CN100336903C - Extracting norfibroase from snake venom and preparation process for liquid drugs injection of norfibroase - Google Patents
Extracting norfibroase from snake venom and preparation process for liquid drugs injection of norfibroase Download PDFInfo
- Publication number
- CN100336903C CN100336903C CNB2004100215584A CN200410021558A CN100336903C CN 100336903 C CN100336903 C CN 100336903C CN B2004100215584 A CNB2004100215584 A CN B2004100215584A CN 200410021558 A CN200410021558 A CN 200410021558A CN 100336903 C CN100336903 C CN 100336903C
- Authority
- CN
- China
- Prior art keywords
- preparation
- fiber eliminating
- eliminating enzyme
- snake venom
- preparing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 85
- 239000003998 snake venom Substances 0.000 title abstract description 32
- 238000002347 injection Methods 0.000 title abstract description 11
- 239000007924 injection Substances 0.000 title abstract description 11
- 239000007788 liquid Substances 0.000 title description 9
- 239000003814 drug Substances 0.000 title description 4
- 229940079593 drug Drugs 0.000 title description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000008215 water for injection Substances 0.000 claims abstract description 11
- 108090000790 Enzymes Proteins 0.000 claims description 74
- 102000004190 Enzymes Human genes 0.000 claims description 74
- 239000000835 fiber Substances 0.000 claims description 72
- 238000001467 acupuncture Methods 0.000 claims description 26
- 239000006166 lysate Substances 0.000 claims description 20
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 12
- 238000012856 packing Methods 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 24
- 239000012043 crude product Substances 0.000 abstract description 13
- 108010027612 Batroxobin Proteins 0.000 abstract description 11
- 239000000243 solution Substances 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 239000003381 stabilizer Substances 0.000 abstract description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 abstract 2
- 239000011780 sodium chloride Substances 0.000 abstract 1
- 238000003860 storage Methods 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 68
- 238000001042 affinity chromatography Methods 0.000 description 33
- 239000000872 buffer Substances 0.000 description 19
- 238000000746 purification Methods 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 14
- 239000000047 product Substances 0.000 description 14
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 13
- 230000000694 effects Effects 0.000 description 12
- 238000005516 engineering process Methods 0.000 description 12
- 239000000284 extract Substances 0.000 description 11
- 239000000203 mixture Substances 0.000 description 10
- 229920002684 Sepharose Polymers 0.000 description 9
- 238000004255 ion exchange chromatography Methods 0.000 description 9
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 239000006285 cell suspension Substances 0.000 description 8
- 210000004408 hybridoma Anatomy 0.000 description 8
- -1 polyoxyethylene Polymers 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 239000000427 antigen Substances 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 229940088623 biologically active substance Drugs 0.000 description 7
- 238000009472 formulation Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 238000004321 preservation Methods 0.000 description 7
- 239000002994 raw material Substances 0.000 description 7
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 241000271042 Gloydius halys Species 0.000 description 6
- 206010035226 Plasma cell myeloma Diseases 0.000 description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 201000000050 myeloid neoplasm Diseases 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 230000002035 prolonged effect Effects 0.000 description 6
- 210000004988 splenocyte Anatomy 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 206010003445 Ascites Diseases 0.000 description 4
- 101000772006 Bombus ignitus Venom serine protease Bi-VSP Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 4
- 239000004480 active ingredient Substances 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 4
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 230000001804 emulsifying effect Effects 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000004448 titration Methods 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 208000007536 Thrombosis Diseases 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000012501 chromatography medium Substances 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 230000002537 thrombolytic effect Effects 0.000 description 3
- 206010002198 Anaphylactic reaction Diseases 0.000 description 2
- 108010001779 Ancrod Proteins 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 208000032109 Transient ischaemic attack Diseases 0.000 description 2
- 230000036783 anaphylactic response Effects 0.000 description 2
- 208000003455 anaphylaxis Diseases 0.000 description 2
- 229960004233 ancrod Drugs 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 206010008118 cerebral infarction Diseases 0.000 description 2
- 208000026106 cerebrovascular disease Diseases 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 208000010125 myocardial infarction Diseases 0.000 description 2
- 210000004180 plasmocyte Anatomy 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 201000010875 transient cerebral ischemia Diseases 0.000 description 2
- 206010059245 Angiopathy Diseases 0.000 description 1
- 208000033386 Buerger disease Diseases 0.000 description 1
