CN101361717B - Batroxobin freeze-dry powder injection and preparation method thereof - Google Patents
Batroxobin freeze-dry powder injection and preparation method thereof Download PDFInfo
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- CN101361717B CN101361717B CN2007101200842A CN200710120084A CN101361717B CN 101361717 B CN101361717 B CN 101361717B CN 2007101200842 A CN2007101200842 A CN 2007101200842A CN 200710120084 A CN200710120084 A CN 200710120084A CN 101361717 B CN101361717 B CN 101361717B
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Abstract
The invention relates to a Batroxobin freeze-dried powder preparation and a preparation method thereof. The Batroxobin freeze-dried powder preparation comprises Batroxobin and freeze-dried protecting agent and excipient which are pharmaceutically acceptable. The ratio of Batroxobin to freeze-dried protecting agent is 10BU:0.01-10mg, and the ratio of Batroxobin to excipient is 10BU:2-200mg. The Batroxobin freeze-dried powder preparation is characterized by simple preparation process, adaptability to scale production, high stability and long-term storage and can be used for intravenous injection due to adding no preservative simultaneously, thus expanding the clinical application range.
Description
Technical field
The present invention relates to batroxobin freeze-dried powder and preparation method thereof.
Background technology
Batroxobin (Batroxobin) is to adopt the modern biotechnology means, from the spearhead agkistrodon halyx pallas venom, extracts the batroxobin of purification.These article are glycoprotein; Its peptide chain is made up of 231 amino acid residues, molecular weight 36000D, and it can single-mindedly act between the A α chain N-terminal arginine and glycine of fibrinogen molecule; Discharge fibrinopeptide A; This is similar with thrombin, but Hageman factor I is not had activation, can not make fibrin be cross-linked into insoluble grumeleuse.This fibrin is unstable, very easily by the fibrinolysin degraded, from blood circulation, removes.So these article athrombia appearance Blood clotting but have and reduce Fibrinogen and anticoagulation.Big quantity research shows that the former effect of batroxobin fibrin degradation is fairly obvious, to blooc coagulation factor, platelet function, thrombinogen, go out clotting time and then influence very for a short time, hepatic and renal function is not also had obvious influence.
Batroxobin can be used as thrombolytic drug clinically, can treat the ischemic diseases that ischemic cerebrovascular, sudden deafness, pulmonary infarction cause, and have outstanding effectiveness and safety.
At present, have only the aqueous injection listing of batroxobin both at home and abroad, and as yet not relevant for the bibliographical information of batroxobin powder injection formulation.The batroxobin aqueous injection has following two open defects:
1, less stable, preservation condition is harsh: principal agent is unstable in the aqueous solution, for keeping stability of formulation; The preservation condition of batroxobin aqueous injection is comparatively harsh, can only under 0～5 ℃ of condition, (can not freeze) to keep in Dark Place, so in transportation, must protect with ice bag; Transport difficulty and cost of transportation have been increased; Also increased patient's financial burden, owing in storage, be prone to degraded, can only in 18 months, use simultaneously;
2, added antibacterial: because these article poor heat stability; Preparation can not be taked final sterilization technology; Added a certain amount of antibacterial-chlorobutanol in the batroxobin aqueous injection preparation prescription of listing; According to " regulation that Chinese pharmacopoeia is 2005 editions, " in venous transfusion and the brain pond, the injection used in the epidural, canalis spinalis all must not add antibacterial.", therefore the batroxobin aqueous injection of this interpolation antibacterial can not be used for venous transfusion, and this has greatly limited the clinical scope of application of batroxobin.
Summary of the invention
In order to overcome the deficiency of prior art, technical problem to be solved by this invention provides a kind of safe, stable, effective batroxobin freeze-dried powder and preparation method thereof, and this batroxobin freeze-dried powder technology is simple; Be fit to large-scale production; Solved active drug composition batroxobin and under normal condition, (comprised and having stored and transportation) a stable difficult problem, reduced cost of transportation and transport difficulty, simultaneously owing to do not add antibacterial; Can be used for venous transfusion; Enlarge the clinical scope of application, also eliminated the potential safety hazard of bringing because of antibacterial, improved the safety of medication.
According to an aspect of the present invention; Batroxobin freeze-dried powder according to the invention comprises batroxobin and pharmaceutically acceptable freeze drying protectant; Said batroxobin is 10BU: 0.01-10mg with the ratio of freeze drying protectant, is preferably 10BU: 0.05～1mg, more preferably 10BU: 0.1～1mg.The used freeze drying protectant of the present invention is used to prevent batroxobin by preparation production equipment and container absorption, and can prevent batroxobin degeneration inactivation in freezing dry process, and can reduce the loss of activity of preparation in transportation and storage process.
