CN114736892A - Process for extracting urokinase from modified silica gel - Google Patents
Process for extracting urokinase from modified silica gel Download PDFInfo
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- CN114736892A CN114736892A CN202210419044.2A CN202210419044A CN114736892A CN 114736892 A CN114736892 A CN 114736892A CN 202210419044 A CN202210419044 A CN 202210419044A CN 114736892 A CN114736892 A CN 114736892A
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical class O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 190
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 title claims abstract description 106
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 title claims abstract description 106
- 229960005356 urokinase Drugs 0.000 title claims abstract description 106
- 238000000034 method Methods 0.000 title claims abstract description 55
- 230000008569 process Effects 0.000 title claims abstract description 48
- 239000000741 silica gel Substances 0.000 claims abstract description 116
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 116
- 238000006277 sulfonation reaction Methods 0.000 claims abstract description 26
- 238000010828 elution Methods 0.000 claims abstract description 24
- 210000002700 urine Anatomy 0.000 claims abstract description 24
- 238000002156 mixing Methods 0.000 claims abstract description 22
- 230000021523 carboxylation Effects 0.000 claims abstract description 21
- 238000006473 carboxylation reaction Methods 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000011259 mixed solution Substances 0.000 claims abstract description 16
- 239000002994 raw material Substances 0.000 claims abstract description 16
- 239000000203 mixture Substances 0.000 claims abstract description 13
- 238000004440 column chromatography Methods 0.000 claims abstract description 11
- 238000001914 filtration Methods 0.000 claims abstract description 11
- 239000008367 deionised water Substances 0.000 claims abstract description 7
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 7
- 238000001179 sorption measurement Methods 0.000 claims abstract description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 22
- 238000006243 chemical reaction Methods 0.000 claims description 18
- 239000000945 filler Substances 0.000 claims description 15
- 239000002245 particle Substances 0.000 claims description 15
- 239000003480 eluent Substances 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 230000004048 modification Effects 0.000 claims description 12
- 238000012986 modification Methods 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 239000006167 equilibration buffer Substances 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 6
- 239000000908 ammonium hydroxide Substances 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 150000002500 ions Chemical class 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims description 5
- NTZRDKVFLPLTPU-UHFFFAOYSA-N CC[Na] Chemical compound CC[Na] NTZRDKVFLPLTPU-UHFFFAOYSA-N 0.000 claims description 5
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 5
- 239000004115 Sodium Silicate Substances 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 5
- 239000008351 acetate buffer Substances 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 150000007942 carboxylates Chemical class 0.000 claims description 5
- 238000005341 cation exchange Methods 0.000 claims description 5
- 239000007979 citrate buffer Substances 0.000 claims description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- 239000011734 sodium Substances 0.000 claims description 5
- 229910052708 sodium Inorganic materials 0.000 claims description 5
- 229910052911 sodium silicate Inorganic materials 0.000 claims description 5
- AKEJUJNQAAGONA-UHFFFAOYSA-N sulfur trioxide Chemical compound O=S(=O)=O AKEJUJNQAAGONA-UHFFFAOYSA-N 0.000 claims description 5
- HIFJUMGIHIZEPX-UHFFFAOYSA-N sulfuric acid;sulfur trioxide Chemical compound O=S(=O)=O.OS(O)(=O)=O HIFJUMGIHIZEPX-UHFFFAOYSA-N 0.000 claims description 5
- XTHPWXDJESJLNJ-UHFFFAOYSA-N sulfurochloridic acid Chemical compound OS(Cl)(=O)=O XTHPWXDJESJLNJ-UHFFFAOYSA-N 0.000 claims description 5
- 235000011149 sulphuric acid Nutrition 0.000 claims description 5
- 229920002684 Sepharose Polymers 0.000 claims description 4
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- 238000005516 engineering process Methods 0.000 abstract description 5
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- 230000000694 effects Effects 0.000 description 12
- 239000000499 gel Substances 0.000 description 9
- 239000000377 silicon dioxide Substances 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 7
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- 239000000047 product Substances 0.000 description 7
- 239000012043 crude product Substances 0.000 description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
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- 108010088842 Fibrinolysin Proteins 0.000 description 3
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- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 3
- 238000012856 packing Methods 0.000 description 3
- 229940012957 plasmin Drugs 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
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- 108010073385 Fibrin Proteins 0.000 description 2