- 206010008088 Cerebral artery embolism Diseases 0.000 description 1
- 206010008132 Cerebral thrombosis Diseases 0.000 description 1
- 241001449342 Chlorocrambe hastata Species 0.000 description 1
- 241001505404 Deinagkistrodon acutus Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010068365 Femoral artery embolism Diseases 0.000 description 1
- 201000001429 Intracranial Thrombosis Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241001474977 Palla Species 0.000 description 1
- 208000006193 Pulmonary infarction Diseases 0.000 description 1
- 208000003782 Raynaud disease Diseases 0.000 description 1
- 208000012322 Raynaud phenomenon Diseases 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 206010061373 Sudden Hearing Loss Diseases 0.000 description 1
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 1
- 206010043540 Thromboangiitis obliterans Diseases 0.000 description 1
- 208000007814 Unstable Angina Diseases 0.000 description 1
- 241000271897 Viperidae Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000002429 anti-coagulating effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000007503 antigenic stimulation Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229960002210 batroxobin Drugs 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N butyl alcohol Substances CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000011221 initial treatment Methods 0.000 description 1
- 201000010849 intracranial embolism Diseases 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000004089 microcirculation Effects 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 230000007575 pulmonary infarction Effects 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention discloses a method for extracting defibrases from snake venom and preparing a defibrase injection preparation. The method for extracting defibrases from snake venom comprises the following steps: preparing monoclonal antibody; preparing defibrase crude products; preparing antibody affinity columns; preparing defibrases. The method for preparing a defibrase injection preparation comprises the following steps: taking purified defibrases; adding the purified defibrases in a proper amount of water for injection for dissolving to obtain a solution; adding 5 to 20g of sodium chloride stabilizing agents in every 1000 ml of the solution; split charging to obtain a defibrase injection preparation. The present invention has the advantages of simple defibrase preparation process, adaption to scale production, high injection preparation stability and long time storage.
Description
Technical field
The present invention relates to a kind of preparation method who from snake venom, extracts fiber eliminating enzyme and fiber eliminating enzyme hydro-acupuncture preparation, relate in particular to a kind of extracting method that the monoclonal antibody affinity chromatography technology carries out the high purity bioactive enzyme and preparation technology of high stability hydro-acupuncture preparation of adopting with thrombolytic effect.
Background technology
Fiber eliminating enzyme is a proteolytic ferment, and the energy thrombus suppresses thrombosis, microcirculation improvement.Be used for acute cerebral infarction clinically, comprise the prevention that cerebral thrombosis, cerebral embolism, transient ischemic attack (TIA) and cerebral infarction recur again; The prevention that myocardial infarction, unstable angina pectoris and myocardial infarction recur again; Limb angiopathy comprises the femoral artery embolism, thromboangiitis obliterans, Raynaud disease; Also be suitable for blood and be high glutinous state, hypercoagulative state, the preceding state of thrombus; Sudden deafness; Diseases such as pulmonary infarction.
The snake venom thrombin-like enzyme preparation of the tool anticoagulant and thrombolytic effect of from various snake venom, extracting, at home, be extensive use of for many years outward.The multistep column chromatography is taked in traditional fiber eliminating enzyme separation and purification; comprise ion exchange chromatography and molecular sieve gel chromatography; even take means such as high performance liquid phase preparation; the medicinal enzyme of obtaining micro-single component like this is feasible; but a large amount of large-scale productions obtain the medicinal enzyme of one-component almost cannot, but also have the low problem of resulting product purity.And adopt polysaccharide is the affinity chromatography of aglucon, and preparation technology can reach tens milligrams of samples, can obtain single component but must take multistep to operate suddenly.
At present, external snake venom thrombin-like enzyme preparation is aqueous injection, because of being subjected to the restriction of preparation level, all contains the sanitas composition in the prescription to keep stability of formulation, as Ancrod, Batroxobin, Japanese DF-521 etc.; The stability of China for guaranteeing to tire in the prolonged preservation process is all made freeze-dried powder, has reduced the specific activity of goods, also increases the possibility that polymer produces simultaneously.
Summary of the invention
The present invention is exactly a kind of preparation method who extracts fiber eliminating enzyme and fiber eliminating enzyme hydro-acupuncture preparation from snake venom who provides in order to solve the problems of the technologies described above, and its technology is simple, is fit to large-scale production, but and prolonged preservation.