Because the dosage of said batroxobin freeze-dried powder is less, therefore also can add excipient, said batroxobin is 10BU: 2～200mg with the ratio of excipient, is preferably 10BU: 5～100mg, more preferably 10BU: 10～80mg.
Owing in the preparation process of batroxobin freeze-dried powder, need usually at first batroxobin to be dissolved in the solution of appropriate pH, in the for example pharmaceutically acceptable buffer, so can contain an amount of buffer salt in the said preparation.
The raw material of the used batroxobin of the present invention can be to obtain through from snake venom, extracting purification, also can be that the method through gene recombinaton prepares.The method that obtains the batroxobin raw material from these two kinds of approach has all had the pertinent literature report.For example, Chinese invention patent application 03116054.9 discloses a kind of batroxobin of gene recombination method preparation.In the present invention, if no special instructions, the batroxobin that is used to prepare freeze-dried powder is the batroxobin of purification.
In a specific embodiment of the present invention, the method for from snake venom, extracting batroxobin can comprise steps such as DEAE-Sepharose Fast Flow chromatography, affinity chromatograph, gel permeation chromatography, lyophilization.It is simple that this method has technology, is fit to the advantage of large-scale industrial production.
The purity of the used batroxobin of the present invention can be preferably more than 98% for more than 95%, and most preferably 100%.
Said snake venom be for extracting various types of snake venom of batroxobin, for example spearhead agkistrodon halyx pallas venom etc.
Particularly, from snake venom, extract the method for batroxobin, can comprise the steps:
1) DEAE-Sepharose Fast Flow chromatography
Take by weighing snake venom,, with 4000rpm/min centrifugal 20 minutes, get supernatant with 0.02mol/L pH7.4Tris-HCl buffer extraction 4 hours; Deposition is extracted once with method again, merges supernatant, and adjustment pH to 7.4 gets extracting solution.With on the extracting solution in the good DEAE-Sepharose Fast Flow chromatographic column of balance; Earlier do not adsorb impurity with 0.02mol/L Tris-HCl buffer solution elution; The buffer solution that reuse contains 0～0.6mol/L NaCl carries out gradient elution, collects the active peak of batroxobin, obtains active component and collects liquid A.
2) affinity chromatograph
Above-mentioned active component is collected liquid A go up in the good affinity column of balance, do not adsorb impurity with containing 1.0mol/LNaCl0.05mol/L Tris-HCl pH7.0 buffer solution elution earlier, the detector baseline is steady after seeing through the peak; Then with containing 0.1mol/L eluant 1.0mol/L NaCl0.05mol/L Tris-HCl pH7.0 buffer solution elution active component, and on the 280nm UV-detector, detect protein peak, collect active component, obtain active component and collect liquid B.
3) gel permeation chromatography
Get active component and collect liquid B, with molecular cut off 10, the bag filter of 000D is concentrated into about 20ml Polyethylene Glycol, pours in the depyrogenation reagent bottle subsequent usely, is concentrated solution B.Getting concentrated solution B goes up in using 0.1mol/L NH
4On the good solvent resistant column of Ac buffer balance, use 0.1mol/L NH then
4The Ac buffer solution elution detects through the 280nm UV-detector, collects the activated protein peak, collects liquid and packs in the depyrogenation bottle, and the Strict aseptic operation obtains active component and collects liquid C.
4) lyophilization
Getting molecular cut off is 10, and the bag filter of 000D boiled 30 minutes with water for injection earlier, then bag filter was taken out; The water for injection of packing into; Hunting leak on filter paper in the sealing two ends, gets intact bag filter and clean three times with water for injection; Above-mentioned active component is collected liquid C pack in the bag filter sealing two ends into.Above step should attend the sterile working at super-clean bench.At last, the bag filter that installs is concentrated into about 50ml to Polyethylene Glycol, to the water for injection dialysis of pre-cooling to 4 ℃ three times, each 2 hours.With 0.22 μ m filter filtration sterilization, the packing lyophilizing gets lyophilized powder with above-mentioned dialysis solution.
Get above-mentioned lyophilized powder,, be the batroxobin crude drug after qualified according to batroxobin quality standard check.
