CN1733913A - High purity venom kininogenase prepartion method and its pharmaceutical formulation - Google Patents
High purity venom kininogenase prepartion method and its pharmaceutical formulation Download PDFInfo
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- CN1733913A CN1733913A CN 200410009422 CN200410009422A CN1733913A CN 1733913 A CN1733913 A CN 1733913A CN 200410009422 CN200410009422 CN 200410009422 CN 200410009422 A CN200410009422 A CN 200410009422A CN 1733913 A CN1733913 A CN 1733913A
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- kininogenase
- snake venom
- preparation
- venom
- active ingredient
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Abstract
The invention relates to a process for preparing high purity snake venom kininogenase, which comprises integrating antigen-antibody reaction with compatible chromatography technology, and isolating and purifying kininogenase from raw material snake venom. The invention also relates to the pharmaceutical preparation from the high purity snake venom kininogenase.
Description
Technical field
The present invention relates to the preparation method of high purity snake venom kininogenase, relate in particular to the high purity snake venom kininogenase that adopts the immune-affinity chromatography preparation.
The invention still further relates to the high purity snake venom kininogenase and the preparation thereof that adopt the inventive method preparation.
Background technology
Kininogenase is the intravital a kind of proteolytic ferment of animal, is present in the tissue of many organs.People understanding the earliest, research is clear that the Pancreatic Kininogenase of snake venom in dirty the most.It is a class endo-type proteolytic ferment that Pancreatic Kininogenase (Pancreatic Kallidinogenase) is called pancreatic kallikrein (Pancreatic Kallikrein) again, is present in the intravital multiple tissue of Mammals, the abundantest with the dirty middle content of snake venom especially.Act on prokinin in vivo, make it discharge kassinin kinin, thereby given play to a series of pharmacological action: make Marjoram Extract, improve peripheral blood circulation; Promote the prostaglandin(PG) secretion, the expansion arteriole increases the kidney vascular flow; Activate the plasmin system, make the scleroproein hydrolysis, blood viscosity lowering prevents thrombosis, thrombus.Be mainly used in ephrosis change and eyeground blood supply disorder, vascular hypertension, cerebral arteriosclerosis and cerebral artery thrombosis, coronary heart disease and other occlusive surrounding blood vessel treatment of diseases that microcirculation disturbance causes clinically.
But snake venom kininogenase preparation, because purity is lower, the restriction on the safety can only be used for oral and intramuscularly, and its range of application, indication and curative effect thereof are very limited.
Summary of the invention
One object of the present invention is to provide a kind of preparation method of high purity snake venom kininogenase, utilizes immune-affinity chromatography to prepare highly purified snake venom kininogenase.
Another object of the present invention is to provide the high purity snake venom kininogenase that uses the inventive method preparation.
A further object of the present invention is to provide the pharmaceutical preparation that contains high purity snake venom kininogenase of the present invention.
According to an aspect of the present invention, prepare the method for high purity snake venom kininogenase, comprise the steps:
1) will contain snake venom raw material preliminary purification, the separation of snake venom kininogenase, obtaining main component is the active ingredient 1 of snake venom kininogenase;
2) described active ingredient 1 is further purified, obtains active ingredient 2;
3) with described active ingredient 2 as antigen, preparation antisnake venom kininogenase specific antibody;
4) the described antisnake venom kininogenase specific antibody for preparing is combined the preparation antibody affinity chromatography with the affinity chromatography carrier;
5) with described antibody affinity chromatography purification procedures 1) in active ingredient 1, obtain high purity snake venom kininogenase.
In the method for the present invention, antigen antibody reaction is combined with the affinity chromatography technology, from the raw material snake venom that contains the snake venom kininogenase,, prepare the high snake venom kininogenase of purity the separation and purification of snake venom kininogenase.In this method, at first need prepare specific antibody, again this specific antibody be combined with the affinity chromatography carrier that suits, be prepared into antibody affinity chromatography at the snake venom kininogenase, utilize this antibody affinity chromatography to carry out purifying, prepare highly purified snake venom kininogenase raw material.
In the methods of the invention, specific antibody at the snake venom kininogenase can adopt the several different methods preparation, in a specific embodiments of the present invention, be the antigen immune mouse at first with the snake venom kininogenase, acquisition contains the mouse resisting anteserum of antisnake venom kininogenase antibody, simultaneously this snake venom kininogenase is combined with the affinity chromatography carrier, be prepared into the antigen affinity column, with antigen affinity column on the mouse resisting anteserum, utilization is incorporated into antigen protein on the affinity chromatography carrier and the antibody specific combination in the serum, obtains the specific antibody of purifying.In addition, also can adopt monoclonal antibody technique to prepare the specific antibody of snake venom kininogenase of the present invention, for example, to merge through mouse boosting cell after the immunity of snake venom kininogenase and myeloma cell, obtain hybridoma, the hybridoma of screening secretion snake venom kininogenase antibody is built strain, can obtain the specific antibody of stable snake venom kininogenase thus.
The antisnake venom kininogenase specific antibody for preparing is combined with the affinity chromatography carrier that suits, be prepared into antibody affinity chromatography, use this antibody affinity chromatography to carry out separation and purification, prepare highly purified snake venom kininogenase raw material.