In order to solve the problems of the technologies described above, the present invention is achieved in that
Extract the method for fiber eliminating enzyme from snake venom, it comprises the steps:
1). MONOCLONAL ANTIBODIES SPECIFIC FOR: behind purifying and the fiber eliminating enzyme (1g/L) that from snake venom, extracts qualitatively and isopyknic Freund's complete adjuvant mixing and emulsifying, give animal immune; Get the splenocyte of the animal of immunity then with the Koher method, the immune animal splenocyte of being got is made cell suspension; With the myeloma cell of the cell suspension of gained and same animals by (8-15): 1 mixed, under the effect of 30-80% polyoxyethylene glycol, merge syzygy; Syzygy is carried out selectivity cultivates, at first sift out the hybridoma that to secrete the monoclonal antibody that reacts with predetermined antigens, and then therefrom sifted out and be scheduled to specific hybridoma, finally select practical use and have stable growth and homogeneous cell clone that function is special, obtain monoclonal antibody with the ascites method, standby behind the purifying;
2). the preparation of fiber eliminating enzyme crude product: get snake venom, snake venom through the ion exchange chromatography preliminary purification, is got the fiber eliminating enzyme crude product of preliminary purification, standby;
3). the preparation of antibody affinity chromatography:
3.1 get the carrier of agarose,, get the activatory agarose with this carrier of an amount of cyanogen bromide-activated as affinity chromatography;
The monoclonal antibody of purifying is dissolved in the borate buffer 3.2 get, filters the back and adds in the affinity column, adds the activatory agarose then, at 5-20 ℃ of following stirring reaction 15-25 hour, must have the separating medium of special affinity;
3.3 the dress post promptly obtains antibody affinity chromatography, and is standby;
4). the preparation of fiber eliminating enzyme:
To join in the antibody affinity chromatography gradually through the fiber eliminating enzyme crude product of ion exchange chromatography preliminary purification, treat that effluent liquid is after drawing stable baseline on the detector, use affinity chromatography elutriant wash-out active ingredient instead, collect elution peak, the desorb from the affinity media of the biologically active substance that is adsorbed is got off, measure enzymic activity, promptly get the fiber eliminating enzyme finished product of purifying after the lyophilize.
A kind of preparation method of fiber eliminating enzyme hydro-acupuncture preparation, it comprises the steps:
Get the fiber eliminating enzyme of purifying, it is joined in the proper amount of water for injection dissolve, get lysate, add 5--20g sodium-chlor stablizer in every 1000ml lysate, packing promptly gets the fiber eliminating enzyme hydro-acupuncture preparation.
The preparation method of described fiber eliminating enzyme hydro-acupuncture preparation, it comprises the steps: to get the fiber eliminating enzyme 5000u of purifying, it is joined in the 1000ml proper amount of water for injection dissolve, get lysate, in every 1000ml lysate, add 9--15g sodium-chlor stablizer, 1000 preparations of aseptic subpackaged one-tenth, every content 5u promptly gets the fiber eliminating enzyme hydro-acupuncture preparation.
Advantage of the present invention and effect:
The applicant is applied to the monoclonal antibody affinity chromatography technology purifying of fiber eliminating enzyme product first, and operation steps is simple, resulting product purity height, and primary treatment snake venom amount is big, can prepare up to a hundred milligrams of fiber eliminating enzyme samples at every turn.It is the affinity chromatography of aglucon that domestic such products production adopts polysaccharide, and preparation technology can reach tens milligrams of samples, can obtain single component but must take multistep to operate suddenly.
Antibody is meant the sphaeroprotein that can have immunologic function with the corresponding antigens specific combination.It is after body immune system is subjected to antigenic stimulation, bone-marrow-derived lymphocyte activation, breed and be divided into plasmocyte, by plasmocyte synthetic and excretory to this antigen have special avidity immunoglobulin (Ig) (immunoglobulin, Ig).
Because immunologic stimulant generally is the antigenic substance that contains multiple antigenic determinant, so these antibody also are the mixtures of multiple antibody, are polyclonal antibody usually.Cross reaction can take place in the polyclonal antibody of this multi-antigenic determinant in antigen antibody reaction, false positive results occurs.Along with the development of modern biotechnology, the revolution on the molecular biology has taken place, monoclonal antibody has appearred.
Monoclonal antibody is that the cell that will produce antibody merges mutually with the myeloma cell with unlimited multiplication capacity, by limiting dilution assay and clone technology, makes the single cell of fusion form monoclonal cell system.This antibody that is produced by monoclonal cell system is the immunocyte excretory at an antigenic determinant, because this immunocyte is the clone that can infinitely go down to posterity that single bone-marrow-derived lymphocyte and myeloma cell clone (fusion) produce, thereby consequent antibody is monoclonal antibody.But monoclonal antibody technique demonstrates important value owing to have specificity, affinity and the industrial mass production of height aspect medical.
The present invention as a kind of biotechnology means, utilizes the characteristics of antibody and antigenic high-affinity and highly selective with monoclonal antibody, and monoclonal antibody is connected on the carrier, object is carried out the specificity affinity chromatography, i.e. the monoclonal antibody affinity chromatography technology.This technology can be from biotoxin or biomaterial separation and purification particular proteins or other be difficult for the material of purifying.
The monoclonal antibody affinity chromatography that the present invention adopts is compared with other bioseparation means of purification has remarkable advantages:
Because the high degree of specificity of monoclonal antibody, thereby seldom cause foreign protein to pollute.
