Process for the production of biodegradeable, functionalised polymer particles, and use thereof as pharmaceutical supports

Abstract

The invention relates to a method for producing biodegradable, functionalised polymer particles, and to the use of the same as medicament carriers.

1 - 29 . (canceled) 
     
     
         30 . A process for producing polymer particles, the process comprising:
 A) providing a cell comprising at least one nucleic acid that codes for a fusion protein, the fusion protein comprising   (a) a polymer synthase, and   (b) at least one protein selected from an oligopeptide, antibody, non-catalytic protein or enzyme, fused with the polymer synthase;   B) cultivating the cell in a culture medium so that the cell produces the fusion protein from the at least one nucleic acid and produces polymer particles; and   C) separating the polymer particles from the cultivated cell to produce a composition comprising polymer particles.   
     
     
         31 . A process according to  claim 30 , wherein the polymer synthase is from  Ralstonia, Alcaligenes, Pseudomonas, Aeromonas  or  Thiocapsa.    
     
     
         32 . A process according to  claim 30 , wherein the culture medium comprises at least one hydroxy fatty acid. 
     
     
         33 . A process according to  claim 30 , wherein the cell is selected from  Escherichia, Ralstonia, Alcaligenes, Pseudomonas, Halobiforma Aeromonas , and  Thiocapsa.    
     
     
         34 . A process according to  claim 30 , wherein the cell is selected from  Ralstonia eutropha, Alcaligenes latus, Escherichia coli, Pseudomonas fragi, Pseudomonas putida, Pseudomonas oleovorans, Pseudomonas aeruginosa, Pseudomonas fluorescens, Halobiforma haloterrestris, Aeromonas punctata  or  Thiocapsa pfennigii.    
     
     
         35 . A process according to  claim 30 , wherein the polymer particles have a diameter of 10 nm to 3 μm. 
     
     
         36 . A process according to  claim 30 , wherein the polymer particles have a diameter of 10 nm to 900 nm. 
     
     
         37 . A process according to  claim 30 , wherein the polymer particles have a diameter of 10 nm to 100 nm. 
     
     
         38 . A process according to  claim 30 , wherein at least one dye is added to the culture medium and incorporated into the particles. 
     
     
         39 . A process according to  claim 30 , further comprising
 D) chemically modifying the polymer synthase by contacting the polymer synthase with a coupling reagent.   
     
     
         40 . A process according to  claim 39 , wherein the coupling reagent is selected from the group consisting of bis(2-oxo-3-oxazolydinyl)phosphonic chloride (BOP—Cl), bromotrispyrrolidinophosphonium hexafluorophosphate (PyBroP), benzotriazol-1-yl-oxy-trispyrrolidinophosphonium hexafluorophosphate (PyBOP), n-hydroxysuccinimide biotin, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), dicyclohexylcarbodiimide, disuccinimidyl carbonate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), bis(2-oxo-3-oxazolydinyl)phosphine, diisopropylcarbodiimide (DIPC), 2-(1H-benzotrioxazolyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(5-norbornene-2,3-dicarboxyimido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), para-nitrophenylchloroformate, and O-(n-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU). 
     
     
         41 . A process according to  claim 30 , further comprising
 D) binding a biologically active substance to the fusion protein, wherein the biologically active substance is selected from   i) dideoxyinosine, floxuridine, 6-mercaptopurine, doxorubicin, daunorubicin, 1-darubicin, cisplatin, methotrexate, taxol, antibiotics, anticoagulants, germicides, antiarrhythmic agents and active ingredient precursors or derivatives thereof, or   ii) insulin, calcitonin, ACTH, glucagons, somatostatin, somatotropin, somatomedin, parathyroid hormone, erythropoietin, hypothalamic release factors, prolactin, thyroid-stimulating hormone, endophins, enkephalins, vasopressins, non-naturally occurring opiates, superoxide dismutase, antibodies, interferons, asparaginase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, trypsin, chymotrypsin or pepsin, or   iii) an oligopeptide, antibody, non-catalytic protein or enzyme.   
     
     
         42 . A process according to  claim 30 , wherein the protein is selected from insulin, calcitonin, ACTH, glucagons, somatostatin, somatotropin, somatomedin, parathyroid hormone, erythropoietin, hypothalamic release factors, prolactin, thyroid-stimulating hormone, endophins, enkephalins, vasopressins, non-naturally occurring opiates, superoxide dismutase, antibodies, interferons, asparaginase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, trypsin, chymotrypsin or pepsin. 
     
     
         43 . A process according to  claim 30 , wherein the protein is an antibody. 
     
     
         44 . A process according to  claim 30 , wherein the cell comprises two or more different nucleic acids that code for different fusion proteins. 
     
     
         45 . A process according to  claim 30 , wherein the cell comprises three or more different nucleic acids that code for different fusion proteins. 
     
     
         46 . A process according to  claim 30 , further comprising removing a surface-bound protein from the polymer particles. 
     
     
         47 . A process according to  claim 30 , wherein the composition consists essentially of polymer particles having surface-bound proteins. 
     
     
         48 . A method of binding a target protein comprising
 A) providing a composition of polymer particles produced by a method according to  claim 30 , wherein optionally a coupling reagent is bound to the fusion protein, and   B) contacting the composition with a sample comprising a target protein selected from an oligopeptide, antibody, non-catalytic protein or enzyme so that the protein or the coupling reagent binds the target protein.   
     
     
         49 . A process according to  claim 30 , wherein the cell further comprises one or more nucleic acids that code for one or more additional fusion proteins, the one or more additional fusion proteins comprising
 (a) a polymer particle binding domain, or   (b) a protein involved in the formation of the polymer particles, the protein comprising a polymer particle binding domain,   the additional fusion protein further comprising   i) at least one protein selected from an oligopeptide, antibody, non-catalytic protein or enzyme, or   ii) at least one binding domain capable of binding one or more proteins or one or more coupling reagents, wherein the protein is selected from an oligopeptide, antibody, non-catalytic protein or enzyme, or   iii) at least one protein and at least one binding domain capable of binding one or more biologically active substances or one or more coupling reagents, wherein the protein is selected from an oligopeptide, antibody, non-catalytic protein or enzyme, or   iv) a combination thereof.   
     
     
         50 . A process according to  claim 30 , wherein the protein involved in the formation of polymer particles is selected from a nucleic acid coding for a polymer depolymerase, a polymer regulator, a polymer synthase, and a particle size-determining protein, or a combination thereof. 
     
     
         51 . A process according to  claim 30 , wherein the polymer synthase is from  Ralstonia eutropha, Pseudomonas oleovorans, Pseudomonas putida, Pseudomonas aeruginosa, Aeromonas punctata  or  Thiocapsa pfennigii.