PROCESS FOR THE CHEMICAL IMMOBILIZATION OF BIOLOGICALLY ACTIVE SUBSTANCES ON SILICA

Abstract: A process is disclosed for the chemical immobilization of biologically active substances having a proteinic structure on SiO₂ supports, which comprises preparing a support of SiO₂ functionalised with organic silanes complying with the following general formula wherein: R₁ and R₂, either equal to, or different from, each other, are Cl, Br, CH₃, NO₂, H or NH₂, R II , R III and R IV , either equal to, or different from, one another, are alkyl or alkoxy groups containing from 1 to 10 carbon atoms, R I is equal to (CH₂) p -X-(CH₂) q , wherein X is CH₂ or a mono- or poly-condensed aromatic group, or NH or O, p and q, either equal to, or different from, each other, are integers which can have values comprised within the range of from 0 to 10, with the value of 0 being excluded when X is either NH or O, and coupling the silica functionalised with the organic silane with a biologically active substance having a proteinic structure, after previously preparing a diazo salt on such a functionalised silica.

Description: The present invention relates to a process for chemically and stably immobilizing biologically active substances such as proteins (enzymes, antibodies, biological receptors, and so forth). Such a process can be defined as economically advantageous and easy to achieve without the use of complex chemical reactions. The use of enzymes (biocatalysts) immobilized on solid matrices is largely used for many industrial applications: e.g., for the production of intermediates, for preparing semi-synthetic penicillins, alimentary products and drugs in general. The main advantage of the use of immobilized biocatalysts, both for continuous and batchwise processes, is that the catalyst can be re-used, with a consequent decrease in production costs. A comprehensive survey about the immobilization of enzymes on solid supports is reported in "Trends in Biotechnology", vol. 3, 11, 1985, 276 Howard, H. Weetall. In particular, as regards the chemical immobilization of biocatalysts on supports of inorganic type, such as silica, the process is carried out according to different routes, by using more or less complex chemical reactions. One largely used from the available methods is the one based on the reaction of coupling, via a diazo-derivative obtained on a silica preliminarily treated with a suitable silane containing an amino-aromatic group. Such a process is based on the formation of a diazo-linkage between the enzyme, which is a protein, and the aryl-diazo group present on the inorganic support, obtained after treatment with aminopropyl-triethoxy-silane and subsequent chemical treatments. The reaction occurs thanks to the L-histidine and L-tyrosine moieties present in the protein. On the market, glass beads already exist, which are treated with organosilanes, and are traded by the Companies PIERCE CHEMICAL and ELECTRONUCLEONICS, both in diazo salt, and in nitro-group bearing form. Another potential application for this type of reaction on silanized silica beads can be as an immuno-adsorbent. In fact, the antibody or antigen, provided that is constituted by a protein immobilized via diazo-linkage on a silanized silica support, is used in the purification on column of the antigen or of the specific antibody, as reported by J. Immunological Methods 50, 57, 1982, Kennedy J. F., Keep P. A. and Catty D. Furthermore, the use of antibodies immobilized on silica could find some applications in the field of the immunodiagnostic tests. In the prior art, the generally used route is illustrated, which provides for 3-aminopropyl-triethoxy-silane to be used as the silica-functionalising agent, and consists of a whole series of steps in order to come to the desired product, according to the following scheme: wherein: Tyr = L-tyrosine residues in the protein. As one can see from the above reaction scheme, in order to achieve the end product five steps are necessary, two of which, are delicate, and namely: the (2) step, wherein hydrochloric acid is released, which has to be removed from the reaction environment; and the (3) step, owing to the presence of a catalyst and the consequent complications inhering it its separation after the reaction, due to the fact that it must operate with solid-state reactants. The possibility exists, that an aminophenyl-trimethoxy-silane may be used (as proposed by Mottola H.A., Analytical Chemistry 55, 2089, 1983): the evident advantage thereof is that it can be directly diazotised and coupled with the protein. But, besides the considerably high cost of this product, the problems exist which arise from the considerable steric hyndrance due to the absence of a "spacer" between the silicon oxide moiety and the aromatic ring, and therefore the