Monoclonal antibody and with isolating protein have special avidity, therefore can in single protein peak, wash isolating protein, avoid the dilution or the sex change of isolate.
Concentration requirement to institute's isolate is low, even this protein only accounts for very low concentration in raw material, also can obtain purifying preferably.
Be fit to large-scale industrial production.
At present, external snake venom thrombin-like enzyme preparation is aqueous injection, China's present stage is because the restriction of preparation level, be the stability that guarantees to tire in the prolonged preservation process, all make freeze-dried powder, external similar commodity have Ancrod, Japan's DF-521 etc., external exploitation snake venom preparation is early than China, the purifying technique of its single component Thrombin-like enzyme also begins to use for a long time, and its preparation level is also apparently higher than China, the formulation of at present external like product mostly is injection liquid, but contain the sanitas composition, life-time service is unfavorable to health of human body, and the succeeding in developing of the applicant's liquid drugs injection formulation shows integrating with and surpassing like product of the preparation level of this product of China and world level.
Through the study on the stability in 3 years of room temperature placement, the result shows the fiber eliminating enzyme hydro-acupuncture preparation that does not add stablizer, the requirement that actual titration result after 3 years does not reach quality standard far away, the fiber eliminating enzyme hydro-acupuncture preparation that adds the 5-20g stablizer, the requirement that the titration result can reach quality standards, along with the increasing of stabilizing agent dosage, tiring of fiber eliminating enzyme hydro-acupuncture preparation is stable more, and the stablizer that adds more than the 20g is consistent with adding 20g stablizer to actual titration.Wherein be the suitableeest scope to add the 9-15g stablizer.
The fiber eliminating enzyme of liquid drugs injection formulation is compared with freeze-dried powder formulation and import hydro-acupuncture preparation DF-521, and is with the obvious advantage, comparing result such as following table:
Fiber eliminating enzyme hydro-acupuncture preparation of the present invention and freeze-dried powder formulation and import hydro-acupuncture preparation DF-521 contrast table
| Preparation | The fiber eliminating enzyme hydro-acupuncture preparation | Freeze-dried powder | DF-521 |
| Raw material sources | The northeast agkistrodon halys ussuriensis | Agkistrodon halys ussuriensis or agkistrodon acutus | Brazil spearhead pallas pit viper |
| The raw material extraction process | The specificity affinity chromatography | Non-specific affinity chromatography | Non-specific affinity chromatography |
| The raw material extracting cycle | 7 days | 15 days | |
| The disposable treatment capacity of raw material | The 20g snake venom | The 5g snake venom | |
| The raw material yield | 400,000 units/g snake venom | 50,000 units/g snake venom | |
| Material purity | ＞95 | ＞90 | |
| Specific activity | ＞1800u/mg | ＞1200u/mg | |
| Vehicle | Dextran | ||
| Protective material | Sodium-chlor | Trichloro-butyl alcohol | |
| Polymer | Do not have | Produce in freeze-drying and in placing | Do not have |
| Sensitization | Do not have | The possibility that generation is arranged | Do not have |
| Stability | Room temperature 3 years | Low temperature 2 years | Low temperature 5 years |
1. protect the fiber eliminating enzyme hydro-acupuncture preparation in the preservation process, do not have polymeric generation, the possibility of better having avoided anaphylaxis to produce;
2. avoided vehicle to the influence of titration and the loss of tiring of freeze-drying process, fiber eliminating enzyme loses about 50% (charging capacity is 160-200% in the freeze-drying) in freeze-drying process, this has reduced the specific activity of goods virtually, increased the patient in using the product process, increasing injected dose to foreign protein, also increase simultaneously the possibility that polymer produces, the anaphylaxis that relatively easily produces large molecular weight protein;
3. the adding of the fiber eliminating enzyme middle and high concentration sodium-chlor of liquid drugs injection formulation is that the preparation that this product adds sanitas such as the trimethyl carbinol has better security and stability, places the generation of no degraded product and polymkeric substance more than 3 years for a long time;
4. raw material derives from agkistrodon halys ussuriensis fully, falls fibre, thrombolytic effect mitigation, and toxicity is low, and security is better.
