacting said crosslinked glycoprotein crystal with least a second crosslinking agent, under conditions sufficient to induce crosslinking of said crystal through one or more carbohydrate moieties of said glycoprotein.    
     
     
         33 . A method for producing a crosslinked glycoprotein crystal comprising the steps of: 
 (a) reacting a glycoprotein crystal with a first crosslinking agent, under conditions sufficient to induce crosslinking through one or more amino acid side chain functional groups of said glycoprotein; and    (b) reacting said crosslinked glycoprotein crystal with a second crosslinking agent and at least a third crosslinking agent, said second crosslinking agent being capable of crosslinking said glycoprotein through one or more amino acid side chain functional groups of said glycoprotein and said third crosslinking agent being capable of crosslinking said glycoprotein through one or more carbohydrate moieties of said glycoprotein.    
     
     
         34 . A method for producing a crosslinked glycoprotein crystal comprising the steps of: 
 (a) reacting a glycoprotein crystal with a first crosslinking agent, under conditions sufficient to induce crosslinking through one or more carbohydrate moieties of said glycoprotein; and    (b) reacting said crosslinked glycoprotein crystal with a second crosslinking agent and at least a third crosslinking agent, said second crosslinking agent being capable of crosslinking said glycoprotein through one or more amino acid side chain functional groups of said glycoprotein and said third crosslinking agent being capable of crosslinking said glycoprotein through one or more carbohydrate moieties of said glycoprotein.    
     
     
         35 . The method for producing a crosslinked glycoprotein crystal according to any one of claims  29 ,  31 ,  32 ,  33 , or  34 , wherein said crosslinking agent capable of crosslinking said glycoprotein through the carbohydrate moieties of said glycoprotein is a multifunctional crosslinking agent.  
     
     
         36 . The method for producing a crosslinked glycoprotein crystal according to any one of claims  29 ,  31 ,  32 ,  33 , or  34  wherein said crosslinking agent capable of crosslinking said glycoprotein through the carbohydrate moieties of said glycoprotein is a diamine crosslinking agent.  
     
     
         37 . The method for producing a crosslinked glycoprotein crystal according to  claim 36 , wherein said diamine crosslinking agent is selected from the group consisting of hexamethylenediamine and diaminooctane, and ethylenediamine.  
     
     
         38 . The method for producing a crosslinked glycoprotein crystal according to any one of claims  29 ,  31 ,  32 ,  33 , or  34 , wherein said crosslinking agent capable of crosslinking said glycoprotein through the amino acid side chains of said glycoprotein is a multifunctional crosslinking agent.  
     
     
         39 . The method for producing a crosslinked glycoprotein crystal according to  claim 38 , wherein said multifunctional crosslinking agent is selected from the group consisting of glutaraldehyde, succinic anhydride, phenylglutaric anhydride, salicylaldehyde, acetimidate, formalin, acrolein, succinic semialdehyde, butyraldehyde, dodecylaldehyde, glyceraldehyde or trans-oct-2-enal.  
     
     
         40 . The method for producing a crosslinked glycoprotein crystal according to any one of claims  29 ,  31 ,  32 ,  33 , or  34 , wherein said glycoprotein is selected from the group consisting of hormones, enzymes, antibodies, viral receptors, viral surface glycoproteins, parasite glycoproteins, parasite receptors, T-cell receptors, MHC molecules, immune modifiers, tumor antigens, mucins, inhibitors, growth factors, trophic factors, cytokines, lymphokines, cytokines, toxoids, nerve growth hormones, blood clotting factors, adhesion molecules, multidrug resistance proteins, therapeutic glycoproteins, adenylate cyclases, bone morphogenic proteins and lectins.  
     
     
         41 . The method according to  claim 40 , wherein said enzyme is selected from the group consisting of hydrolases, isomerases, lyases, ligases, transferases and oxidoreductases.  
     
     
         42 . The method according to  claim 41 , wherein said enzyme is selected from the group consisting of lipases and oxidases.  
     
     
         43 . The method according to  claim 41 , wherein said enzyme is glucose oxidase.  
     
     
         44 . The method according to  claim 41 , wherein said enzyme is  Candida rugosa  lipase.  
     
     
         45 . The method for producing a crosslinked glycoprotein crystal according to any one of claims  29 ,  31 ,  32 ,  33 , or  34 , wherein, prior to reacting said glycoprotein crystal with said crosslinking agent, said method further comprises the step of crystallizing said glycoprotein.  
     
     
         46 . The method according to  claim 45 , wherein the step of crystallizing said glycoprotein is carried out without deglycosylating said glycoprotein.  
     
     
         47 . The method according to  claim 46 , wherein the step of crystallizing said glycoprotein is carried out in the presence of a water miscible solvent and polyethylene glycol.  
     
     
         48 . The method for producing a crosslinked glycoprotein crystal according to any one of claims  29 ,  31 ,  32 ,  33 , or  34 , wherein, prior to reacting said glycoprotein crystal with said crosslinking agent, said method further comprises the step of oxidizing the carbohydrate moieties of said glycoprotein.  
     
     
         49 . A protein delivery system, said system comprising crosslinked glycoprotein crystals according to  claim 15 .  
     
     
         50 . A pharmaceutical controlled release formulation comprising crosslinked glycoprotein crystals according to  claim 15 .  
     
     
         51 . A vaccine comprising crosslinked glycoprotein crystals according to  claim 1  or  5 .  
     
     
         52 . A biosensor for detecting an analyte of interest in a fluid, comprising: 
 (a) a crosslinked glycoprotein crystal according to  claim 1  or  5 , wherein said glycoprotein has the activity of acting on the analyte of interest or on a reactant in a reaction in which the analyte of interest participates;    (b) a retaining means for said crosslinked glycoprotein crystal, said retaining means consisting of a material which allows contact between said crosslinked glycoprotein crystal and a fluid, said fluid containing either (1) the analyte upon which the glycoprotein acts or (2) a reactant in a reaction in which the analyte participates; and    (c) a signal transducer which produces a signal in the presence of the analyte.    
     
     
         53 . A method for detecting the presence of a substance in a sample comprising the steps of: 
 (a) contacting the sample with a crosslinked glycoprotein crystal according to  claim 1  or  5 , wherein said glycoprotein has the activity of acting on the substance or on a reactant in a reaction in which the substance participates to cause a detectable change, under conditions and for a time sufficient to permit said activity; and    (b) using a detecting means to detect the detectable change.    
     
     
         54 . A method for producing a selected product using a glycoprotein comprising the steps of: 
 (a) combining at least one substrate with at least one glycoprotein which acts upon the substrate, said glycoprotein being a crosslinked glycoprotein crystal according to  claim 1  or  5 ; and    (b) maintaining the combination produced in step (a) under conditions which permit said glycoprotein to act upon the substrate, thereby producing the selected product.    
     
     
         55 . A method for crystallizing a glycoprotein without removing the carbohydrate moieties of said glycoprotein comprising the steps of: 
 (a) concentrating pure glycoprotein to produce glycoprotein concentrate;    (b) contacting said glycoprotein concentrate with polyethylene glycol to produce glycoprotein polyethylene glycol concentrate;    (c) contacting said glycoprotein polyethylene glycol concentrate with a miscible organic solvent to produce a glycoprotein polyethylene glycol organic solvent concentrate;    (d) contacting said glycoprotein polyethylene glycol organic solvent concentrate with an acid to adjust the pH to between 4.0 and 5.0; and    (e) cooling the pH adjusted concentrate to a temperature between 4 and 6° C. for a period of time sufficient to induce crystallization.