Wherein, said freeze drying protectant can be and is selected from aminoacid, saccharide, polyhydroxy-alcohol, protein and the perfluorooctane sulfonate one or more, preferred polyhydroxy-alcohol.
Said aminoacid can be and is selected from glycine, proline, L-tryptophan, sodium glutamate, alanine, lysine hydrochloride, sarcosine, L-tyrosine, phenylalanine, the arginine one or more.
Preferably, said saccharide can be and is selected from glucose, fructose, lactose, mannose, sucrose, trehalose, maltose, cyclodextrin and derivant thereof, the dextran one or more.
Said polyhydroxy-alcohol can be and is selected from glycerol, mannitol, sorbitol, inositol and the Polyethylene Glycol one or more.
Said protein can be and is selected among bovine serum albumin and the human serum albumin one or both.
Said excipient can be and is selected from mannitol, dextran, glycine, alanine, arginine and the lysine one or more.Said excipient concentration can be 0.2%～20%, is preferably 1%～10% (g/ml).
According on the other hand, the method for preparing of batroxobin freeze-dried powder according to the invention comprises the steps:
1) gets batroxobin, it is joined in an amount of buffer solution dissolve, get lysate;
2) in lysate, add an amount of freeze drying protectant and excipient;
3) packing, lyophilization promptly gets the batroxobin freeze-dried powder.Said freeze drying protectant comprises one or more in aminoacid, saccharide, polyhydroxy-alcohol, protein and the perfluorooctane sulfonate.
Wherein, wherein, said freeze drying protectant can be and is selected from aminoacid, saccharide, polyhydroxy-alcohol, protein and the perfluorooctane sulfonate one or more.
Said aminoacid can be and is selected from glycine, proline, L-tryptophan, sodium glutamate, alanine, lysine hydrochloride, sarcosine, L-tyrosine, phenylalanine, the arginine one or more.
Said saccharide can be and is selected from glucose, fructose, lactose, mannose, sucrose, trehalose, maltose, cyclodextrin and derivant thereof, the dextran one or more.
Said polyhydroxy-alcohol can be and is selected from glycerol, mannitol, sorbitol, inositol and the Polyethylene Glycol one or more.
Said protein can be and is selected among bovine serum albumin and the human serum albumin one or both.
The concentration of said freeze drying protectant in lysate is 0.05～1mg/ml.
Said buffer solution can be the buffer of pharmaceutically acceptable proper pH value; For when preparing injectable powder; Can dissolve batroxobin and additives and keep it stable; The concentration of said buffer solution can be 1mmol/L～20mmol/L, and pH value can be 4.8～6.0, for example acetate buffer, citrate buffer etc.
Said excipient can be and is selected from mannitol, dextran, glycine, alanine, arginine and the lysine one or more.
When batroxobin freeze-dried powder that preparation 5BU/ props up, the batroxobin 8000～13000BU of desirable purification joins it in the buffer solution of 1000ml and to dissolve; Get lysate; In every 1000ml lysate, add freeze drying protectant 0.01～10g and excipient 2～200g, 2000 of aseptic subpackaged one-tenth, every content 5BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
When batroxobin freeze-dried powder that preparation 10BU/ props up, the batroxobin 8000～12500BU of desirable purification joins it in the buffer solution of 1000ml and to dissolve; Get lysate; In every 1000ml lysate, add freeze drying protectant 0.01～10g and excipient 2～200g, 1000 of aseptic subpackaged one-tenth, every content 10BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
Batroxobin freeze-dried powder of the present invention has good stability, and any antibacterial is not added in convenient transportation and storage, can enlarge advantages such as the clinical scope of application.Said preparation has effects such as the fibre of falling, thrombolytic, microcirculation improvement; Be used to treat acute cerebral infarction clinically; Improve the ischemic symptom that various occlusive vascular diseases (like thromboangiitis obliterans, deep phlebitis, pulmonary infarction etc.) cause, improve tip and microcirculation disturbance (like sudden deafness, motion sickness) etc.
Long-time stability through room temperature was placed 2 years are investigated, and the result shows the requirement that the batroxobin hydro-acupuncture preparation has not reached quality standard 6th month titration result; And the batroxobin freeze-dried powder, the basic no change of titration result, and after placing 2 years, the requirement that its titration result can also reach quality standards.
Below will combine the specific embodiment to set forth the present invention in detail, following examples are illustrative fully, only are used for the present invention is specifically described, and are not to be understood that and are limitation of the present invention.