Embodiment
Embodiment one
The method of extracting fiber eliminating enzyme from snake venom comprises the steps:
1). MONOCLONAL ANTIBODIES SPECIFIC FOR: behind purifying and the fiber eliminating enzyme (1g/L) that extracts from snake venom qualitatively and isopyknic freund's adjuvant mixing and emulsifying, to the each 0.2ml of Balb/C mouse subcutaneous injection in 7 ages in week, 2 weeks 1 time, immunity is 3 times altogether; Merge preceding 3 days with the 0.2ml intravenous injection; Get immune mouse spleen cell with the Koher method then, the immune mouse spleen cell of being got is added RPMI-1640, get cell suspension; With the cell suspension of gained and mouse SP2/10 myeloma cell by 10: 1 mixed, under 50% polyoxyethylene glycol effect, merge syzygy; Syzygy is carried out selectivity cultivates, at first sift out the hybridoma that to secrete the monoclonal antibody that reacts with predetermined antigens, and then therefrom sifted out and be scheduled to specific hybridoma, finally select practical use and have stable growth and homogeneous cell clone that function is special, obtain monoclonal antibody with the ascites method, standby behind the purifying;
2). the preparation of fiber eliminating enzyme crude product: get by the snake venom that extracts in the agkistrodon halys ussuriensis, snake venom through the ion exchange chromatography preliminary purification, is got the fiber eliminating enzyme crude product of preliminary purification, standby;
3). the preparation of antibody affinity chromatography:
3.1,, get the activatory sepharose 4B with this carrier of cyanogen bromide-activated with the carrier of sepharose 4B as affinity chromatography;
3.2 get 100mg the monoclonal antibody of purifying be dissolved in the 20ml borate buffer, filter, filtrate adds in the affinity column, adds above-mentioned activatory sepharose 4B then, 10 ℃ of following stirring reactions 18 hours, must have the separating medium of special affinity;
3.3 with the borate buffer of the pH10.2 of 10 times of column volumes with 5ml/min flow velocity washing affinity column cylinder, and then use the ethanolamine solutions of pH10.0,0.1mol/L of 5 times of column volumes and the borate buffer thorough washing of pH8.0,0.1mol/L successively, use the 0.1mol/L phosphate buffered saline buffer balance of pH7.4 at last, promptly obtain antibody affinity chromatography, standby.This post is reusable more than 200 times, but with 20% ethanol prolonged preservation;
4). the preparation of fiber eliminating enzyme:
To join in the antibody affinity chromatography gradually through the fiber eliminating enzyme crude product of ion exchange chromatography preliminary purification, through above-mentioned borate buffer dissolving, after the phosphate buffered saline buffer balance, utilize the specific selectivity absorption biologically active substance of the separating medium with special affinity of antibody affinity chromatography, because impurity and chromatography media do not have affinity interaction, wash-out is removed so can not be adsorbed; Treat that effluent liquid is after drawing stable baseline on the detector, use affinity chromatography elutriant (pH7.8 instead, the 0.1%Tris-Hcl damping fluid) wash-out active ingredient, collect elution peak, the desorb from the affinity media of the biologically active substance that is adsorbed is got off, measure enzymic activity, promptly get the fiber eliminating enzyme finished product of purifying after the lyophilize;
5). the preparation of fiber eliminating enzyme hydro-acupuncture preparation:
Get the fiber eliminating enzyme 5000u of above-mentioned purifying, it joined in the water for injection of 1000ml dissolve, lysate, adding 10g sodium-chlor in lysate is stablizer, 1000 preparations of aseptic subpackaged one-tenth, every content 5u, the actual measurement 4.94u that tires.
This preparation room temperature was placed 3 years, measured the 4.82u that tires.
Embodiment two
The method of extracting fiber eliminating enzyme from snake venom comprises the steps:
1). MONOCLONAL ANTIBODIES SPECIFIC FOR: behind purifying and the fiber eliminating enzyme (1g/L) that from snake venom, extracts qualitatively and isopyknic freund's adjuvant mixing and emulsifying, to mouse immune; Get the splenocyte of above-mentioned immunity then with the Koher method, the splenocyte of being got is added RPMI-1640, get cell suspension; With the cell suspension of gained and SP2/10 myeloma cell by 8: 1 mixed, under 80% polyoxyethylene glycol effect, merge syzygy; Syzygy is carried out selectivity cultivates, at first sift out the hybridoma that to secrete the monoclonal antibody that reacts with predetermined antigens, and then therefrom sifted out and be scheduled to specific hybridoma, finally select practical use and have stable growth and homogeneous cell clone that function is special, obtain monoclonal antibody with the ascites method, standby behind the purifying;
2). the preparation of fiber eliminating enzyme crude product: get by the snake venom that extracts in the agkistrodon halys ussuriensis, snake venom through the ion exchange chromatography preliminary purification, is got the fiber eliminating enzyme crude product of preliminary purification, standby;
3). the preparation of antibody affinity chromatography:
3.1 sepharose 4B as the carrier of affinity chromatography, with this carrier of cyanogen bromide-activated, gets the activatory sepharose 4B;
3.2 get 100mg the monoclonal antibody of purifying be dissolved in the 18ml borate buffer, filter, filtrate adds in the affinity column, adds above-mentioned activatory sepharose 4B then, at 20 ℃ of following stirring reactions hour, must have the separating medium of special affinity;
3.3 with the borate buffer of the pH10.2 of 10 times of column volumes with 6ml/min flow velocity washing affinity column cylinder, and then use the ethanolamine solutions of pH10.0,0.1mol/L of 5 times of column volumes and the borate buffer thorough washing of pH8.0,0.1mol/L successively, use the 0.1mol/L phosphate buffered saline buffer balance of pH7.4 at last, promptly obtain antibody affinity chromatography, standby, this post is reusable more than 200 times, but with 20% ethanol prolonged preservation.
