CN100424171C - High purity venom fibrinolysin prepartion method and its pharmaceutical formulation - Google Patents
High purity venom fibrinolysin prepartion method and its pharmaceutical formulation Download PDFInfo
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- CN100424171C CN100424171C CNB200410009433XA CN200410009433A CN100424171C CN 100424171 C CN100424171 C CN 100424171C CN B200410009433X A CNB200410009433X A CN B200410009433XA CN 200410009433 A CN200410009433 A CN 200410009433A CN 100424171 C CN100424171 C CN 100424171C
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- fibrinolytic enzyme
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- nevin fibrinolytic
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Abstract
The present invention relates to a method for preparing high-purity venom plasmin, which comprises the steps: combining an antigen-antibody reaction and an affinity chromatography technique; separating and purifying venin plasmin from raw material venin containing venin plasmin; preparing venin plasmin with high purity. The invention also relates to the high-purity venin plasmin and a medicinal preparation thereof which are prepared by the method of the present invention. The obtained product is an ideal thrombolytic which integrates the advantages of high purity, moderate selectivity, long half life, inhibit resistance, high efficiency and no toxic or side effect.
Description
Technical field
The present invention relates to the preparation method of highly purified nevin fibrinolytic enzyme, more particularly relate to adopt immunochromatographic method to prepare highly purified nevin fibrinolytic enzyme.
The invention still further relates to nevin fibrinolytic enzyme and preparation thereof with the inventive method preparation.
Background technology
In the current mankind disease, the M ﹠ M of cardiovascular disorder all accounts for the first place, and wherein thrombotic disease can make patient's quality of life descend or be dead.Thrombotic disease mainly comprises three types: cause the coronary artery disease of myocardial infarction, cause the cerebrovascular disease and the phlebothrombosis disease that is easy to cause pulmonary infarction of apoplexy.These diseases all are to cause the vitals ischemic to cause by the thrombosis in the blood vessel of key position.The thrombolysis medicine is the choice drug of these diseases of treatment.
The thrombolysis medicine through decades research, roughly can be divided into three phases.
First-generation PgA (plasminogen activator)
Mainly form by streptokinase SK and urokinase u-PA.Though first-generation PgA has certain thrombolytic effect, exist a lot of defectives.Show as:
1) transformation period is very short in vivo, needs continuous injection can reach therapeutic purpose.Therefore, have more side effect, when treating, almost caused the formation of anti-freezing state bar none as use u-PA.
2) inject the interior back of body and easily produce antibody, become the inhibitor of medicine itself, limited the performance of thrombolytic effect, even caused hemorrhagic syndrome and inspire the platelet aggregation that SK mediates.
3) scleroproein poor selectivity, the Pg (Fibrinogen) in activating thrombus, the also same Pg that activates in the blood, thus activated fibrinolytic system, make blood coagulation system be badly damaged.
S-generation PgA
With tissue-type plasminogen activator (t-PA), Single-chain Urokinase-type Plasminogen Activator (scu-PA) and formylation Profibrinolysin-streptokinase mixture (APSAC) is representative.Significantly different with first-generation Pg A is that s-generation PgA has very high scleroproein selectivity.Therefore, its local fibrinolytic effect in thrombus is strong.But s-generation PgA still exists some shortcoming and defect:
1) t-PA and the scu-PA transformation period in vivo very short, have only continuous injection can reach therapeutic purpose.For dissolving the required dosage of coronary artery thrombosis as early as possible, be enough to cause a large amount of losses of Pg and Fg, thereby cause bleeding tendency.
2) clinical observation, the blood vessel of treatment back mediation is prone to once more and stops up.
3) though the APSAC transformation period longer, if administration can cause the activation of ypotension and systemic fibrinolytic system too soon.
In recent years, on the basis of above-mentioned thrombolytics, improved at its deficiency, developed the combination of mutant, mosaic, monoclonal antibody and the PgA of some PgA with molecular biology and chemical process, obtained certain effect, but cost an arm and a leg, and still can run into following problems in the thromboembolism treatment:
1) the Acute Myocardial Infarction patient of 20%-30% is reactionless to thrombolytic drug, and all undesirable for the result of treatment of old thrombus.
2) acute obstruction again appears in 5% patient.
3) mortality ratio is still very high, reaches 10%.
The incidence of 4) intracranialing hemorrhage is 1%.