Brief description of drawings
Fig. 1 is the electrophoretogram of the batroxobin that prepared by the embodiment of the invention 1;
Fig. 2 A is the HPLC figure of the batroxobin that prepared by the embodiment of the invention 1;
Fig. 2 B is the HPLC figure of batroxobin reference substance.
The specific embodiment
Agents useful for same of the present invention and adjuvant if no special instructions, are commercially available purchase product.
The preparation of batroxobin
1, DEAE-Sepharose Fast Flow chromatography
Take by weighing the spearhead agkistrodon halyx pallas venom (Bothrops atrox moojeni, Sigma reagent catalog number: V5500) 6g, extracted 4 hours with 0.02mol/L pH7.4Tris-HCl buffer 200ml, with 4000rpm/min centrifugal 20 minutes, get supernatant; Deposition is extracted once with method with 0.02mol/L pH7.4Tris-HCl buffer 50ml again, merges supernatant, and adjustment pH to 7.4 gets extracting solution.With on the extracting solution in the good DEAE-Sepharose Fast Flow chromatographic column of balance; Earlier do not adsorb impurity with 0.02mol/L Tris-HCl buffer solution elution; The buffer solution that reuse contains 0～0.6mol/L NaCl carries out gradient elution; Collect the active peak of batroxobin, obtain active component and collect liquid A300ml.
2, affinity chromatograph
Above-mentioned active component is collected liquid A go up in the good affinity column of balance, do not adsorb impurity with containing 1.0mol/LNaCl0.05mol/L Tris-HCl pH7.0 buffer solution elution earlier, the detector baseline is steady after seeing through the peak; Then with containing 0.1mol/L Arg1.0mol/L NaCl0.05mol/L Tris-HCl pH7.0 buffer solution elution active component, and on the 280nm UV-detector, detect protein peak, collect active component, obtain active component and collect liquid B100ml.
3. gel permeation chromatography
Get active component and collect liquid B, with molecular cut off 10, the bag filter of 000D is concentrated into about 20ml Polyethylene Glycol, pours in the depyrogenation reagent bottle subsequent usely, is concentrated solution B.Getting concentrated solution B goes up on the good solvent resistant column of 0.1mol/L NH4Ac buffer balance; Use 0.1mol/L NH4Ac buffer solution elution then; Detect through the 280nm UV-detector, collect the activated protein peak, collection liquid is packed in the depyrogenation bottle; The Strict aseptic operation obtains active component and collects liquid C150ml.
4. lyophilization
Getting molecular cut off is 10, and the bag filter of 000D boiled 30 minutes with water for injection earlier, then bag filter was taken out; The water for injection of packing into; Hunting leak on filter paper in the sealing two ends, gets intact bag filter and clean three times with water for injection; Above-mentioned active component is collected liquid C pack in the bag filter sealing two ends into.Above step should attend the sterile working at super-clean bench.At last, the bag filter that installs is concentrated into about 50ml to Polyethylene Glycol, again to the water for injection dialysis of pre-cooling to 4 ℃ three times, each 2 hours.With 0.22 μ m filter filtration sterilization, the packing lyophilizing gets lyophilized powder with above-mentioned dialysis solution.
Get above-mentioned lyophilized powder,, be the batroxobin crude drug after qualified according to batroxobin quality standard check.
With SDS-PAGE method and HPLC method the gained batroxobin is detected.The HPLC chromatographic condition is: use gel chromatographic columns; (get Na with the 0.1mol/L phosphate buffer
2HPO
412H
2O16.58g, NaH
2PO
42H
2O8.38g, Na
2SO
414.20g, be dissolved in water and be diluted to 1000ml) and be mobile phase; Flow velocity is per minute 1ml; The detection wavelength is 280nm.Number of theoretical plate calculates by the batroxobin peak should be lower than 3000.Algoscopy is: get these article with mobile phase be diluted to contain 10BU among every 1ml solution as need testing solution; Get need testing solution 20 μ l and inject chromatograph of liquid; The record chromatogram calculates by area normalization method, and the relative percentage composition of main peak must not be lower than 90.0% in the test sample.
Fig. 1 is the electrophoretogram of gained batroxobin, and Fig. 2 A and Fig. 2 B are respectively the HPLC collection of illustrative plates of gained batroxobin and batroxobin reference substance (purchase the Control in National Institute for Biological Standards and, Britain, the standard Article Number is 93/526).
The gained batroxobin is 36000 ± 5000D through SDS-PAGE method determining molecular weight, and it is 100% that the HPLC method is measured purity, than living greater than 1000BU/mgPr.