4). the preparation of fiber eliminating enzyme:
To join in the antibody affinity chromatography gradually through the fiber eliminating enzyme crude product of ion exchange chromatography preliminary purification, through above-mentioned borate buffer dissolving, after the phosphate buffered saline buffer balance, utilize the specific selectivity ground absorption biologically active substance of the separating medium with special affinity of antibody affinity chromatography, because impurity and chromatography media do not have affinity interaction, wash-out is removed so can not be adsorbed; Treat that effluent liquid is after drawing stable baseline on the detector, use affinity chromatography elutriant (pH7.8 instead, the 0.1%Tris-Hcl damping fluid) wash-out active ingredient, collect elution peak, the desorb from the affinity media of the biologically active substance that is adsorbed is got off, measure enzymic activity, promptly get the fiber eliminating enzyme finished product of purifying after the lyophilize;
5), the preparation of fiber eliminating enzyme hydro-acupuncture preparation:
Get fiber eliminating enzyme 5000u, it joined in the water for injection of 1000ml dissolve, lysate, adding 9g sodium-chlor in lysate is stablizer, 1000 preparations of aseptic subpackaged one-tenth, every content 5u, the actual measurement 4.97u that tires.
This preparation room temperature was placed 3 years, measured the 4.69u that tires.
Embodiment three
The method of extracting fiber eliminating enzyme from snake venom comprises the steps:
1). MONOCLONAL ANTIBODIES SPECIFIC FOR: behind purifying and the fiber eliminating enzyme (1g/L) that from snake venom, extracts qualitatively and isopyknic freund's adjuvant mixing and emulsifying, to mouse immune; Get the splenocyte of above-mentioned mice immunized then with the Koher method, the splenocyte of the immune mouse got added RPMI-1640, cell suspension; With the cell suspension of gained and mouse SP2/10 myeloma cell by 15: 1 mixed, under 30% polyoxyethylene glycol effect, merge syzygy; Syzygy is carried out selectivity cultivates, at first sift out the hybridoma that to secrete the monoclonal antibody that reacts with predetermined antigens, and then therefrom sifted out and be scheduled to specific hybridoma, finally select practical use and have stable growth and homogeneous cell clone that function is special, obtain monoclonal antibody with the ascites method, standby behind the purifying;
2). the preparation of fiber eliminating enzyme crude product: get by the snake venom that extracts in the agkistrodon halys ussuriensis, snake venom through the ion exchange chromatography preliminary purification, is got the fiber eliminating enzyme crude product of preliminary purification, standby;
3). the preparation of antibody affinity chromatography:
3.1 sepharose 4B as the carrier of affinity chromatography, with this carrier of cyanogen bromide-activated, gets the activatory sepharose 4B;
3.2 get 100mg the monoclonal antibody of purifying be dissolved in the 18ml borate buffer, filter, filtrate adds in the affinity column, adds above-mentioned activatory sepharose 4B then, at 20 ℃ of following stirring reactions hour, must have the separating medium of special affinity;
3.3 with the borate buffer of the pH10.2 of 10 times of column volumes with 6ml/min flow velocity washing affinity column cylinder, and then use the ethanolamine solutions of pH10.0,0.1mol/L of 5 times of column volumes and the borate buffer thorough washing of pH8.0,0.1mol/L successively, use the 0.1mol/L phosphate buffered saline buffer balance of pH7.4 at last, promptly obtain antibody affinity chromatography, standby.This post is reusable more than 200 times, but with 20% ethanol prolonged preservation;
4). the preparation of fiber eliminating enzyme:
To join in the antibody affinity chromatography gradually through the fiber eliminating enzyme crude product of ion exchange chromatography preliminary purification, through above-mentioned borate buffer dissolving, after the phosphate buffered saline buffer balance, utilize the specific selectivity ground absorption biologically active substance of the separating medium with special affinity of antibody affinity chromatography, because impurity and chromatography media do not have affinity interaction, wash-out is removed so can not be adsorbed; Treat that effluent liquid is after drawing stable baseline on the detector, use affinity chromatography elutriant (pH7.8 instead, the 0.1%Tris-Hcl damping fluid) wash-out active ingredient, collect elution peak, the desorb from the affinity media of the biologically active substance that is adsorbed is got off, measure enzymic activity, promptly get the fiber eliminating enzyme finished product of purifying after the lyophilize.