5) for short thrombolytics of transformation period, need frequent drug administration during treatment, make the treatment cost higher.
Third generation thrombolytics---plasmin
Middle 1950s Didisheim and Lewis have reported in many snake venom and have found fibrinolytic, for the first time proposing the snake venom fibrinoclase is insensitive theory to human serum protein's enzyme inhibition factor, for the people's blood clot that dissolves under the pathological conditions with the purifying reptilase lays the foundation.
After in human body, injecting plasmin, the lys of its single-minded ground cracking scleroproein A α-chain
113-leu
114Key, thus play the effect of thrombolysis, evidence, plasmin all has good thrombolysis effect in external or body.Simultaneously, clinically, do not find that so far it has bleeding, this is because plasmin has the Substratspezifitaet of height.Use the octapeptide of Regular Insulin oxidation of beta chain and synthetic and found out that these enzymes of decision are to lys
113-leu
114The single-minded cracked reason of key.The result shows that the same with other enzyme, what determine its cracking site is that sequence is decisive, and nonbonding is decisive.At least with lys
113-leu
114Eight amino-acid sequences on every side are relevant.
Summary of the invention
One object of the present invention is to provide a kind of preparation method of high purity nevin fibrinolytic enzyme, utilizes immune-affinity chromatography to prepare highly purified nevin fibrinolytic enzyme.
Another object of the present invention is to provide the high purity nevin fibrinolytic enzyme that uses the inventive method preparation.
A further object of the present invention is to provide the pharmaceutical preparation that contains high purity nevin fibrinolytic enzyme of the present invention.
According to an aspect of the present invention, prepare the method for highly purified nevin fibrinolytic enzyme, comprise the steps:
1) contain snake venom raw material preliminary purification, the separation of nevin fibrinolytic enzyme, obtaining main component is the active ingredient 1 of nevin fibrinolytic enzyme;
2) described active ingredient 1 is further purified, obtains active ingredient 2;
3) with described active ingredient 2 as antigen, preparation antisnake venom plasmin specific antibody;
4) the described nevin fibrinolytic enzyme specific antibody for preparing is combined the preparation antibody affinity chromatography with the affinity chromatography carrier;
5) with described antibody affinity chromatography purification step 1) active constituent 1, obtain the high purity nevin fibrinolytic enzyme.
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 nevin fibrinolytic enzyme,, prepare the high nevin fibrinolytic enzyme of purity the nevin fibrinolytic enzyme separation and purification.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 nevin fibrinolytic enzyme, utilize this antibody affinity chromatography to carry out purifying, prepare highly purified nevin fibrinolytic enzyme raw material.
In the methods of the invention, specific antibody at nevin fibrinolytic enzyme can adopt the several different methods preparation, in a specific embodiments of the present invention, it at first is the antigen immune mouse with the nevin fibrinolytic enzyme, acquisition contains the mouse resisting anteserum of antisnake venom plasmin antibody, simultaneously this nevin fibrinolytic enzyme 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 nevin fibrinolytic enzyme of the present invention, for example, to merge through mouse boosting cell after the nevin fibrinolytic enzyme immunity and myeloma cell, obtain hybridoma, the hybridoma of screening secretion nevin fibrinolytic enzyme antibody is built strain, can obtain the specific antibody of stable nevin fibrinolytic enzyme thus.
The antisnake venom plasmin 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 nevin fibrinolytic enzyme raw material.
In the methods of the invention, in order to obtain highly purified nevin fibrinolytic enzyme, 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 through antibody affinity chromatography purifying of the present invention, can obtain highly purified nevin fibrinolytic enzyme again.
Preferably use the higher nevin fibrinolytic enzyme of purity as antigenic nevin fibrinolytic enzyme, 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 nevin fibrinolytic enzyme, the nevin fibrinolytic enzyme 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 another aspect of the present invention, the high purity nevin fibrinolytic enzyme by the inventive method preparation is provided, it has following characteristic:
Specific activity is more than the 5000U/mg albumen;
High-efficient liquid phase chromatogram technique analysis, single component collection of illustrative plates, main peak area are greater than 95%, and retention time is 8.3 ± 0.5min;
SDS one polyacrylamide gel electrophoresis shows a band, and molecular weight is 31000 ± 2000 dalton;
In accordance with a further aspect of the present invention, the present invention is provided highly purified nevin fibrinolytic enzyme pharmaceutical preparation, requirement according to medicine registration management way and Good Manufacturing Practice and Quality Control of Drug, by nevin fibrinolytic enzyme is efficacy component, add pharmaceutically acceptable pharmaceutical excipient and prepare the various pharmaceutical preparations that are suitable for clinical application, the formulation of these preparations includes but not limited to plasmin injection liquid and injectable fibrinolysin.