relative closeness of the amino group to the siliceous matrix. To the above, the fact has to be added that the commercial product is constituted by a mixture of the three isomers. Other methods for activating silica via diazo compounds are reported on Talanta 32, 8B, 763, 1985 and Fres. Z. Anal. Chem. 322, 47, 1985, but anyway with very relative advantages. EP-A-291 130 discloses a biosensor having an enzymatic membrane containing a layer of silicon oxide adhering to the membrane by means of a polysiloxanic matrix. It is therein excluded the use of a monomeric silane. It has been surprisingly ascertained that, by using certain organosilanes, to be defined hereinafter, the immobilization of proteinous biologically active substances can be obtained with a few steps, higher yields than with the prior art procedures, and cheaply. The invention, therefore, provides a process for chemically immobilizing on a silica support a proteinous biologically active substance by functionalizing said silica support with an organic silane, characterized in that it comprises the steps of: 1) Functionalizing the silica support with an exclusively monomeric organic silane selected from those having the general formula: wherein: R₁ and R₂, equal to, or different from one another, are selected from -Cl, -Br, -CH₃, -NO₂, -H and -NH₂; R¹ is (CH₂) p -X-(CH₂) q , X is selected from -CH₂, monocondensed aromatic groups, polycondensed aromatic groups, =NH, and -O-, and p and q , equal to, or different from one another, are integers from 0 to 10, the 0 value being excluded when X is either =NH or is -O-; R II , R III and R IV , equal to, or different from each other, are selected from the C₁-C₁₀ alkyl- and alkoxy groups; 2) Diazotizing the functionalized silica by preparing a diazo salt thereon, and 3) Contacting the proteinous biologically active substance concerned with the functionalized and diazotized silica to couple them chemically together. The exclusively monomeric organosilanes of the general formula (I) are prepared, as disclosed, for example, in EP-A-86201103.8, or a derivative thereof, with an amino-silane of the general formula: according to the following reaction scheme: wherein R₁, R₂, R I , R II , R III and R IV have the same meanings as defined hereinabove. The preferred aminosilanes are: 3-aminopropyl-triethoxy-silane H₂N-(CH₂)₃-Si-(OC₂H₅)₃, aminomethyl-triethoxysilane H₂N-CH₂-Si-(OC₂H₅)₃, N-(2-aminoethyl)-aminopropyl-trimethoxy-silane H₂N-(CH₂)₂-NH-(CH₂)₃-Si-(OCH₃)₃, N-(2-aminoethyl)-aminopropyl-triethoxy-silane H₂N-(CH₂)₂-NH-(CH₂)₃-Si-(OC₂H₅)₃, N-(2-aminoethyl)-aminopropyl-dimethoxy-methyl-silane The reaction of synthesis is carried out by gradually adding isatoic anhydride to the amino-silane, in the presence or in the absence of a solvent. The solvents are alcohols, ethers, chlorinated solvents and are water-free. The reaction is carried out under room pressure and at room temperature. The functionalisation of the silica with the organo-silanes of general formula (I) can be accomplished by placing the same silica into contact with the selected organo-silane under reaction conditions well-known to those skilled in the art. Once that the functionalised silica is prepared, and the coupling-capable biologically active substance is selected, the functionalised silica is diazotised. A second method for functionalising silica consists in placing silica into contact with aminopropyl-triethoxy-silane (AAPS) and then carrying out the reaction with isatoic anhydride under heterogeneous phase conditions. The diazotisation is carried out with nitrous acid in a known way, at a temperature comprised within the range of from 0°C to 25°C in water-alcoholic solution (methyl-, ethyl-, propyl alcohol-water solution), at an acidic pH value. The coupling between the functionalised and diazotised silica and the biologically active substance is carried out by placing into intimate contact the two species, and operating at a temperature comprised within the range of from o°C to 25°C, and at a pH comprised within the range of from 5 to 8. By considering 2-amino-N(3-triethoxy-silyl-propyl)benzamide (anthranylamido-propyl-triethoxy-silane) of formula: as an exemplifying functionalising agent for the silica, and, as the substance, an enzyme or an antibody, the reaction scheme is as follows: wherein: Tyr = L-Tyrosine residues in the protein. The fact that, notwithstanding the -NH₂ group is in the ortho-position relatively to the carbonyl in the group the -NH₂ function, as its diazo salt, is reactive towards the biological active substance, is quite surprising. The above disclosed process of chemical immobilization can be also carried out on other types of solid inorganic supports, such as, e.g., supports of TiO₂, SnO₂, and so forth. Some examples are now given, which have the