5). the preparation of fiber eliminating enzyme hydro-acupuncture preparation:
Get fiber eliminating enzyme 5000u, it joined in the water for injection of 1000ml dissolve, lysate, adding 5g sodium-chlor in lysate is stablizer, 1000 preparations of aseptic subpackaged one-tenth, every content 5u, the actual measurement 4.89u that tires.
This preparation room temperature was placed 3 years, measured the 4.28u that tires.
Embodiment four
The preparation of fiber eliminating enzyme hydro-acupuncture preparation: get fiber eliminating enzyme 5000u, it joined in the water for injection of 1000ml dissolve, lysate, adding 15g sodium-chlor in lysate is stablizer, 1000 preparations of aseptic subpackaged one-tenth, every content 5u, the actual measurement 4.92u that tires.
This preparation room temperature was placed 3 years, measured the 4.93u that tires.
Embodiment five
The preparation of fiber eliminating enzyme hydro-acupuncture preparation: get fiber eliminating enzyme 5000u, it joined in the water for injection of 1000ml dissolve, lysate, adding 12g sodium-chlor in lysate is stablizer, 1000 preparations of aseptic subpackaged one-tenth, every content 5u, the actual measurement 4.91u that tires.
This preparation room temperature was placed 3 years, measured the 4.88u that tires.
Embodiment six
The preparation of fiber eliminating enzyme hydro-acupuncture preparation: get fiber eliminating enzyme 5000u, it joined in the water for injection of 1000ml dissolve, lysate, adding 20g sodium-chlor in lysate is stablizer, 1000 preparations of aseptic subpackaged one-tenth, every content 5u, the actual measurement 4.99u that tires.
This preparation room temperature was placed 3 years, measured the 4.96u that tires.
Claims (2)
1. the preparation method of a fiber eliminating enzyme hydro-acupuncture preparation, it comprises the steps:
Get the fiber eliminating enzyme of purifying, it is joined in the proper amount of water for injection dissolve, get lysate, add 5--20g sodium-chlor stablizer in every 1000ml lysate, packing promptly gets the fiber eliminating enzyme hydro-acupuncture preparation.
2. the preparation method of fiber eliminating enzyme hydro-acupuncture preparation according to claim 1, it is characterized in that getting the fiber eliminating enzyme 5000u of purifying, it is joined in the 1000ml proper amount of water for injection dissolve, get lysate, in every 1000ml lysate, add 9--15g sodium-chlor stablizer, 1000 preparations of aseptic subpackaged one-tenth, every content 5u promptly gets the fiber eliminating enzyme hydro-acupuncture preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100215584A CN100336903C (en) | 2004-07-27 | 2004-07-27 | Extracting norfibroase from snake venom and preparation process for liquid drugs injection of norfibroase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100215584A CN100336903C (en) | 2004-07-27 | 2004-07-27 | Extracting norfibroase from snake venom and preparation process for liquid drugs injection of norfibroase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1597945A CN1597945A (en) | 2005-03-23 |
| CN100336903C true CN100336903C (en) | 2007-09-12 |
Family
ID=34663356
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100215584A Expired - Lifetime CN100336903C (en) | 2004-07-27 | 2004-07-27 | Extracting norfibroase from snake venom and preparation process for liquid drugs injection of norfibroase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100336903C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104558170A (en) * | 2014-12-22 | 2015-04-29 | 广西大学 | Preparation process of antivenin |
| CN110791491A (en) * | 2019-12-11 | 2020-02-14 | 昆明龙津药业股份有限公司 | Method for extracting defibrase from snake venom |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1065680A (en) * | 1992-04-09 | 1992-10-28 | 中国科学院动物研究所 | Purifying method for agkistrodon acutus venin defibering enzyme |
| 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 |
| CN1432406A (en) * | 2002-12-20 | 2003-07-30 | 周宇 | Application of snake venom protein hydrolase in preparing medicine |
-
2004
- 2004-07-27 CN CNB2004100215584A patent/CN100336903C/en not_active Expired - Lifetime
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1065680A (en) * | 1992-04-09 | 1992-10-28 | 中国科学院动物研究所 | Purifying method for agkistrodon acutus venin defibering enzyme |
| 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 |
| CN1432406A (en) * | 2002-12-20 | 2003-07-30 | 周宇 | Application of snake venom protein hydrolase in preparing medicine |