In the methods of the invention, in order to obtain highly purified snake venom kininogenase, can adopt the method for raw material being carried out the substep purifying, for example, in a specific embodiments of the present invention, at first the raw material snake venom is dialysed and hold back the composition of certain molecular weight, then the sample that obtains is carried out preliminary purification by ion exchange chromatography, obtain active ingredient 1, this active ingredient 1 can obtain highly purified snake venom kininogenase again through antibody affinity chromatography purifying of the present invention.
Preferably use the higher snake venom kininogenase of purity as antigenic snake venom kininogenase, in the methods of the invention, should can further obtain through the separation of preparative high performance liquid chromatography by the active ingredient 1 with above-mentioned preliminary purification as antigenic snake venom kininogenase, the snake venom kininogenase of Huo Deing (active ingredient 2) can use as the antigen of immune mouse and the antigen of preparation antigen affinity column like this.
The material that is used as the carrier of affinity column in the inventive method can use various suitable solid support materials, preferred solid support material can use polysaccharide matrix, for example: Sepharose 4B sepharose, diethylin dextrane gel or CM-sephadex, can earlier carrier be activated before use, in embodiment of the present invention, use cyanogen bromide as activator, the antigen for preparing or specific antibody are coupled on the carrier that has activated, can prepare antigen affinity column and antibody affinity chromatography.
According to a further aspect in the invention, provide the high purity snake venom kininogenase by the inventive method preparation, it has following characteristic:
Specific activity is more than every milligram of albumen 800 units;
High performance liquid chromatography detects, and single component collection of illustrative plates, main peak area are greater than 95%, and retention time is 7.8 ± 0.5min;
Show a band on the SDS-polyacrylamide gel electrophoresis, molecular weight is 34～44 kilodaltons;
Amino acid sequence analysis, terminal 15 aminoacid sequences of N-are:
Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His-Arg-Phe-Leu。
In accordance with a further aspect of the present invention, the pharmaceutical preparation that contains high purity snake venom kininogenase of the present invention is provided, with high purity snake venom kininogenase of the present invention is effective component, add pharmaceutically acceptable pharmaceutical excipient and prepare and be suitable for the various pharmaceutical preparations of clinical application, the formulation of these preparations includes but not limited to tablet, capsule, small-volume injection, lyophilized injectable powder and specializes in intravenous injection and infusion solutions or freeze-dried preparation that intravenous drip is used.For example, venin for injection kininogenase (freeze-drying used for intravenous injection snake venom kininogenase), snake venom kininogenase injection liquid (used for intravenous injection snake venom kininogenase), snake venom kininogenase enteric coated capsule and snake venom kininogenase enteric coated tablet etc.
The present invention adopts the immunoaffinity chromatography technology, and the kininogenase purity height that extracts from snake venom, the product of acquisition are higher than living, purity is high, does not contain or only contain small amount of impurities, and is safe in utilization, can reach the requirement of intravenously administrable.Compare with intramuscular administration with oral, intravenously administrable can make the effective constituent of medicine all directly enter blood, shorten absorption process and reduce loss, thereby drug effect is rapid, and shorten the course of treatment, reduces the painful of patient and can save medical expenses; Be particularly useful for the rescue of acute cardiovascular and cerebrovascular embolism class diseases, can win valuable therapeutic time, improve curative ratio and reduce case fatality rate.The exploitation of high purity snake venom kininogenase of the present invention has enlarged the indication and the use range of these product.
Brief description of drawings
Fig. 1 is the anion exchange chromatography ultraviolet absorpting spectrum of preparation active ingredient 1 in the inventive method;
Fig. 2 is an antisnake venom kininogenase specific antibody purge process registering instrument collection of illustrative plates in the inventive method;
Fig. 3 is the affinity chromatography process recording instrument collection of illustrative plates of preparation high purity snake venom kininogenase in the inventive method;
Fig. 4 is a high purity snake venom kininogenase SDS-polyacrylamide gel electrophoresis collection of illustrative plates of the present invention;
Among the figure: swimming lane 1,2,4,5,6,7,9,10 is the snake venom kininogenase;
Fig. 5 is the high purity snake venom kininogenase high-efficient liquid phase chromatogram spectrum of the present invention's preparation;
Fig. 6 is the high purity snake venom kininogenase high-efficient liquid phase chromatogram spectrum of another batch of the present invention's preparation;
Fig. 7 is another batch high purity snake venom kininogenase high-efficient liquid phase chromatogram spectrum of the present invention's preparation.
The embodiment of invention
The preparation of high purity snake venom kininogenase
1, the processing of snake venom crude product
Accurately take by weighing viper venom (Liaoning radically reform She Chang produce), Tris-HCl damping fluid with 0.02mol/L PH7.8 extracts its dissolving, got supernatant in centrifugal 20 minutes with 4000r/min then, precipitation is got supernatant with a spot of damping fluid recentrifuge, merges supernatant liquor dress dialysis tubing (component of molecular weight cut-off more than 10000) and treats sample in 12 hours in the dialysis of the Tris-HCl of 0.02mol/L PH7.8 damping fluid.
2, ion exchange chromatography preliminary purification
After sample has been dialysed with anion-exchange column (DEAE-Sepharose) on the flow velocity of 6.0ml/min, the foreign protein that does not adsorb with the Tris-HCL buffer solution elution of 0.02mol/L PH7.8 after last sample finishes, when waiting to see through the peak down to baseline, the damping fluid that contains 0.05mol/L and 0.115mol/L sodium-chlor with 0.02mol/L Tris-HCl carries out gradient elution respectively, collect active peak, make active ingredient 1.The main component of active ingredient 1 is the snake venom kininogenase, and tool snake venom kininogenase activity is identical with the snake venom kininogenase according to the molecular weight that the main band of SDS-polyacrylamide gel electrophoresis calculates; This flow process detects with the 280nm Ultraviolet Detector, uses the registering instrument record simultaneously, as shown in Figure 1.