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- 102100023038 WD and tetratricopeptide repeats protein 1 Human genes 0.000 description 2
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- 241000124008 Mammalia Species 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
- 108010051456 Plasminogen Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 208000034700 Vitreous opacities Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
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- 230000001093 anti-cancer Effects 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000005303 weighing Methods 0.000 description 1
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
- C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
- C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21068—Tissue plasminogen activator (3.4.21.68), i.e. tPA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
Abstract
The invention provides a process for extracting urokinase by modified silica gel, which comprises the following steps: the method comprises the following steps: selecting a silica gel raw material; step two: HF treatment of silica gel; step three: performing sulfonation treatment on silica gel; step four: performing silica gel carboxylation treatment; step five: mixing sulfonated silica gel and carboxylated silica gel; step six: mixing the silica gel mixed solution with urine; step seven: carrying out filtration and adsorption on the mixed solution; step eight: and (4) eluting and obtaining urokinase. The invention extracts modified silica gel, utilizes column chromatography technology to carry out column operation, the process is circulated and reciprocated for 3-5 times, the collected silica gel mixture is washed for three times by equilibrium buffer solution, and is washed for three times by deionized water, urokinase can be obtained by elution, and the loss of urokinase can be reduced; can separate urokinase better, can effectively improve the urokinase content, and the yield of liquid urokinase can reach 80-92 percent, thereby obtaining urokinase with higher purity, and the purity of the collected urokinase is more than 45 percent.
Description
Technical Field
The invention belongs to the technical field of separation and extraction, and particularly relates to a process for extracting urokinase from modified silica gel.
Background
Urokinase is an alkaline protease, is produced by kidney, is mainly present in urine of human bodies and mammals, has normal functions of human bodies when blood coagulates, but forms ascorbic acid in blood vessels under certain pathological conditions to cause serious consequences when the blood circulation is blocked.
Urokinase is a kind of protein hydrolase with strong specificity, and has esterase activity, no antigenicity, no toxicity and other side effects, and may be used clinically in treating pulmonary thrombus, coronary heart disease, cerebral thrombus, myocardial infarction, vitreous opacity, eyeground hemorrhage, intracranial hemorrhage, venous embolism, acute and chronic glomerulonephritis, etc. and also in treating fibrin deposition caused diseases and as supplementary anticancer medicine.
Urokinase is an enzyme protein isolated from healthy human urine, or obtained from human kidney tissue culture. Consists of two parts with the molecular weights of 33000(LMW-tcu-PA) and 54000 (HMW-tcu-PA). Urokinase acts directly on the endogenous fibrinolysis system and can catalyze and crack plasminogen to form plasmin, and the plasmin can degrade fibrin clots and fibrinogen, blood coagulation factor V, blood coagulation factor VIII and the like in blood circulation, thereby playing a role in thrombolysis. Has quick response and good effect on newly formed thrombus. Can also improve the activity of vascular ADP enzyme, inhibit platelet aggregation induced by ADP, and prevent thrombosis. After the urokinase is instilled in a vein, the fibrinolytic enzyme activity in the body of a patient is obviously improved; after several hours of withdrawal, plasmin activity returns to its original level.
In addition, the Chinese patent publication No. CN111647586A provides a method for adsorbing urokinase in urine by using resin, belonging to the technical field of separation and extraction. The method comprises the following steps: (1) preparing a urokinase solution; (2) preparing urokinase freeze-dried powder; in the preparation process of urokinase, the D6246 resin bag is used for adsorbing urokinase in urine without other treatment of the urine, and then the resin bag is loaded into a chromatographic column and eluted to obtain the urokinase.
However, the existing preparation process of urokinase has the problems of low utilization rate of urine, low extraction rate of urokinase and low purity and activity of the extracted urokinase.
Therefore, the invention of a process for extracting urokinase by using modified silica gel is very necessary.
Disclosure of Invention
In order to solve the technical problems, the invention provides a process for extracting urokinase by modified silica gel, which aims to solve the problems of low urokinase extraction rate caused by low urine utilization rate and low purity and activity of the extracted urokinase in the existing preparation process of urokinase.