The present invention combines traditional purifying technique and immunoaffinity chromatography technology, is purified into a kind of plasmin from viper venom.And utilize this bulk drug to make to supply the preparation of number of ways administrations such as intramuscular injection, intravenous injection and intravenous infusion.The characteristics of this technology are that the specificity of enzyme is strong, through the high performance liquid chromatography inspection is one-component, on the SDS-polyacrylamide gel electrophoresis, show a band, prove through clinical trial, this product has unique curative effect in thromboembolism treatment, having overcome the shortcoming and defect that the first-generation and s-generation PgA exist, is a new milestone of thrombolytic drug.Show as:
1) can directly act on thrombosed scleroproein, it is degraded, and the old thrombus is had unique curative effect.
2) degradable plasma fibrinogen in vivo, blood viscosity lowering, the antagonism zymoplasm, thus reduce the sickness rate of hypertension, atherosclerosis, ischemic heart disease.
3) transformation period in vivo longer, experiment showed, that administration got blood in 24 hours, hematological indices (as plasma fibrinogen content, plasma viscosity etc.) still can obviously change.
4) fibrinolytic system in the body is not had obvious activation, therefore do not have bleeding tendency, avoided the generation of complication.
5) because this product purity height, so toxic side effect reduces greatly, uses safer.
6) very strong Substratspezifitaet is arranged, effect is rapid, good effect.
In sum, nevin fibrinolytic enzyme provided by the invention is a collection selectivity appropriateness, long half time, and anti-the inhibition, render a service height, have no side effect in the perfect solution suppository of one.
Brief description of drawings
Fig. 1 is the ultraviolet absorpting spectrum of the anion exchange chromatography record of preparation active ingredient 1 in the inventive method;
Fig. 2 is an antisnake venom plasmin 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 nevin fibrinolytic enzyme in the inventive method;
Fig. 4 is a high purity nevin fibrinolytic enzyme 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 a nevin fibrinolytic enzyme;
Wherein, 1,4,9 is 040312 batch of plasmin sample
2,5,10 is 040315 batch of plasmin sample
6,7 is 040317 batch of plasmin sample
Fig. 5 is the high purity nevin fibrinolytic enzyme high-efficient liquid phase chromatogram spectrum of the present invention's preparation;
Fig. 6 is the high purity nevin fibrinolytic enzyme high-efficient liquid phase chromatogram spectrum of another batch of the present invention's preparation;
Fig. 7 is another batch high purity nevin fibrinolytic enzyme high-efficient liquid phase chromatogram spectrum of the present invention's preparation.
The embodiment of invention
The preparation of highly purified nevin fibrinolytic enzyme
1. the processing of snake venom crude product
Accurately take by weighing viper venom (Liaoning radically reform She Chang produce), the slow middle liquid of Tris-HCl with 0.02mol/L pH7.8 fills dissolving with it, got supernatant in centrifugal 20 minutes with 4000r/min then, precipitation is dissolved the centrifuging and taking supernatant once more with a spot of damping fluid, merge supernatant liquor dress dialysis tubing (molecular weight cut-off is the component 10000 or more) to the slow middle liquid of Tris-HCl of 0.02mol/L pH7.8 dialyse 12 hours standby.
2. ion exchange chromatography purifying
The sample of dialysing is 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 then, when seeing through the peak down to baseline, carry out gradient elution with the 0.02mol/L Tris-HCl damping fluid that contains 0.05mol/L and 0.2mol/L sodium-chlor, collect active peak, make active ingredient 1.The main component of active ingredient 1 is a nevin fibrinolytic enzyme, and tool nevin fibrinolytic enzyme activity is identical with nevin fibrinolytic enzyme 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 nevin fibrinolytic enzyme
4. the preparation of antigen affinity chromatography
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/L NaCl) pH8.3 damping fluid.Above-mentioned Sepharose-4B is mixed with active ingredient 2 solution, at stirring at room 2h, with the Na of 0.1mol/LpH8.3
2CO
3Behind (containing 0.5mol/LNaCl) damping fluid thorough washing, use 0.1mol/L Tris-HCl damping fluid (pH8.3) 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 the 0.2ml stimulator in the mouse tail vein injection booster immunization in preceding 3 days in blood sampling.