Batroxobin freeze-dried powder system each (5BU/ props up)
Get batroxobin crude drug (preparing by embodiment 1, down together) 10000BU, it is joined dissolve among 5mmol/L citrate buffer (pH5.0) 1000ml; Get lysate, in lysate, add sucrose 1g, mannitol 80g; Stirring and dissolving also shakes up, aseptic filtration, 2000 of the aseptic subpackaged one-tenth of filtrating; Every content 5BU, lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 4.5BU that tires.
Embodiment 3
Get batroxobin crude drug 10000BU, it is joined among 10mmol/L citrate buffer (pH5.4) 1000ml dissolve, get lysate; In lysate, add glycine 0.5g, dextran 50g, stirring and dissolving also shakes up; Aseptic filtration, 2000 of the aseptic subpackaged one-tenth of filtrating, every content 5BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 4.4BU that tires.
Embodiment 4
Get batroxobin crude drug 10000BU, it is joined among 20mmol/L citrate buffer (pH5.8) 1000ml dissolve, get lysate; In lysate, add cyclodextrin 0.1g, Dextran 10 g, stirring and dissolving also shakes up; Aseptic filtration, 2000 of the aseptic subpackaged one-tenth of filtrating, every content 5BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 4.7BU that tires.
Embodiment 5
Get batroxobin crude drug 10000BU, it is joined among 5mmol/L acetate buffer (pH5.0) 1000ml dissolve, get lysate; In lysate, add sucrose 1g, mannitol 50g, stirring and dissolving also shakes up; Aseptic filtration, 2000 of the aseptic subpackaged one-tenth of filtrating, every content 5BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 4.6BU that tires.
Embodiment 6
Get batroxobin crude drug 10000BU, it is joined among 10mmol/L acetate buffer (pH5.4) 1000ml dissolve, get lysate; In lysate, add glycine 0.5g, dextran 30g, stirring and dissolving also shakes up; Aseptic filtration, 2000 of the aseptic subpackaged one-tenth of filtrating, every content 5BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 4.2BU that tires.
Embodiment 7
Get batroxobin crude drug 10000BU, it is joined among 20mmol/L acetate buffer (pH5.8) 1000ml dissolve, get lysate; In lysate, add cyclodextrin 0.1g, Dextran 10 g, stirring and dissolving also shakes up; Aseptic filtration, 2000 of the aseptic subpackaged one-tenth of filtrating, every content 5BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 4.6BU that tires.
Embodiment 8
Get batroxobin crude drug 10000BU, it is joined among 5mmol/L acetate buffer (pH5.0) 1000ml dissolve, get lysate; In lysate, add sucrose 0.05g, mannitol 200g, stirring and dissolving also shakes up; Aseptic filtration, 2000 of the aseptic subpackaged one-tenth of filtrating, every content 5BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 4.6BU that tires.
Embodiment 9
Get batroxobin crude drug 10000BU, it is joined among 10mmol/L acetate buffer (pH5.4) 1000ml dissolve, get lysate; In lysate, add glycine 10g, dextran 5g, stirring and dissolving also shakes up; Aseptic filtration, 2000 of the aseptic subpackaged one-tenth of filtrating, every content 5BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 4.2BU that tires.
Batroxobin freeze-dried powder preparation (10BU/ props up)
Get batroxobin crude drug 10000BU, it is joined among 5mmol/L citrate buffer (pH5.0) 1000ml dissolve, get lysate; In lysate, add sucrose 1g, mannitol 80g, stirring and dissolving also shakes up; Aseptic filtration, 1000 of the aseptic subpackaged one-tenth of filtrating, every content 10BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 9.3BU that tires.
Embodiment 11
Get batroxobin crude drug 10000BU, it is joined among 10mmol/L citrate buffer (pH5.4) 1000ml dissolve, get lysate; In lysate, add glycine 0.5g, dextran 50g, stirring and dissolving also shakes up; Aseptic filtration, 1000 of the aseptic subpackaged one-tenth of filtrating, every content 10BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 9.0BU that tires.
Get batroxobin crude drug 10000BU, it is joined among 20mmol/L citrate buffer (pH5.8) 1000ml dissolve, get lysate; In lysate, add cyclodextrin 0.1g, Dextran 10 g, stirring and dissolving also shakes up; Aseptic filtration, 1000 of the aseptic subpackaged one-tenth of filtrating, every content 10BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 9.5BU that tires.