A process for extracting urokinase by modified silica gel specifically comprises the following steps:
the method comprises the following steps: selecting a silica gel raw material;
step two: HF treatment of silica gel;
step three: performing sulfonation treatment on silica gel;
step four: performing silica gel carboxylation treatment;
step five: mixing sulfonated silica gel and carboxylated silica gel;
step six: mixing the silica gel mixed solution with urine;
step seven: carrying out filtration and adsorption on the mixed solution;
step eight: and (4) eluting and obtaining urokinase.
Preferably, in the step one, silica gels with different specific surface areas and particle size ranges are selected for sulfonic acid modification, and silica gels with different specific surface areas are selected for carboxylation modification.
Preferably, in the first step, the silica gel raw material has a silica gel specific surface area of 400 square meters per gram and a silica gel particle size range of 38-1000 um; the silica gel specific surface area of the silica gel raw material for carboxylation is 200-400 square meters per gram, and the particle size range is 200-400 micrometers.
Preferably, in the second step, 1kg of silica gel is put into a 5L reaction kettle, 45L of 20-1000ppm of HF aqueous solution is refluxed for 10-30 hours, after the reaction is finished, a sand core is filtered, water is washed to be neutral, and the silica gel is dried at 100-200 ℃.
Preferably, in the third step, the sulfonating agent used for sulfonating is SO3 or H2SO4 or fuming sulfuric acid or ClSO2OH, the silica gel and the sulfonating agent are added into a reaction kettle for mixing and sulfonating, and the sulfonating time is 30-40 minutes.
Preferably, in step four, the silica gel carboxylation process: putting 180ml ethanol and 420ml water into a 1000ml three-neck bottle, adding 100g silica gel and 24ml sodium carboxylate ethyl sodium silicate with the concentration of 50%, stirring and reacting for 1-10 hours, filtering by a sand core, and drying in a vacuum drier at 90-120 ℃ for 10-20 hours.
Preferably, in the fifth step, the sulfonated silica gel and the carboxylated silica gel in the third step and the fourth step are mixed according to the proportion of 1: 1-3: 1, so that a modified silica gel mixture is obtained.
Preferably, in the sixth step, the male urine and the silica gel mixed solution in the fifth step are mixed in a ratio of 1:5, and the mixture is stirred uniformly to obtain the urokinase-containing silica gel.
Preferably, in the seventh step, the urokinase-containing silica gel in the sixth step is subjected to column chromatography, the process is circulated for 3-5 times, the collected silica gel mixture is washed with the equilibrium buffer solution for three times and then is washed with deionized water for three times until the urine is colorless, so that a crude urokinase product is obtained, and the pH value of the crude urokinase product solution is 4.5-7.5; further, the pH is 4.5-6.5; the filler filled in the cation exchange chromatographic column is high-resolution ion chromatographic filler, and can be selected from one of Capto SimPopt, SPsepharose HP and source 30S; the equilibration buffer is selected from: one of acetate buffer, citrate buffer, phosphate buffer and Tris-HCl buffer; and the equilibration buffer contains 150-500mM sodium chloride.
Preferably, in step eight, the elution process of the crude urokinase is divided into two times: the first elution was performed with 2% ammonium hydroxide and 1M sodium chloride eluent at 35% volume under stirring for 1 hour, the second elution was performed with the same volume of eluent for 30 minutes, and urokinase collection was started.
In addition, in the process for extracting urokinase by using modified silica gel, in the third step, the used sulfonation uses an element analyzer to measure sulfur, the sulfonation uses a COOH substitution degree test, and the specific operations are as follows:
COOH degree of substitution test
1. The application range is as follows: the method is suitable for testing the degree of substitution of COOH filler.
2. The content is as follows:
2.1 preparation of reagents:
0.1mol/l NaOH standard solution, methyl red indicator (0.1g methyl red dissolved in 60ml ethanol, water added to 100ml), 0.1mol/l HCl standard solution
2.2 testing principle:
by reaction of NaOH with carboxyl groups in the packing，Replacement of H+The packing is washed with water and excess OH is titrated with HCl-The degree of substitution of the filler can be calculated
2.3 test methods:
weighing bottle about 3.5g of the filler was weighed, about 1.5g was weighed by subtractive method into an ion exchange column and the weight was recorded (to the nearest 0.0001g), and the weight loss on drying was calculated after the remaining filler had been dried in an oven at 105 ℃ for 2 h.