6. prepare antisnake venom plasmin 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 (0.05mol/L Tris-HCl damping fluid, pH=7.8) 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 (0.02mol/L Tris-HCl damping fluid, pH=7.8, contain 0.5mol/L NaCl, 7%Arg) wash-out is collected active ingredient, as shown in Figure 2.After the active ingredient collected concentrated polyoxyethylene glycol with dialysis tubing (molecular weight cut-off is 10000), lyophilize was antisnake venom plasmin 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 10.0 0.1mol/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 nevin fibrinolytic enzyme
The preparation of [embodiment 1] immune antibody affinity column
With high-efficient liquid phase chromatogram purification and through nevin fibrinolytic enzyme (active ingredient 2) 100 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 5 times altogether.Got the 0.2ml stimulator in the 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 nevin fibrinolytic enzyme 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 nevin fibrinolytic enzyme (active ingredient 2) 200 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 5 times altogether.Got the 0.2ml stimulator in the mouse tail vein injection booster immunization in preceding 3 days at collection serum.With antibody affinity chromatography purifying nevin fibrinolytic enzyme antibody.
With cyanogen bromide-activated diethylin dextrane gel, the nevin fibrinolytic enzyme 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 nevin fibrinolytic enzyme (active ingredient 2) 400 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 5 times altogether.Before gathering serum, got the 0.2ml stimulator in the mouse tail vein injection booster immunization in preceding 3 days.With antibody affinity chromatography purifying nevin fibrinolytic enzyme antibody.
Use the cyanogen bromide-activated CM-sephadex, the nevin fibrinolytic enzyme antibody coupling that obtains to polysaccharide matrix, thereby make immune affinity chromatographic column.
The preparation of [embodiment 4] high purity nevin fibrinolytic enzyme bulk drug
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 dissolved the centrifuging and taking supernatant once more with a spot of damping fluid, and merging supernatant liquor dress dialysis tubing delays middle liquid dialysis 12 hours to the Tris-HCl of 0.02mol/L pH7.8.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 (0.05mol/L Tris-HCl damping fluid, pH=7.8 contains 0.5mol/L 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, (0.05mol/LTris-HCl damping fluid, pH=7.8 contain 0.5mol/L 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, detect with high performance liquid chromatography, obtain the single component collection of illustrative plates, its retention time is 8.477, is 30800 dalton with SDS-polyacrylamide gel electrophoresis determining molecular weight.Pyrogen, hemorrhage poison, neural poison, the irritated inspection, all up to specification.
The preparation of [embodiment 5] high purity nevin fibrinolytic enzyme bulk drug
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 dissolved the centrifuging and taking supernatant once more with a spot of damping fluid, and merging supernatant liquor dress dialysis tubing delays middle liquid dialysis 10 hours to the Tris-HCl of 0.02mol/L pH7.8.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 (0.05mol/L Tris-HCl damping fluid, pH=7.8 contains 0.5mol/L 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, (0.05mol/LTris-HCl damping fluid, pH7.8 contain 0.5mol/L 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, detect with high performance liquid chromatography, obtain the single component collection of illustrative plates, its retention time is 8.482, is 30500 dalton with SDS-polyacrylamide gel electrophoresis determining molecular weight.Pyrogen, hemorrhage poison, neural poison, the irritated inspection, all up to specification.
The preparation of [embodiment 6] high purity nevin fibrinolytic enzyme bulk drug
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 dissolved the centrifuging and taking supernatant once more with a spot of damping fluid, 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 0.115mol/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 dialysis, last antibody affinity chromatography through ion-exchange purification.With affinity chromatography balance liquid (0.05mol/L Tris-HCl damping fluid, pH7.8, contain 0.5mol/L NaCl) balance, treat that effluent liquid after drawing stable baseline on the Protein Detection instrument, uses affinity chromatography elutriant (0.05mol/LTris-HCl damping fluid, pH7.8 instead, contain 0.5mol/L NaCl, 7%Arg) wash-out is collected elution peak, measures the activity of its enzyme.