purpose of better illustrating the present invention, it being understood that the same invention has not to be considered as being limited by them or to the. Example 1 Trypsin Immobilization a) Reaction of silica with silane 2 g of SiO₂ (Merck 60, 70-230 mesh, 0.063-0.200 mm) is treated with 2.0 ml of anthranylamide-propyl-triethoxy-silane (AAPS) in 20 ml of anhydrous toluene. The reaction system is maintained 16 hours under refluxing condition sunder vacuum, with mechanical stirring. The treated silica is filtered, and washed a plurality of times with hot toluene, and finally with absolute ethanol. The silica charged to the oven in order to be dried weighed 2.4 g. b) Diazo salt formation To a small jacketed reactor, 1 g of SiO₂ treated with silane is charged, and to it 15 ml of 2 N HCl is added. With mechanical stirring, and at the temperature of 5°C, 5 l of H₂O containing 1 g of NaNO₂ is added dropwise. The overall reaction time is of 2 hours. The reaction product is subsequently repeatedly washed on a porous septum with 0.1 M phosphate buffer at pH 7. c) Immobilization of trypsin via diazo-coupling To a small jacketed reactor, 1 g of treated SiO₂ as a diazo salt, 10 ml of 50 mM phosphate buffer at pH 7.5, 20 mg of trypsin (Boehringer Biochemia Robin) and 0.015 M CaCl₂ are charged. The solution is kept with slow mechanical stirring at the temperature of 5°C for about 2 hours. The silica is subsequently washed on a porous septum with buffer solution. The treated and washed silica is stored in 0.046 M Tris buffer at pH 8.1 at 5°C, 50 mM CaCl₂. Hydrolytic Activity of the Trypsin in a Closed-Loop Flow-System and in an Open-Loop Flow System The system inside which the measurement tests are carried out is constituted by a peristaltic pump, a spectrophotometer containing a temperature-controlled cuvette, and a small reactor inside which the enzyme immobilized on silica is present. The whole system is connected to a series of rubber hoses as shown in Figure 1, wherein: A = substrate; W = drain; P = peristaltic pump; V = valves for closed/open-loop system; R = small reactor containing SiO₂ + linked enzyme; S = spectrophotometer; C = temperature-controlled cuvette; E = estinction. The solution which contains the substrate is constituted by 46 mM Tris buffer pH 8.1, 100 mg of CaCl₂, 0.5 mM, 1 mM, 2 mM benzoyl-L-arginine-ethyl-ester (BAEE). The hydrolytic activity is checked by measuring the extinction at 254 nm of the solution after the passage of the immobilized enzyme. In Figure 2, the reaction kinetics are plotted as a function of the concentration of the substrate in a closed-loop-flow system (a: BAEE = 2 mM - T = 25°C; b: BAEE = 1 mM - T = 25°C; c: BAEE = 0.5 mM - T = 25°C). In Figure 3, the same reaction kinetics are reported for an open-loop-flow system (a: BAEE = 2 mM - T = 25°C; b: BAEE = 1 mM - T = 25°C; c: BAEE = 0.5 mM - T = 25°C). In Figure 4, the values of maximum activity of the enzyme (closed-loop system) are reported, wherein the activity is expressed as the extinction values as a function of substrate concentration. A good linearity can be observed, even if in the presence of a possible inhibition of the substrate. Example 2 Immobilization of Chymotrypsin The (a) and (b) steps are the same as of Example 1. e) Immobilization of chymotrypsin via diazo-coupling. To a small jacketed reactor, 1 g of treated SiO₂ as a diazo salt, 10 ml of 0.1 M phosphate buffer at pH 7.4, 2 mg of indole, 25 mg of chymotrypsin (Boehringer Biochemia Robin) are added. The solution is maintained with slow mechanical stirring, and at the temperature of 5°C for about 2 hours. Finally, the silica is washed a plurality of times with buffer solution on a porous septum. The silica is stored in 0.1 M Tris buffer at pH 7.8, 50 mM CaCl₂, at 5°C. In Table 1, the amounts of the residual aminoacids are reported as % by weight, after treatment of the enzyme-containing silica in 6 M HCl at 100°C for 18 hours. The values were obtained with an automatic aminoacid analyser. Example 3 Immobilization of Thermolysin The (a) and (b) steps are the same as of Example 1. e) Immobilization of thermolysin via diazo-coupling. To a small jacketed reactor, 1 g of treated SiO₂ as a diazo salt, and a solution containing 15 ml of phosphate buffer at pH 7.5, 30 mg of thermolysin (Agrade, Lot 100540, Calbiochem, Calif.), 10 mM CaCl₂ are added. The solution is maintained with slow mechanical stirring at the temperature of 5°C for about 2 hours. The silica is finally washed with buffer solution on a porous septum. The silica is stored in Tris buffer - 50 m HCl at 5°C. In Table 2, the amounts of the residual aminoacids are reported as % by weight, after treatment in 6 N HCl at 100°C for 18 hours of the enzyme-containing silica. The values were obtained with an automatic aminoacid analyser.