Transferring 50ml of 0.1mol/l NaOH standard solution into an exchange column tube by using a transfer pipette (controlling the flow rate to 3-5ml/min), dividing by 3 times by using pure water, washing the ion column tube by 10ml each time, collecting eluent, adding a methyl red indicator into the eluent, titrating to orange by using 0.1mol/ml HCl standard solution, and keeping the color unchanged for 15s to obtain an end point.
2.4 calculation method:
V1- -volume of NaOH added to the ion exchange column
C1- -concentration of NaOH added to the ion exchange column
V2Volume of HCl used for titration to endpoint
C2- -concentration of HCl used for titration
W- -weight of exchanged packing in ion exchange column
Loss on drying of X- - -filler
Taking the arithmetic mean value of the two parallel measurements as an analysis result, wherein the mean deviation of the two parallel measurements is less than or equal to 5%, and two effective figures are reserved in the result.
Compared with the prior art, the invention has the beneficial effects that:
urokinase exhibits specific adsorption; the selectivity is good, and the sample loading amount is high; the extracted urokinase has high purity and the extraction method is simple;
the urokinase product obtained by the process for extracting urokinase by using the modified silica gel has high content of macromolecular urokinase, high activity yield and better clinical use effect;
the process for extracting urokinase by using the modified silica gel does not need to treat urine, column chromatography technology is used for column chromatography operation, the process is circulated and repeated for 3-5 times, the collected silica gel mixture is washed for three times by using the equilibrium buffer solution, then is washed for three times by using deionized water, and the urokinase can be obtained by elution, so that the operation is simple, and the loss of urokinase can be reduced.
The elution process of the crude urokinase product is divided into two times: performing primary elution, namely stirring and eluting for 1 hour by using 2% ammonium hydroxide and 1M sodium chloride eluent according to the volume of 35%, performing secondary elution, eluting for 30 minutes by using the same volume of the eluent, and starting to collect urokinase; can separate urokinase better, can improve the urokinase content effectively, the yield of the liquid urokinase can reach 80-92 percent, thus obtaining the urokinase with higher purity, and the purity of the collected urokinase is more than 45 percent.
Drawings
FIG. 1 is a process flow chart of the modified silica gel extraction of urokinase.
Detailed Description
The invention is further described below with reference to the accompanying drawings:
example 1:
as shown in the attached figure 1, the invention provides a process for extracting urokinase by modified silica gel, which specifically comprises the following steps:
a process for extracting urokinase by modified silica gel specifically comprises the following steps:
s101: selection of silica gel raw materials: selecting silica gels with different specific surface areas and particle size ranges for sulfonic acid modification, and selecting silica gels with different specific surface areas for carboxylation modification; the silica gel raw material has a silica gel specific surface area of 400 square meters per gram and a silica gel particle size range of 200 um; the silica gel raw material for carboxylation has a silica gel specific surface area of 200 square meters per gram and a particle size range of 200 um.
S102: HF treatment of silica gel: 1kg of silica gel is put into a 5L reaction kettle, 45L of 500ppm HF aqueous solution is refluxed for 10 hours, a sand core is filtered after the reaction is finished, the silica gel is washed to be nearly neutral, and the silica gel is dried at 100 ℃.
S103: and (3) performing silica gel sulfonation treatment: the sulfonation reagent used in the sulfonation is SO3 or H2SO4 or fuming sulfuric acid or ClSO2OH, the silica gel and the sulfonation reagent are added into a reaction kettle for mixing sulfonation, and the sulfonation time is 30 minutes.
S104: and (3) performing silica gel carboxylation treatment: the silica gel carboxylation process comprises the following steps: putting 180ml ethanol and 420ml water into a 1000ml three-neck bottle, adding 100g silica gel and 24ml sodium carboxylate ethyl sodium silicate with the concentration of 50%, stirring and reacting for 2 hours, filtering by a sand core, and drying in a vacuum drier at 90 ℃ for 15 hours.