Non-Patent Citations (2)
| Title |
|---|
| 蛇毒纤溶酶注射液的临床研究 姜桂荣，魏化伟，首都医药，第4期 2004 * |
| 蛇毒纤溶酶的分离纯化及其溶栓作用 马骉，张骞，宋梦薇，中国生化药物杂志，第25卷第3期 2004 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1597945A (en) | 2005-03-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3016729B1 (en) | Affinity chromatography matrix | |
| CN101059487A (en) | Immunoaffinity chromatography column and its uses in purifying quinolone analogue drug | |
| Miller et al. | Human thrombin: isolation and stability | |
| FR3008097A1 (en) | AFFINITY CHROMATOGRAPHY MATRIX | |
| CN100336903C (en) | Extracting norfibroase from snake venom and preparation process for liquid drugs injection of norfibroase | |
| CN1500873A (en) | Nereides protease, separating and purifying method and application thereof | |
| CN1603405A (en) | Preparation method for high purity lumbrukinase and pharmaceutical preparation made therefrom | |
| CN101063117A (en) | White-browed snake venom blood coagulation enzyme and its extraction method and application | |
| CN1242059C (en) | Production of rDSPA 'alpha' I | |
| CN1159438C (en) | Ahylysantinfarctase thrombase and its production process | |
| CN102199192A (en) | Method for separating functional polypeptide from tortoise-shell glue | |
| CN111748031B (en) | High-purity antivenin and preparation method thereof | |
| CN104498464A (en) | Method for extracting agkistrodon snake venom hemocoagulase from gloydius blomhoffi brevicaudus venom | |
| JPH03169893A (en) | Method for refining | |
| JP6442409B2 (en) | Method for producing porous cellulose beads | |
| CN104672306A (en) | Preparation method and application of peptide with anti-thrombosis activity | |
| US4960756A (en) | Lectin like protein substance, method of obtaining same and anti-tumor agent comprising same | |
| US20200190166A1 (en) | Polyvalent immunotherapeutics of high specificty based on modified antibodies and a lyophilized injectable formulation highly safe and effective | |
| CN105586330B (en) | A kind of Halase and preparation method thereof | |
| CN1159437C (en) | Ahylysantinfarctase thrombase and its production process | |
| CN1846713A (en) | Snake venom extract for preventing and treating pyemia | |
| CN1258594C (en) | Long-noded pit viper poison dissolving fiber protein No.2 gene and use thereof | |
| CN1248735C (en) | Preparing method and usage of cardiotoxin | |
| CN100365020C (en) | Protein from snake venom for preventing and treating pyemia | |
| CN1288245C (en) | Long-noded pit viper dissolving fiber protein No.4 gene and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Assignee: BEIJING SAISHENG PHARMACEUTICAL Co.,Ltd. Assignor: SHENYANG SIJIA TECHNOLOGY DEVELOPMENT Co.,Ltd. Contract fulfillment period: 2008.10.31 to 2010.10.31 Contract record no.: 2008210000079 Denomination of invention: Extracting norfibroase from snake venom and preparation process for liquid drugs injection of norfibroase Granted publication date: 20070912 License type: General permission Record date: 20081209 |
|
| LIC | Patent licence contract for exploitation submitted for record |
Free format text: COMMON LICENSE; TIME LIMIT OF IMPLEMENTING CONTACT: 2008.10.31 TO 2010.10.31; CHANGE OF CONTRACT Name of requester: BEIJING SAISHENG PHARMACY CO., LTD. Effective date: 20081209 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20090313 Address after: No. 8 prosperous street, Beijing economic and Technological Development Zone, Beijing Patentee after: BEIJING SAISHENG PHARMACEUTICAL Co.,Ltd. Address before: Liaoning city of Shenyang province Shenyang Hunnan Industrial Zone Century Road No. 1, block A No. 2006 Patentee before: SHENYANG SIJIA TECHNOLOGY DEVELOPMENT Co.,Ltd. |
|
| ASS | Succession or assignment of patent right |
Owner name: BEIJING SAISHENG PHARMACY CO., LTD. Free format text: FORMER OWNER: SHENYANG SIJIA TECHNOLOGY DEVELOPMENT CO., LTD. Effective date: 20090313 |
|
| C56 | Change in the name or address of the patentee | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 100176 No. 8 prosperous street, Beijing economic and Technological Development Zone, Beijing Patentee after: Beijing Saisheng Pharmaceutical Co.,Ltd. Address before: 100176 No. 8 prosperous street, Beijing economic and Technological Development Zone, Beijing Patentee before: BEIJING SAISHENG PHARMACEUTICAL Co.,Ltd. |
|
| CX01 | Expiry of patent term | ||
| CX01 | Expiry of patent term |
Granted publication date: 20070912 |