3, be used as the preparation of antigenic snake venom kininogenase
The active ingredient 1 dress molecular weight cut-off that ion exchange chromatography is collected is that 10000 dialysis tubing concentrates, and separates with preparative high performance liquid chromatography, collects active ingredient, and qualitative, snake venom kininogenase (active ingredient 2).Its tool snake venom kininogenase activity, the SDS-polyacrylamide gel electrophoresis is shown as a band, and the molecular weight of calculating and snake venom kininogenase standard substance coincide; HPLC detects and is one-component, and retention time and snake venom kininogenase standard substance coincide.
4, the preparation of antigen affinity column
Claim 2g Sepharose-4B through cyanogen bromide-activated, the HCL that adds 300ml 1mol/L fully stirs and makes it to expand, and other gets the above-mentioned active ingredient 2 of 30mg and is dissolved in 0.1mol/L Na
2CO
3In (containing 0.5mol/lNaCL) pH8.3 damping fluid.Above-mentioned Sepharose-4B is mixed with these active ingredient 2 solution,, use 0.1mol/L pH8.3Na at stirring at room 2h
2CO
3Behind (containing 0.5mol/lNaCL) damping fluid thorough washing, use 0.1mol/L Tris-HCL damping fluid (pH8.30) to wash again once again, add same damping fluid, in stirred overnight at room temperature.The dress post prepares the antigen affinity column.
5, mouse resisting anteserum preparation
With above-mentioned active ingredient 2 (50～500 μ g/ml) and equal-volume Freund's complete adjuvant mixing and emulsifying as stimulator, to the BALB/c mouse in 7 ages in week (middle inspection institute Experimental Animal Center) subcutaneous injection immunity, each 0.2ml, 1 time weekly, immunity is 4～6 times altogether.Got 0.2ml stimulator mouse tail vein injection booster immunization in preceding 3 days at collection serum.
6, preparation antisnake venom kininogenase specific antibody
Get above-mentioned immune serum, with antigen affinity column on the serum sample, combine with antibodies specific in the sample with the antigen protein that is combined on the gel, purifying obtains specific antibody.Concrete steps are: with balance liquid (0.02MTris-HCL damping fluid, pH=7.2) behind the balance antigen affinity column, with antigen affinity column on the serum sample, with the balance liquid balance to effluent liquid after drawing stable baseline on the Protein Detection instrument, with elutriant (0.02MTris-HCL damping fluid, pH=7.2, contain 0.5M Nacl, 7%Arg) wash-out is collected active ingredient, as shown in Figure 2.After the active ingredient collected concentrated with dialysis tubing (molecular weight cut-off is 10000, and polyoxyethylene glycol is concentrated), lyophilize was antisnake venom kininogenase specific antibody.
7, the preparation of antibody affinity chromatography
With Sepharose 4B sepharose, diethylin dextrane gel or CM-sephadex carrier, with this carrier of cyanogen bromide-activated as affinity chromatography; The specific antibody of getting the above-mentioned preparation of 100mg is dissolved in the 20ml borate buffer, filter, filtrate adds in the activatory carrier, 10 ℃ of following stirring reactions 18 hours, adorn post next day, pH 10.2 borate buffers with 10 times of column volumes wash cylinder with 5～6ml/min flow velocity, collect effluent liquid, and then use the ethanolamine solutions of pH 10.00.1mol/L of 5 times of column volumes and borate buffer (pH 8.0) thorough washing of 0.1mol/L successively, with the 0.1mol/L phosphoric acid buffer washing balance of pH 7.4, promptly obtain antibody affinity chromatography at last.This post is reusable more than 200 times, but with 20% ethanol prolonged preservation.
8, the preparation of high purity snake venom kininogenase
Below, by detailed description, describe the present invention in detail to specific embodiment:
The preparation of [embodiment 1] immune antibody affinity column
With high-efficient liquid phase chromatogram purification and through snake venom kininogenase (active ingredient 2) 50 μ g/ml and equal-volume Freund's complete adjuvant mixing and emulsifying qualitatively as stimulator, to the BALB/c mouse subcutaneous injection immunity in 7 ages in week, each 0.2ml, 1 time weekly, immunity is 6 times altogether.Got 0.2ml stimulator mouse tail vein injection booster immunization in preceding 3 days at collection serum.With antigen affinity column antibody purification.
With cyanogen bromide-activated 4B-Sepharose sepharose, the snake venom kininogenase antibody coupling that obtains to polysaccharide matrix, thereby make immune affinity chromatographic column.
The preparation of [embodiment 2] immune antibody affinity column
With high-efficient liquid phase chromatogram purification and through snake venom kininogenase (active ingredient 2) 250 μ g/ml and equal-volume Freund's complete adjuvant mixing and emulsifying qualitatively as stimulator, to the BALB/c mouse subcutaneous injection immunity in 7 ages in week, each 0.2ml, 1 time weekly, immunity is 6 times altogether.Got 0.2ml stimulator mouse tail vein injection booster immunization in preceding 3 days at collection serum.With antibody affinity chromatography purifying snake venom kininogenase antibody.