S105: mixing of sulfonated silica gel and carboxylated silica gel:
s106: mixing the silica gel mixed solution with urine: and (5) mixing the male urine with the silica gel mixed solution in the fifth step in a mixing ratio of 1:5, and uniformly stirring to obtain the urokinase-containing silica gel.
S107: and (3) dripping, filtering and adsorbing the mixed solution: performing column chromatography on the urokinase-containing silica gel in the step six by utilizing a column chromatography technology, wherein the process is circularly repeated for 3-5 times, washing the collected silica gel mixture for three times by using an equilibrium buffer solution, and then washing for three times by using deionized water until urine is colorless, so as to obtain a urokinase crude product, wherein the pH value of a urokinase crude product solution is 4.5; further, the pH was 4.5; the filler filled in the cation exchange chromatographic column is high-resolution ion chromatographic filler, and can be selected from one of Capto SimPopt, SP sepharose HP and source 30S; the equilibration buffer is selected from: one of acetate buffer, citrate buffer, phosphate buffer and Tris-HCl buffer; and the equilibration buffer contains 150-500mM sodium chloride.
S108: elution of urokinase to obtain: the elution process of the crude urokinase product is divided into two times: the first elution was performed with 2% ammonium hydroxide and 1M sodium chloride eluent at 35% volume for 1 hour with stirring, the second elution was performed with the same volume of eluent for 30 minutes, and urokinase collection was started.
Example 2:
as shown in the attached figure 1, the invention provides a process for extracting urokinase by modified silica gel, which specifically comprises the following steps:
a process for extracting urokinase by modified silica gel specifically comprises the following steps:
s101: selection of silica gel raw materials: selecting silica gels with different specific surface areas and particle size ranges for sulfonic acid modification, and selecting silica gels with different specific surface areas for carboxylation modification; the silica gel raw material has a silica gel specific surface area of 500 square meters per gram and a silica gel particle size range of 800 micrometers; the silica gel raw material for carboxylation has a silica gel specific surface area of 250 square meters per gram and a particle size range of 220 um.
S102: HF treatment of silica gel: 1kg of silica gel is put into a 5L reaction kettle, 45L of 600ppm HF aqueous solution is refluxed and reacted for 12 hours, a sand core is filtered after the reaction is finished, the silica gel is washed to be nearly neutral, and the silica gel is dried at 110 ℃.
S103: and (3) performing silica gel sulfonation treatment: the sulfonation reagent used in the sulfonation is SO3 or H2SO4 or fuming sulfuric acid or ClSO2OH, the silica gel and the sulfonation reagent are added into a reaction kettle for mixing sulfonation, and the sulfonation time is 35 minutes.
S104: and (3) performing silica gel carboxylation treatment: the silica gel carboxylation process comprises the following steps: 180ml of ethanol and 420ml of water are placed in a 1000ml three-necked flask, 100g of silica gel and 24ml of 50% sodium carboxylate ethyl sodium silicate are added and stirred for reaction for 2 hours, and then the mixture is filtered by a sand core and dried in a vacuum dryer at 90 ℃ for 15 hours.
S105: mixing of sulfonated silica gel and carboxylated silica gel:
s106: mixing the silica gel mixed solution with urine: and D, mixing the male urine with the silica gel mixed solution in the step five in a mixing ratio of 1:5, and uniformly stirring to obtain the urokinase-containing silica gel.
S107: and (3) dripping, filtering and adsorbing the mixed solution: performing column chromatography on the urokinase-containing silica gel in the step six by utilizing a column chromatography technology, wherein the process is circularly repeated for 3-5 times, washing the collected silica gel mixture for three times by using an equilibrium buffer solution, and then washing for three times by using deionized water until urine is colorless, so as to obtain a urokinase crude product, wherein the pH value of a urokinase crude product solution is 5; further, the pH is 5.5; the filler filled in the cation exchange chromatographic column is high-resolution ion chromatographic filler, and can be selected from one of Capto SimPopt, SP sepharose HP and source 30S; the equilibration buffer is selected from: one of acetate buffer, citrate buffer, phosphate buffer and Tris-HCl buffer; and the equilibration buffer contains 150-500mM sodium chloride.