Detect through activity, the component of collecting has very high fibrinolytic, gets this component and suitably dilutes, detect with high performance liquid chromatography, obtain the single component collection of illustrative plates, its retention time is 8.498, is 30150 dalton with SDS-polyacrylamide gel electrophoresis determining molecular weight.
Pyrogen, hemorrhage poison, neural poison, the irritated inspection, all up to specification.
The assessment of performance of high purity nevin fibrinolytic enzyme
1. than the mensuration of vigor
(1) preparation of reagent
Tris buffer (pH7.8) is got Tutofusin tris 2.42g, and sodium-chlor 4.06g adds water 400ml dissolving, transfers pH to 7.8 with the 4mol/L hydrochloric acid soln, adds water to 500ml.
The experiment damping fluid is got trihydroxy methyl aminomethane buffer solution (pH7.8), 0.7% calcium chloride solution and 0.9% sodium chloride solution with 1: 2: 4 volume mixing promptly.
Thrombin solution is got zymoplasm (Nat'l Pharmaceutical ﹠ Biological Products Control Institute), adds trihydroxy methyl aminomethane buffer solution (pH7.8) and dissolves and be diluted to the solution that contains 5 units among every 1ml.
Fibrinogen solution is got Fibrinogen (Nat'l Pharmaceutical ﹠ Biological Products Control Institute), adds trihydroxy methyl aminomethane buffer solution (pH7.8) dissolving and quantitatively is diluted to the solution that contains the about 5mg of solidifiable albumen among every 1ml.
(2) trial-product (the high purity nevin fibrinolytic enzyme of embodiment 4～6 preparation is as described above got in the preparation of need testing solution, lot number is 040312,040315 and 040317, down together) an amount of, add trihydroxy methyl aminomethane buffer solution (pH7.8) dissolving and quantitatively be diluted to the solution that contains 15 units among every 1ml.
(3) assay method is got centrifuge tube, each accurate experiment damping fluid 0.2ml that adds, need testing solution 0.1ml, put 37 ℃ of water-bath preheatings 2 minutes, add thrombin solution 0.1ml, fibrinogen solution 0.1ml, the several seconds of jolting immediately, mixing, put 37 ℃ of water-bath accurate responses 30 minutes, put termination reaction in the ice-water bath, centrifugal (14000 rev/mins) 4 minutes are with method operation 3 times, carefully liquid is blotted, the accurate 0.2mol/L sodium hydroxide solution 3ml that adds in residue, heating in water bath 8 minutes treats that scleroproein dissolves fully, after the cooling, with the 0.2mol/L sodium hydroxide solution is blank, according to spectrophotometry (two appendix IVA of Chinese Pharmacopoeia version in 2000), measures optical density A at 280nm wavelength place.Replace need testing solution to record As with tris buffer (pH7.8) 0.1ml simultaneously, as blank with the method operation.Be calculated as follows the unit of activity of trial-product
Trial-product vigor (U/mg)=(As-A) * Ws * N/ of every 1mg (optical density of As-blank in As * 0.1 * 30 * W) formula
The optical density of A-need testing solution
Contain the proteic micrograms of solidifiable among the Ws-fibrinogen solution 0.1mg
N-trial-product extension rate
0.1-the need testing solution volume, ml
The 30-reaction times, minute
W-trial-product sampling amount, mg
The definition of plasmin unit of activity: under these conditions, 1 minute dissolves 1 microgram scleroproein (solidifiable albumen) is a plasmin unit of activity.
It is an amount of that protein content is got trial-product, add water and make and contain the proteic solution of 0.15mg among every 1ml approximately, as need testing solution, precision is measured need testing solution 1.0ml, measure according to " forint phenol assay method " (seeing notes), calculate the protein content (mg) in every 1mg trial-product.
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 XVB of Chinese Pharmacopoeia version in 2000 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 IVB of Chinese Pharmacopoeia version in 2000) 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.
Be calculated as follows than living
2. molecular weight determination
Get this product, measure according to electrophoretic method (two appendix VF of Chinese Pharmacopoeia version in 2000 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 damping fluid (water intaking 4.8ml, tris buffer (pH6.8) 1.2ml, glycerine 1.0ml, 10% sodium lauryl sulphate 2.0ml, 0.5% tetrabromophenol sulfonphthalein solution 0.5ml, beta-mercaptoethanol 0.5ml, mixing) make 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 trial-product damping fluid (with the preparation of standard protein solution) and makes and contain the proteic solution of 1 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.