With cyanogen bromide-activated diethylin dextrane gel, the snake venom kininogenase antibody coupling that obtains to polysaccharide matrix, thereby make immune affinity chromatographic column.
The preparation of [embodiment 3] immune antibody affinity column
With high-efficient liquid phase chromatogram purification and through snake venom kininogenase (active ingredient 2) 500 μ g/ml and equal-volume Freund's complete adjuvant mixing and emulsifying qualitatively as stimulator, to the BALB/c mouse subcutaneous injection immunity in 7 ages in week, each 0.2ml, 1 time weekly, immunity is 6 times altogether.Got 0.2ml stimulator mouse tail vein injection booster immunization in preceding 3 days at collection serum.With antibody affinity chromatography purifying snake venom kininogenase antibody.
Use the cyanogen bromide-activated CM-sephadex, the snake venom kininogenase antibody coupling that obtains to polysaccharide matrix, thereby make immune affinity chromatographic column.
The preparation of [embodiment 4] high purity snake venom kininogenase raw material medicine
Accurately take by weighing viper venom 15g, the slow middle liquid 60ml of Tris-HCl with 0.02mol/L PH7.8 extracts its dissolving, got supernatant in centrifugal 20 minutes with 4000r/min then, precipitation is got supernatant with a spot of damping fluid recentrifuge, merges supernatant liquor dress dialysis tubing sample was treated in the slow middle liquid dialysis of Tris-HCl of 0.02mol/L PH7.8 in 12 hours.After sample has been dialysed with anion-exchange column (DEAE-Sepharose) on the flow velocity of 6.0ml/min, last sample finishes foreign protein that the back do not adsorb with the Tris-HCL buffer solution elution of 0.02mol/L PH7.8 when waiting to see through the peak and arriving baseline down, and the damping fluid that contains 0.05mol/L and 0.115mol/L sodium-chlor with 0.02mol/L Tris-HCL carries out gradient elution and collects active peak (active ingredient 1) respectively.With above-mentioned active ingredient 1 usefulness balance liquid (0.02MTris-HCL damping fluid, pH=7.8 contain 0.5M Nacl) dialysis, last antibody affinity chromatography through ion-exchange purification.With affinity chromatography balance liquid balance, treat effluent liquid after drawing stable baseline on the Protein Detection instrument, (0.02MTris-HCL damping fluid, pH=7.2 contain 0.5M Nacl, 7%Arg) wash-out to use the affinity chromatography elutriant instead.Collect elution peak, measure the activity of its enzyme.
Detect through activity, the component of collecting has very high fibrinolytic, gets this component and suitably dilutes, and detects with high performance liquid chromatography, obtains the single component collection of illustrative plates, and its retention time is 7.782, and pyrogen, hemorrhage poison, neural poison, the irritated inspection are all up to specification.
The preparation of [embodiment 5] high purity snake venom kininogenase raw material medicine
Accurately take by weighing viper venom 30g, the slow middle liquid 100ml of Tris-HCl with 0.02mol/L PH7.8 extracts its dissolving, got supernatant in centrifugal 20 minutes with 4000r/min then, precipitation is got supernatant with a spot of damping fluid recentrifuge, merges supernatant liquor dress dialysis tubing sample was treated in the slow middle liquid dialysis of Tris-HCl of 0.02mol/L PH7.8 in 10 hours.After sample has been dialysed with anion-exchange column (DEAE-Sepharose) on the flow velocity of 8.0ml/min, last sample finishes foreign protein that the back do not adsorb with the Tris-HCL buffer solution elution of 0.02mol/L PH7.8 when waiting to see through the peak and arriving baseline down, and the damping fluid that contains 0.05mol/L and 0.110mol/L sodium-chlor with 0.02mol/L Tris-HCL carries out gradient elution and collects active peak (active ingredient 1) respectively.With above-mentioned active ingredient 1 usefulness balance liquid dialysis, last antibody affinity chromatography through ion-exchange purification.With affinity chromatography balance liquid balance, treat that effluent liquid after drawing stable baseline on the Protein Detection instrument, uses affinity chromatography elutriant wash-out instead.Collect elution peak, measure the activity of its enzyme.
Detect through activity, the component of collecting has very high fibrinolytic, gets this component and suitably dilutes, and detects with high performance liquid chromatography, obtains the single component collection of illustrative plates, and its retention time is 7.768, is 39500 dalton with the sds polyacrylamide determining molecular weight.
Pyrogen, hemorrhage poison, neural poison, the irritated inspection, all up to specification.
The preparation of [embodiment 6] high purity snake venom kininogenase raw material medicine
Accurately take by weighing viper venom 20g, the slow middle liquid 100ml of Tris-HCl with 0.02mol/L PH7.8 extracts its dissolving, got supernatant in centrifugal 20 minutes with 4000r/min then, precipitation is got supernatant with a spot of damping fluid recentrifuge, merges supernatant liquor dress dialysis tubing sample was treated in the slow middle liquid dialysis of Tris-HCl of 0.05mol/L PH7.8 in 10 hours.After sample has been dialysed with anion-exchange column (DEAE-Sepharose) on the flow velocity of 8.0ml/min, last sample finishes foreign protein that the back do not adsorb with the Tris-HCL buffer solution elution of 0.05mol/L PH7.8 when waiting to see through the peak and arriving baseline down, and the damping fluid that contains 0.05mol/L and 0.115mol/L sodium-chlor with 0.05mol/L Tris-HCL carries out gradient elution and collects active peak (active ingredient 1) respectively.With above-mentioned active ingredient 1 usefulness balance liquid dialysis, last antibody affinity chromatography through ion-exchange purification.With affinity chromatography balance liquid balance, treat that effluent liquid after drawing stable baseline on the Protein Detection instrument, uses affinity chromatography elutriant wash-out instead.Collect elution peak, measure the activity of its enzyme.