S108: elution of urokinase to obtain: the elution process of the crude urokinase product is divided into two times: the first elution was performed with 2% ammonium hydroxide and 1M sodium chloride eluent at 35% volume under stirring for 1 hour, the second elution was performed with the same volume of eluent for 40 minutes, and urokinase collection was started.
Example 3:
as shown in the attached figure 1, the invention provides a process for extracting urokinase by modified silica gel, which specifically comprises the following steps:
a process for extracting urokinase by modified silica gel specifically comprises the following steps:
s101: selection of silica gel raw materials: selecting silica gels with different specific surface areas and particle size ranges for sulfonic acid modification, and selecting silica gels with different specific surface areas for carboxylation modification; the silica gel raw material has a silica gel specific surface area of 800 square meters per gram and a silica gel particle size range of 600 micrometers; the silica gel raw material for carboxylation has a silica gel specific surface area of 300 square meters per gram and a particle size range of 300 micrometers.
S102: HF treatment of silica gel: 1kg of silica gel is put into a 5L reaction kettle, 45L of 500ppm HF aqueous solution is put into reflux reaction for 25 hours, after the reaction is finished, a sand core is filtered, the silica gel is washed to be nearly neutral, and the silica gel is dried at 160 ℃.
S103: and (3) performing silicagel sulfonation treatment: the sulfonation reagent used in the sulfonation is SO3 or H2SO4 or fuming sulfuric acid or ClSO2OH, the silica gel and the sulfonation reagent are added into a reaction kettle for mixing sulfonation, and the sulfonation time is 35 minutes.
S104: and (3) performing silica gel carboxylation treatment: the silica gel carboxylation process comprises the following steps: putting 180ml ethanol and 420ml water into a 1000ml three-neck bottle, adding 100g silica gel and 24ml sodium carboxylate ethyl sodium silicate with the concentration of 50%, stirring and reacting for 2 hours, filtering by a sand core, and drying in a vacuum drier at 90 ℃ for 12 hours.
S105: mixing of sulfonated silica gel and carboxylated silica gel:
s106: mixing the silica gel mixed solution with urine: and D, mixing the male urine with the silica gel mixed solution in the step five in a mixing ratio of 1:5, and uniformly stirring to obtain the urokinase-containing silica gel.
S107: and (3) drop filtration and adsorption of the mixed solution: carrying out column chromatography operation on the urokinase-containing silica gel in the sixth step by utilizing a column chromatography technology, wherein the process is circulated and repeated for 3-5 times, the collected silica gel mixture is washed for three times by using a balanced buffer solution, and then is washed for three times by using deionized water until urine is colorless, so that a urokinase crude product is obtained, and the pH value of a urokinase crude product solution is 4.5; further, the pH is 5; the filler filled in the cation exchange chromatographic column is high-resolution ion chromatographic filler, and can be selected from one of Capto SimPopt, SP sepharose HP and source 30S; the equilibration buffer is selected from: one of acetate buffer, citrate buffer, phosphate buffer and Tris-HCl buffer; and the equilibration buffer contains 150-500mM sodium chloride.
S108: elution of urokinase to obtain: the elution process of the crude urokinase product is divided into two times: the first elution was performed with 2% ammonium hydroxide and 1M sodium chloride eluent at 35% volume with stirring for 1 hour, the second elution was performed with the same volume of eluent for 40 minutes, and urokinase collection was started.
The production efficiency and the purity of urokinase obtained by combining other processes in the field are compared as shown in the following table.
TABLE 1 Effect of pretreatment Process on modification
TABLE 2
Comparative data 2
Effect of different silica gels on urokinase adsorption
The weight of urokinase adsorbed by 100g of silica gel can averagely reach more than 10 percent, and the extraction efficiency is effectively improved.
From the data in the table, it can be seen that the adsorption amount of the sulfonated and carboxylated modified silica gel to urokinase is much higher than that of the unmodified silica gel; meanwhile, it was found in the experiment that urokinase adsorbed on unmodified silica gel was difficult to elute, and the number of elution times was increased. Compared with the single modified silica gel for adsorbing urokinase, the hybrid modified silica gel obtained by the technical scheme has higher absorption efficiency.