Claims: Process for chemically immobilizing on a silica support a proteinous biologically active substance by functionalizing said silica support with an organic silane, characterized in that it comprises the steps of: 1) Functionalizing the silica support with an exclusively monomeric organic silane selected from those having the general formula: wherein: R₁ and R₂, equal to, or different from one another, are selected from -Cl, -Br, -CH₃, -NO₂, -H and -NH₂; R¹ is (CH₂) p -X-(CH₂) q , X is selected from -CH₂, monocondensed aromatic groups, polycondensed aromatic groups, =NH, and -O-, and p and q , equal to, or different from one another, are integers from 0 to 10, the 0 value being excluded when X is either =NH or is -O-; R II , R III and R IV , equal to, or different from each other, are selected from the C₁-C₁₀ alkyl- and alkoxy groups; 2) Diazotizing the functionalized silica by preparing a diazo salt thereon, and 3) Contacting the proteinous biologically active substance concerned with the functionalized and diazotized silica to couple them chemically together. Process according to Claim 1, wherein step 2) is carried out with nitrous acid, at a temperature of from 0°C to 25°C, in a water-alcoholic solution at an acidic pH. Process according to Claim 1, wherein step 3) is carried out at a temperature of from 0°C to 25°C and at a pH of from 5 to 8. Process according to Claim 1, wherein said exclusively monomeric organic silane is the product of the reaction of the isatoic acid anhydride, or a derivative thereof, with: 3-aminopropyl-triethoxy-silane, of formula: H₂N-(CH₂)₃-Si-(OC₂H₅)₃ Process according to Claim 1, wherein said exclusively monomeric organic silane is the product of the reaction of the isatoic acid anhydride, or a derivative thereof, with: aminomethyl-triethoxysilane, of formula: H₂N-CH₂-Si-(OC₂H₅)₃ Process according to Claim 1, wherein said exclusively monomeric organic silane is the product of the reaction of the isatoic acid anhydride, or a derivative thereof, with: (N-(2-aminoethyl)-aminopropyl-trimethoxy silane) of formula: H₂N-(CH₂)₂-NH-(CH₂)₃-Si-(OCH₃)₃ Process according to Claim 1, wherein said exclusively monomeric organic silane is the product of the reaction of the isatoic acid anhydride, or a derivative thereof, with: (N-(2-aminoethyl)-aminopropyl-triethoxy silane) of formula: H₂N-(CH₂)₂-NH-(CH₂)₃-Si-(OC₂H₅)₃ Process according to Claim 1, wherein said exclusively monomeric organic silane is the product of the reaction of the isatoic acid anhydride, or a derivative thereof, with: (N-(2-aminoethyl)-aminopropyl-dimethoxy methyl silane) of formula: Process according to Claim 1, wherein step 1) is carried out by contacting the silica with the selected exclusively monomeric organic silane. Process according to Claim 1, wherein step 1) is carried out by first contacting the silica with aminopropyl-triethoxy-silane, and reacting the resultant product with the isatoic acid anhydride under heterogeneous phase conditions thereafter.

Inventors: BARBARO, AURELIO, BATTILOTTI, MASSIMO, COLAPICCHIONI, CLAUDIO, FIORINI, MARIO
Applicants: ENIRICERCHE S.P.A.
Application year: 1988
Main classification: C12N11
IPC codes: G01N33, C12N11