Detect through activity, the component of collecting has very high fibrinolytic, gets this component and suitably dilutes, and detects with high performance liquid chromatography, obtains the single component collection of illustrative plates, and its retention time is 7.698, is 39800 dalton with the sds polyacrylamide determining molecular weight.
Pyrogen, hemorrhage poison, neural poison, the irritated inspection, all up to specification.
The assessment of performance of high purity snake venom kininogenase
1, high purity snake venom kininogenase is to the mensuration of N-benzoyl-L-arginine ethyl ester hydrochloride substrate hydrolysis speed
Method: get trial-product (according to the high purity snake venom kininogenase of previous embodiment 4～6 preparations, lot number is 040302,040305 and 040307, down together) an amount of, add phosphate buffered saline buffer (pH7.0) and (get Sodium phosphate dibasic 10.9g, SODIUM PHOSPHATE, MONOBASIC 2.3g adds the about 700ml of water and makes dissolving, adjust pH to 7.0, thin up is to 1000ml again) make dissolving, make the solution that every 1ml contains 10 units.Measure 4.0ml, put in the 10ml measuring bottle, add proteinase inhibitor solution (take by weighing proteinase inhibitor (Amresco) 5mg, add phosphate buffered saline buffer (pH7.0) and dissolve and be diluted to 10ml) 1ml, use phosphate buffered saline buffer (pH7.0) to be diluted to scale again, be need testing solution.
Measure at 5 minutes substrate solution of 30 ± 0.5 ℃ of preheatings and (get N-benzoyl-L-arginine ethyl ester hydrochloride 17.7mg, add tris buffer (0.1M, pH8.0) to 100ml, 4 ℃ of preservations) 2.9ml puts in the 1cm colorimetric pool, add need testing solution 1.0ml, mixing, timing immediately, under 30 ± 0.5 ℃, shine spectrophotometry (two appendix IV of Chinese Pharmacopoeia version in 2000 A) and measure 4 minutes absorbancy (A at 253nm wavelength place
4) and 6 minutes absorbancy (A
6).Other gets above-mentioned echidnotoxin enzyme inhibitors 1ml, adds above-mentioned phosphate buffered saline buffer (pH7.0) and is diluted to 10ml, measures 1.0ml and puts in the 1cm colorimetric pool, adds 5 minutes substrate solution 2.9ml of 30 ± 0.5 ℃ of preheatings, and mixing is blank; Try to achieve A value [(A
6-A
4)/2], be calculated as follows.
R value (hydrolysis rate) should be 0.12～0.17.
In the formula: a is the mg number among the every 1ml of need testing solution;
B is the potency unit number among the every 1ml of trial-product.
2, purity testing
(1) high performance liquid chromatography (2000 editions appendix VI of Chinese Pharmacopoeia H) is measured
Chromatographic condition and system suitability test:
Instrument model SHIMADZU (Tianjin, island)
SPD-10A vp detector
SCL-10A vp controller
LC-AT vp infusion pump
FCV-10AL vp solvent switch
The PGU-14A de-aerator
The Class-vp6.10 chromatographic working station
Chromatographic column is TSK-2000 gel chromatographic columns 300 * 7.5mm, 5 μ m; Moving phase 0.2mol/L phosphate buffered saline buffer (pH6.8); Detect wavelength 280nm; Flow velocity 1.0ml/min; 25 ℃ of detected temperatures;
Measurement result: should be not less than 3000 by the calculating of snake venom kininogenase peak.
Assay method: get trial-product and make the solution that every 1ml contains 10 units, get 20 μ l and inject liquid chromatograph, the record color atlas with moving phase, calculate by area normalization method, the main peak area should be greater than 95%, and retention time should be 7.8 ± 0.5min, and the HPLC measurement result of three batches of samples is seen Fig. 5～7.
(2) the SDS-polyacrylamide gel electrophoresis is measured (seeing molecular weight determination for details), the results are shown in Figure 4.
3, molecular weight determination
Method: electrophoretic method (two appendix V of Chinese Pharmacopoeia version in 2000 F the 5th method SDS-polyacrylamide gel electrophoresis)
It is an amount of with standard protein (molecular weight ranges 1～100,000) that molecular weight determination is got in the preparation of standard protein solution, add trial-product (according to the high purity snake venom kininogenase of previous embodiment 4～6 preparations, damping fluid (water intaking 4.8ml down together), tris buffer pH6.81.2ml, glycerine 1.0ml, 10% sodium lauryl sulphate 2.0ml, 0.5% tetrabromophenol sulfonphthalein solution 0.5ml, beta-mercaptoethanol 0.5ml, mixing) makes and contain the proteic solution of 1 μ g among every 1ml.Face with 5min in the preposition boiling water bath cooling.
Trial-product an amount of (press protein content calculates) is got in the preparation of need testing solution, adds the trial-product damping fluid and makes and contain the proteic solution of 2 μ g among per 1 μ l, faces with 5min in the preposition boiling water bath, cools off.