In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", and the like, indicate orientations and positional relationships based on those shown in the drawings, and are used only for convenience of description and simplicity of description, and do not indicate or imply that the equipment or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be considered as limiting the present invention.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The process for extracting urokinase from modified silica gel is characterized by comprising the following steps:
the method comprises the following steps: selecting a silica gel raw material;
step two: HF treatment of silica gel;
step three: performing sulfonation treatment on silica gel;
step four: performing silica gel carboxylation treatment;
step five: mixing sulfonated silica gel and carboxylated silica gel;
step six: mixing the silica gel mixed solution with urine;
step seven: carrying out filtration and adsorption on the mixed solution;
step eight: and (4) eluting and obtaining urokinase.
2. The process for extracting urokinase from modified silica gel as claimed in claim 1, wherein in the first step, silica gel with different specific surface area and particle size range is selected for sulfonic acid modification, and silica gel with different specific surface area is selected for carboxylation modification.
3. The process for extracting urokinase from modified silica gel as claimed in claim 1, wherein in the step one, the specific surface area of silica gel used for sulfonating the silica gel raw material is 400-800m2The particle size of the silica gel is 38-1000 um; the silica gel specific surface area of the silica gel raw material for carboxylation is 200-400m2The particle size range is 200- & lt 400 & gtum.
4. The process for extracting urokinase from modified silica gel as claimed in claim 1, wherein in step two, the silica gel HF treatment process comprises placing 1kg of silica gel in a 5L reaction kettle, refluxing 45L of 20-1000ppm HF aqueous solution for 10-30 hours, filtering with sand core after the reaction is finished, washing with water to near neutrality, and drying at 100-200 ℃.
5. The process for extracting urokinase from modified silica gel as claimed in claim 1, wherein in step three, the sulfonating agent used in sulfonation is SO3 or H2SO4 or fuming sulfuric acid or ClSO2OH, the silica gel and the sulfonating agent are added into the reaction kettle for mixed sulfonation, and the sulfonation time is 30-40 minutes.
6. The process for extracting urokinase from modified silica gel according to claim 1, wherein in step four, the silica gel carboxylation process: putting 180ml ethanol and 420ml water into a 1000ml three-neck bottle, adding 100g silica gel and 24ml sodium carboxylate ethyl sodium silicate with the concentration of 50%, stirring and reacting for 1-10 hours, filtering by a sand core, and drying in a vacuum drier at 90-120 ℃ for 10-20 hours.
7. The process for extracting urokinase from modified silica gel as claimed in claim 1, wherein in the fifth step, the sulfonated silica gel and the carboxylated silica gel in the third step and the fourth step are mixed according to a ratio of 1: 1-3: 1, so as to obtain a modified silica gel mixture.
8. The process for extracting urokinase from modified silica gel according to claim 1, wherein in the sixth step, the male urine and the silica gel mixture in the fifth step are mixed in a weight ratio of 1:5, and the mixture is stirred uniformly to obtain the urokinase-containing silica gel.
9. The process for extracting urokinase from modified silica gel as claimed in claim 1, wherein in step seven, the urokinase-containing silica gel in step six is subjected to column chromatography, the process is repeated 3-5 times, the collected silica gel mixture is washed with equilibration buffer solution for three times and then with deionized water for three times until the urine is colorless, so as to obtain crude urokinase, and the pH of the crude urokinase solution is 4.5-7.5; further, the pH is 4.5-6.5; the filler filled in the cation exchange chromatographic column is high-resolution ion chromatographic filler, and can be selected from one of Capto SimPopt, SP sepharose HP and source 30S; the equilibration buffer is selected from: one of acetate buffer, citrate buffer, phosphate buffer and Tris-HCl buffer; and the balance buffer contains 150-500mM sodium chloride.
10. The process for extracting urokinase from modified silica gel as claimed in claim 1, wherein in step eight, the elution process of crude urokinase is divided into two times: the first elution was performed with 2% ammonium hydroxide and 1M sodium chloride eluent at 35% volume under stirring for 1 hour, the second elution was performed with the same volume of eluent for 30 minutes, and urokinase collection was started.
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