Measurement result: be determined as a band, molecular weight is 31000 ± 2000 dalton, the results are shown in Figure 4.
3. purity test
(1) as above on the SDS-polyacrylamide gel electrophoresis, should show a band (seeing molecular weight determination for details).
(2) according to high effective liquid chromatography for measuring (two appendix VIH of Chinese Pharmacopoeia version in 2000), trial-product should be one-component.
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
TSK-2000 (300 * 7.5mm, 5 μ m) gel chromatographic columns; Moving phase 0.2mol/L phosphate buffered saline buffer (pH6.8); Detect wavelength 280nm; Flow velocity 1.0ml/min; Temperature: 25 ℃; Number of theoretical plate calculates by the plasmin peak should be not less than 3000.
Assay method: get trial-product (according to the high purity nevin fibrinolytic enzyme of previous embodiment 4～6 preparations) and make the solution that every 1ml contains 100 units with moving phase, get 20 μ l and inject liquid chromatograph, the record color atlas, calculate by area normalization method, the main peak area should be greater than 95%, and retention time should be 8.3 ± 0.5min; Before the main peak high molecular impurity must not be arranged, the HPLC measurement result of three batches of samples is seen Fig. 5～7.
4. pyrogen is got trial-product (according to the high purity nevin fibrinolytic enzyme of previous embodiment 4～6 preparations, down together), make the solution that contains 2 units among every 1ml, check (two appendix XID of 2000 years versions of Chinese Pharmacopoeia) in accordance with the law with sodium chloride injection, exempt from the every 1kg injection of body weight 10ml by family, should be up to specification.
5. hemorrhage poison is got trial-product, adds the chlorination sodium injection and makes solution that every 1ml contains 4 units as need testing solution, gets 5 of the small white mouses of body weight 18～22g, back subcutaneous injection need testing solution 0.1ml, inject and put to death animal in back 24 hours, peeling is observed, and mouse back must not have bleeding.
6. neural poison is got trial-product, add the chlorination sodium injection and make the solution that contains 100 units among every 1ml, as need testing solution, get 3 of the doves of body weight 300～500g, dosage is by the every 1kg injection of body weight need testing solution 0.5ml, intravenously administrable, observed 24 hours, animal must not be had a convulsion, death, has a convulsion or death as there being one in 3 doves, 5 retrials of dove should be got in addition, all death must not be occurred.
7. to get trial-product an amount of for hypersensitive test, make with sodium chloride injection the need testing solution that contains 2 units among every 1ml as sensitization liquid with attack liquid.
Get 6 of body weight 250-350g cavys, every other day abdominal injection need testing solution 0.5ml, 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.The assay of three batches of samples is listed as follows:
Can reach a conclusion by verification result: utilization immunoaffinity chromatography technology, the plasmin purity height that from snake venom, extracts, the product that obtains is higher than living, reach more than the 5000U/mg albumen, 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 29～33KD, safe in utilization, can reach the requirement of intravenously administrable.
The pharmaceutical preparation that contains the high purity nevin fibrinolytic enzyme
[embodiment 7] injectable fibrinolysin
At first, in the local laminar flow clean area by preparation prescription with plasmin raw material medicine (according to previous embodiment preparation of the present invention, and qualified, down with) and the accurate weighings of auxiliary material such as albumin, sodium-chlor, phosphoric acid salt, dextran through every detection in liquid dispensing container; Add the injection water and fully dissolve or dilute, making phosphatic concentration is 0.03mol/L, and sodium chloride concentration is 0.9%, and 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 warehouse-in.
[embodiment 8] plasmin injection liquid and used for intravenous injection plasmin preparation
At first, in the local laminar flow clean area by preparation prescription with accurate weighings of auxiliary material such as plasmin raw material medicine and albumin, sodium-chlor, phosphoric acid salt in liquid dispensing container; Add the injection water and fully dissolve or dilute, making phosphatic concentration is 0.03mol/L, and sodium chloride concentration is 0.9%, and 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, with the intermediate after filtering by loading amount pack into antibiotic bottle tamponade simultaneously, roll lid and get product.In minute process of assembling, should carry out 4 times loading quantity inspection at least, guarantee that the dose of packing in each bottle is accurate.At last, through final product quality after the assay was approved, pack, go into stockyard and deposit.