Assay method adopts vertical slab electrophoresis according to above-mentioned electrophoretic method test, and every hole adds 5～10 μ l need testing solutions or standard protein solution, coomassie brilliant blue staining.Logarithm with the standard protein molecular weight is an ordinate zou, is that X-coordinate carries out straight-line regression with the mobility, by the molecular weight of regression equation calculation trial-product.
Mensuration should be a band, and molecular weight should be 39000 ± 5000 dalton, the results are shown in Figure 4.
4, determined amino acid sequence
Through Peking University's school of life and health sciences amino acid sequence analysis, the-terminal amino acid sequence is: Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His-Arg-Phe-Leu with the high purity snake venom kininogenase of previous embodiment 4～6 preparation.
5, tire and compare vitality test
It is an amount of that snake venom kininogenase protein standard substance (institute is examined in middle pharmaceutical biological product inspection) is got in the preparation of standardized solution, precision is weighed, add phosphate buffered saline buffer (pH7.0) and (get Sodium phosphate dibasic 10.9g, SODIUM PHOSPHATE, MONOBASIC 2.3g, add the about 700ml of water and make dissolving, transfer pH to 7.0, thin up is to 1000ml again) dissolve and make the solution that contains 10 units among every 1ml.
It is an amount of that trial-product is got in the preparation of need testing solution, and precision is weighed, and adds above-mentioned phosphate buffered saline buffer (pH7.0) and make the solution that contains 10 units among every approximately 1ml approximately.
N-benzoyl-L-arginine ethyl ester hydrochloride 17.7mg is got in the preparation of substrate solution, the accurate title, decide, add 0.1mol/L Tris-HCl damping fluid (pH8.0) and (take by weighing Tutofusin tris 12.114g, add the about 800ml dissolving of water, with 6mol/L hydrochloric acid soln adjust pH to 8.0, add water to 1000ml) to 100ml, 4 ℃ of preservations.
The substrate solution 2.5ml that assay method is measured preheating in 25 ± 0.5 ℃ of water-baths puts in the 1cm colorimetric pool, accurate adding need testing solution or standard solution 0.1ml, mixing, timing immediately.According to ultraviolet spectrophotometry (two appendix IV of Chinese Pharmacopoeia version in 2000 A), measure in 253nm wavelength place at 25 ± 0.5 ℃, be blank with the substrate solution, accurately read 1 minute absorbance A
1Absorbance A with 3 minutes
3Standard substance and trial-product replicate(determination) are averaged for 3 times, calculate A value (A=A
3-A
1), be calculated as follows tiring of every 1mg trial-product:
It is an amount of that protein content is got trial-product, accurate claims surely, adds water and make and contain the proteic solution of 0.15mg among every 1ml approximately, and precision is measured trial-product 1.0ml.Measure according to forint phenol assay method (seeing notes), calculate every 1mg for the protein content (mg) in the examination.
Be calculated as follows than living:
Annotate: forint phenol assay method
Reagent: the alkaline copper test solution is got sodium hydroxide 10g, and sodium sulfate 50g adds water 400ml and makes its dissolving, as first liquid; Get soluble tartrate 0.5g, add water 50ml and make its dissolving, other gets copper sulfate 0.25g and adds water 30ml and make its dissolving, and two liquid are mixed as second liquid.
Before facing usefulness, merge first, second two liquid, and add water to 500ml.
The bovine serum albumin reference substance is got in the preparation of reference substance solution, adds water and makes the solution that contains 0.3mg among every 1ml.
It is an amount of that trial-product is got in the preparation of need testing solution, accurate claims surely, adds water and make and contain the proteic solution of 0.15mg among every 1ml approximately.
The preparation precision of typical curve is measured reference substance solution 0.0ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, puts respectively in the tool plug test tube, respectively adds water to 1.0ml.Add alkaline copper test solution 1.0ml more respectively, shake up; Each adds forint test solution (two appendix XV of Chinese Pharmacopoeia version in 2000 B got forint test solution stock solution, by 1: 15 dilute with water) 4.0ml, capping plug, mixing immediately.Putting accurate response 5min in 55 ℃ of water-baths, put 10min in the cooling bath, is blank according to spectrophotometry (two appendix IV of Chinese Pharmacopoeia version in 2000 B) with No. 0 pipe, measures absorbancy at 650nm wavelength place.With reference substance solution concentration optical density A corresponding with it
ContrastCarry out straight-line regression, obtain regression equation.
The assay method precision is measured need testing solution 1.0ml, and the method under the sighting target directrix curve preparation from " adding the alkaline copper test solution " operation, records the absorbance A of its sample
Sample, from the proteic content of regression equation calculation, and multiply by extension rate, promptly.
6, hypersensitive test
It is an amount of to get trial-product, add 0.9% sodium chloride injection make need testing solution that every 1ml contains 100 units as sensitization liquid with attack liquid.
Get 6 of body weight 250-350g cavys, every other day abdominal injection need testing solution 0.5ml sensitization makes sensitization continuous 3 times.Animal is divided into two groups then, 3 every group, attacks in back the 14th day of injection for the first time and intravenous injection need testing solution 1.0ml on the 21st respectively.Examine the reaction of cavy in the 15min of injection back, all anaphylaxis must not occur.If any two or more person in perpendicular hair, expiratory dyspnea, sneeze, retch or the phenomenon such as cough 3; Or one of hello sound, tic, collapse or phenomena of mortality person, should be judged to the positive.
7, undue toxicity is got trial-product, adds 0.9% sodium chloride injection and makes the solution that every 1ml contains 10 units, checks in accordance with the law and (2000 editions two appendix XI C of Chinese Pharmacopoeia) presses the administration of vein method, and is up to specification.
The verification result of three batches of samples is listed as follows:
Can reach a conclusion by verification result: utilization immunoaffinity chromatography technology, the kininogenase purity height that from snake venom, extracts, the product that obtains is higher than living, reach more than every milligram of albumen 800 units, check to be one-component through HPLC, show a band on the SDS-polyacrylamide gel electrophoresis, it is free from foreign meter that molecule measuring is decided to be 34～44KD, safe in utilization, can reach the requirement of intravenously administrable.
The pharmaceutical preparation that contains high purity snake venom kininogenase
[embodiment 7] venin for injection kininogenase and freeze-drying used for intravenous injection snake venom kininogenase preparation
At first, in the local laminar flow clean area, snake venom kininogenase raw material medicine (is prepared according to previous embodiment of the present invention by the preparation prescription, and it is qualified through every detection, down with) with the accurate weighings of auxiliary material such as phosphoric acid salt, sodium-chlor, dextran in liquid dispensing container, add the injection water and fully dissolve or dilute, making phosphatic concentration is 0.03mol/L, sodium chloride concentration is 0.9%, dextran concentration is 3%, behind the stirring and evenly mixing, obtains the preparation intermediate; Sterile Filtration, reply Sterile Filtration system carries out integrity test before and after wherein filtering.Then, the intermediate after filtering by the loading amount antibiotic bottle of packing into, should be carried out 4 times loading quantity inspection at least in minute process of assembling, guarantee that each dose of only packing in the bottle is accurate.At last, the antibiotic bottle sign indicating number of the medicine of will packing into carries out quick-frozen to subzero 20～30 ℃ according to the kind freeze-drying curve on the dividing plate of each layer of freeze drying box, kept 8 hours; Vacuumize then, under vacuum state, slowly be warming up to 35～40 ℃, keep certain hour, take out after reducing to room temperature again.Tamponade, roll cover finished product.Through final product quality after the assay was approved, pack, go into stockyard and deposit.
[embodiment 8] snake venom kininogenase enteric coated capsule preparation.
At first, with accurate weighings of auxiliary material such as snake venom kininase raw material medicine and starch, lactose, ratio of adjuvant is a starch: lactose is 1: 1 in 100,000 grades of clean areas, and in the preparation container, thorough mixing is even, obtains the preparation intermediate.Then, get outward appearance, length, thickness, stink, moisture, friability, thawing time limit, residue on ignition and microbiological examination capsule shell all up to specification, fill intermediate medicinal powder, obtain work in-process.Cover joint on the capsule at last, flatten polishing, make finished product.Through final product quality after the assay was approved, with vial or Plastic Bottle airtight package, go into stockyard, be placed on shady and cool dry place, temperature must not surpass 25 ℃, and relative humidity must not surpass 45%.
[embodiment 9] snake venom kininogenase enteric coated tablet
At first, (starch: carboxy-propyl cellulose sodium=1: 1) in the preparation container, thorough mixing is even, obtains the preparation intermediate with accurate weighings of auxiliary material such as snake venom kininogenase raw material medicine and starch, carboxy-propyl cellulose sodium in 100,000 grades of clean areas.Then, granulate and compressing tablet, in the compressing tablet process, note the outward appearance of tablet at any time, regularly spot-check weight differential, hardness and the disintegration of tablet.At last, the enteric coated finished product that obtains.Through final product quality after the assay was approved, pack, go into stockyard, be stored in cool place, ventilation, dry place, note preventing to make moist, mouldy, rotten.
By above each embodiment as seen, method of the present invention has prepared highly purified snake venom kininogenase from the snake venom raw material, with this highly purified snake venom kininogenase is raw material, cooperate with auxiliary materials such as various pharmaceutically acceptable carriers and vehicle, can prepare and be suitable for clinical application, particularly be suitable for snake venom kininogenase preparation by the intravenous route administration.
Above detailed description of the present invention does not limit the present invention, and those skilled in the art can make various changes and distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the defined scope of claims of the present invention.
Claims (10)
1, prepares the method for high purity snake venom kininogenase, comprise the steps:
1) will contain snake venom raw material preliminary purification, the separation of snake venom kininogenase, obtaining main component is the active ingredient 1 of snake venom kininogenase;
2) described active ingredient 1 is further purified, obtains active ingredient 2;
3) with described active ingredient 2 as antigen, preparation antisnake venom kininogenase specific antibody;
4) the described antisnake venom kininogenase specific antibody for preparing is combined the preparation antibody affinity chromatography with the affinity chromatography carrier;
5) with described antibody affinity chromatography purification procedures 1) in active ingredient 1, obtain high purity snake venom kininogenase.
2, the described method of claim 1 is characterized in that the antisnake venom of preparation described in step 3) kininogenase specific antibody comprises the steps:
3a) with described active ingredient 2 as antigen, immune mouse, preparation antiserum(antisera);
3b) described active ingredient 2 is combined preparation antigen affinity column with the affinity chromatography carrier;
3c) with antigen affinity column on the described antiserum(antisera), separate obtaining antisnake venom kininogenase specific antibody.