By above each embodiment as seen, method of the present invention has prepared highly purified nevin fibrinolytic enzyme from the snake venom raw material, with this highly purified nevin fibrinolytic enzyme is raw material, cooperate with auxiliary materials such as various pharmaceutically acceptable carriers and vehicle, can prepare the pharmaceutical preparation that is suitable for clinical application.
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 (6)
1. prepare the method for high purity nevin fibrinolytic enzyme, comprise the steps:
1) the snake venom raw material that will contain nevin fibrinolytic enzyme is by following step preliminary purification, separation, and obtaining main component is the active ingredient 1 of nevin fibrinolytic enzyme, and described step is:
1a) with the snake venom raw material through the dialysis, molecular weight cut-off 10000 is with top;
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/LpH7.8, 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, collects active peak;
2) described active ingredient 1 is further separated through preparative high performance liquid chromatography, preparation active ingredient 2 nevin fibrinolytic enzymes;
3) with active ingredient 2 as antigen, prepare antisnake venom plasmin specific antibody by following step, described step is:
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 plasmin specific antibody;
4) the described antisnake venom plasmin 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 the high purity nevin fibrinolytic enzyme.
2. the described method of claim 1 is characterized in that in step 3), prepares antisnake venom plasmin specific antibody with following method, and described step comprises:
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 plasmin antibody is expressed in screening, sets up cell strain;
3iv) cultivate described cell strain and obtain described antisnake venom plasmin specific antibody.
3. claim 1 or 2 described methods is characterized in that described affinity chromatography carrier is Sepharose4B sepharose, diethylin dextrane gel or CM-sephadex.
4. the high purity nevin fibrinolytic enzyme of claim 1 method preparation is characterized in that described high purity nevin fibrinolytic enzyme has following characteristic:
Specific activity is more than the 5000U/mg albumen;
High performance liquid chromatography detects, and single component collection of illustrative plates, main peak area are greater than 95%, and retention time is 8.3～8.8min;
Show a band on the SDS-polyacrylamide gel electrophoresis, molecular weight is 31000 ± 2000 dalton.
5. a pharmaceutical preparation contains described high purity nevin fibrinolytic enzyme of claim 4 and pharmaceutically acceptable pharmaceutical excipient.
6. the described pharmaceutical preparation of claim 5 is characterized in that described pharmaceutical preparation is the intravenously administrable formulation.
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| CN100457897C (en) * | 2006-03-28 | 2009-02-04 | 中国海洋大学 | Prepn. process of and use of thrombus dissolving enzyme |
| WO2008020742A2 (en) * | 2006-08-16 | 2008-02-21 | Wisdom Asset Company | Method for extracting fibrinogenic/fibrinolytic protein from agkistrodon blomhoffii ussuriensis venom |
| CN102372770B (en) * | 2010-08-13 | 2014-04-30 | 兆科药业（广州）有限公司 | Angiogenesis inhibitant, purification method and medicinal composition therewith |
| CN102080093B (en) * | 2010-12-01 | 2012-09-05 | 安徽丰原药业股份有限公司 | Mutant venin fibrinolytic enzyme gene and preparation method thereof |
| CN102080099B (en) * | 2010-12-01 | 2012-10-10 | 安徽丰原药业股份有限公司 | Mutant Alfimeprase engineering strain, and preparation method and applications thereof |
| WO2014123516A1 (en) * | 2013-02-06 | 2014-08-14 | Nc Medical Research Inc | Cell therapy for the treatment of neurodegeneration |
| CN108918749B (en) * | 2018-07-17 | 2020-05-05 | 北京赛升药业股份有限公司 | Reversed phase HPLC detection method of snake venom fibrinolytic enzyme |
| CN113049837A (en) * | 2021-04-01 | 2021-06-29 | 捷和泰（北京）生物科技有限公司 | Whole blood PCT immunoturbidimetric reagent |
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| CN1120070A (en) * | 1994-12-26 | 1996-04-10 | 合肥兆峰科大药业有限公司 | Point pallas pit viper poisonous fibre-dissolving enzyme and its purifying method |
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| CN1120070A (en) * | 1994-12-26 | 1996-04-10 | 合肥兆峰科大药业有限公司 | Point pallas pit viper poisonous fibre-dissolving enzyme and its purifying method |
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| c2erbBO2 多克隆抗体的制备及特性分析. 顾斐等.复旦学报(医学版)，第29卷第6期. 2002 |
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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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