3, the described method of claim 1 is characterized in that the antisnake venom of preparation described in step 3) kininogenase specific antibody comprises the steps:
3i) with described active ingredient 2 as antigen, immune mouse, preparation immune mouse spleen cell;
3ii) described immune mouse spleen cell and murine myeloma cell are merged the hybridoma that preparation is merged;
3iii) hybridoma of antisnake venom kininogenase antibody is expressed in screening, sets up cell strain;
3iv) cultivate described cell strain and obtain described antisnake venom kininogenase specific antibody.
4, claim 1,2 or 3 described methods is characterized in that the described snake venom raw material of step 1) preliminary purification, separation comprise the steps:
1a) with the snake venom raw material through the dialysis, molecular weight cut-off is 10000 parts;
1b) the above-mentioned part of holding back is adsorbed through the DEAE-Sepharose ion exchange column, behind the foreign protein that does not adsorb with the Tris-HCl buffer solution elution of 0.02mol/L pH7.8, the damping fluid that contains 0.05mol/L and 0.115mol/L sodium-chlor with the Tris-HCl of 0.02mol/L carries out gradient elution respectively, collect active peak, preparation active ingredient 1.
5, claim 1,2 or 3 described methods is characterized in that described affinity chromatography carrier is Sepharose 4B sepharose, diethylin dextrane gel or CM-sephadex.
6, claim 1,2 or 3 described methods is characterized in that described step 2) in be further purified active ingredient 1 method for active ingredient 1 is separated through preparative high performance liquid chromatography, preparation snake venom kininogenase is active ingredient 2.
7, the high purity snake venom kininogenase of claim 1 method preparation.
8, the described high purity snake venom of claim 7 kininogenase is characterized in that described high purity snake venom kininogenase has following characteristic:
Specific activity is more than every milligram of albumen 800 units;
High performance liquid chromatography detects, and single component collection of illustrative plates, main peak area are greater than 95%, and retention time is 7.8 ± 0.5min;
Show a band on the SDS-polyacrylamide gel electrophoresis, molecular weight is 34～44 kilodaltons;
Amino acid sequence analysis, terminal 15 aminoacid sequences of N-are:
Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His-Arg-Phe-Leu。
9, a kind of pharmaceutical preparation contains claim 7 described high purity snake venom kininogenase and pharmaceutically acceptable pharmaceutical excipient.
10, the described pharmaceutical preparation of claim 9 is characterized in that described pharmaceutical preparation is the intravenously administrable formulation.
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| CNB2004100094221A CN100395331C (en) | 2004-08-11 | 2004-08-11 | High purity venom kininogenase prepartion method and its pharmaceutical formulation |
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| WO2012019423A1 (en) * | 2010-08-13 | 2012-02-16 | 兆科药业(香港)有限公司 | Angiogenesis inhibitor, method for purifying thereof and pharmaceutical composition comprising thereof |
| CN102813637A (en) * | 2012-08-31 | 2012-12-12 | 济南维尔康生化制药有限公司 | Kallidinogenase enteric coated tablet and preparation method thereof |
| CN103645328A (en) * | 2013-12-17 | 2014-03-19 | 中国计量学院 | Method for preparing peanut allergic protein Arah2 standard |
| CN104558170A (en) * | 2014-12-22 | 2015-04-29 | 广西大学 | Preparation process of antivenin |
| CN111487352A (en) * | 2020-05-28 | 2020-08-04 | 海南热带海洋学院 | Method for detecting titer of anti-snake venom serum |
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2004
- 2004-08-11 CN CNB2004100094221A patent/CN100395331C/en not_active Expired - Lifetime
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012019423A1 (en) * | 2010-08-13 | 2012-02-16 | 兆科药业(香港)有限公司 | Angiogenesis inhibitor, method for purifying thereof and pharmaceutical composition comprising thereof |
| CN102813637A (en) * | 2012-08-31 | 2012-12-12 | 济南维尔康生化制药有限公司 | Kallidinogenase enteric coated tablet and preparation method thereof |
| CN102813637B (en) * | 2012-08-31 | 2013-08-21 | 济南维尔康生化制药有限公司 | Kallidinogenase enteric coated tablet and preparation method thereof |
| CN103645328A (en) * | 2013-12-17 | 2014-03-19 | 中国计量学院 | Method for preparing peanut allergic protein Arah2 standard |
| CN103645328B (en) * | 2013-12-17 | 2016-01-13 | 中国计量学院 | A kind of preparation method of Major Peanut Allergens with IgE-binding Arah2 standard items |
| CN104558170A (en) * | 2014-12-22 | 2015-04-29 | 广西大学 | Preparation process of antivenin |
| CN111487352A (en) * | 2020-05-28 | 2020-08-04 | 海南热带海洋学院 | Method for detecting titer of anti-snake venom serum |
| CN111487352B (en) * | 2020-05-28 | 2023-08-18 | 海南热带海洋学院 | Method for detecting antivenom serum titer |
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|---|---|
| CN100395331C (en) | 2008-06-18 |
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Address after: 100078 No. 8 prosperous street, Beijing economic and Technological Development Zone Patentee after: Beijing Saisheng Pharmaceutical Co.,Ltd. Address before: 100078 No. 8 prosperous street, Beijing economic and Technological Development Zone Patentee before: BEIJING SAISHENG PHARMACEUTICAL Co.,Ltd. |
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