Embodiment 13
Get batroxobin crude drug 10000BU, it is joined among 5mmol/L acetate buffer (pH5.0) 1000ml dissolve, get lysate; In lysate, add sucrose 1g, mannitol 50g, stirring and dissolving also shakes up; Aseptic filtration, 1000 of the aseptic subpackaged one-tenth of filtrating, every content 10BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 9.4BU that tires.
Get batroxobin crude drug 10000BU, it is joined among 10mmol/L acetate buffer (pH5.4) 1000ml dissolve, get lysate; In lysate, add glycine 0.5g, dextran 30g, stirring and dissolving also shakes up; Aseptic filtration, 1000 of the aseptic subpackaged one-tenth of filtrating, every content 10BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 9.1BU that tires.
Get batroxobin crude drug 10000BU, it is joined among 20mmol/L acetate buffer (pH5.8) 1000ml dissolve, get lysate; In lysate, add cyclodextrin 0.01g, dextran 2g, stirring and dissolving also shakes up; Aseptic filtration, 1000 of the aseptic subpackaged one-tenth of filtrating, every content 10BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 9.6BU that tires.
Get batroxobin crude drug 10000BU, it is joined among 20mmol/L citrate buffer (pH5.8) 1000ml dissolve, get lysate; In lysate, add cyclodextrin 1g, Dextran 10 0g, stirring and dissolving also shakes up; Aseptic filtration, 1000 of the aseptic subpackaged one-tenth of filtrating, every content 10BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 9.5BU that tires.
Embodiment 17
Get batroxobin crude drug 10000BU, it is joined among 5mmol/L acetate buffer (pH5.0) 1000ml dissolve, get lysate; In lysate, add sucrose 1g, mannitol 50g, stirring and dissolving also shakes up; Aseptic filtration, 1000 of the aseptic subpackaged one-tenth of filtrating, every content 10BU; Lyophilization promptly gets the batroxobin freeze-dried powder.
This preparation room temperature was placed 2 years, measured the 9.4BU that tires.
The stability of batroxobin freeze-dried powder and hydro-acupuncture preparation relatively
Embodiment 18
(Japanese eastern Pedicellus et Pericarpium Trapae pharmaceutical industries Zhu Shi commercial firm produces with batroxobin freeze-dried powder (by embodiment 4 preparation) and hydro-acupuncture preparation; Trade name DF-521) placed simultaneously at normal temperatures 2 years; Carry out study on the stability, tire respectively at adopting under the batroxobin injection national drug standards titration item method to measure in 0,1,2,3,6,9,12,18,24 month.Comparing result is seen table 1.
The result shows the requirement that the batroxobin hydro-acupuncture preparation has not reached quality standard 6th month titration result; And the batroxobin freeze-dried powder, the basic no change of titration result, and after placing 2 years, the requirement that its titration result can also reach quality standards.
Table 1, batroxobin freeze-dried powder and aqueous injection titration result
| ? | Freeze-dried powder (BU/ props up) | Hydro-acupuncture preparation (BU/ props up) |
| 0 month | 5.1 | 5.2 |
| 1 month | 5.0 | 4.9 |
| 2 months | 5.1 | 4.6 |
| 3 months | 5.2 | 4.2 |
| 6 months | 5.1 | 3.1 |
| 9 months | 5.1 | 2.1 |
| 12 months | 5.0 | --- |
| 18 months | 4.9 | --- |
| 24 months | 4.7 | --- |
Although describe the present invention in detail through specific embodiment, those skilled in the art are easy to make according to the present invention some modification or carry out some replacement and equivalent process, so these variations should be included within the scope of the present invention.
Claims (7)
1. batroxobin freeze-dried powder; It is characterized in that; Said batroxobin freeze-dried powder comprises batroxobin and pharmaceutically acceptable freeze drying protectant, and said batroxobin is 10BU: 0.01～10mg with the ratio of freeze drying protectant, and batroxobin is 10BU: 2～200mg with the ratio of excipient; Said batroxobin is a kind of batroxobin that from the spearhead agkistrodon halyx pallas venom, extracts purification;
Said batroxobin lyophilized injectable powder prepares through following method:
1) gets batroxobin, it is joined in the buffer solution dissolve, get lysate;
2) in lysate, add freeze drying protectant and excipient;
3) packing, lyophilization promptly gets the batroxobin freeze-dried powder;
The pH value of wherein said buffer is 4.8～6.0.
2. batroxobin freeze-dried powder according to claim 1 is characterized in that, said batroxobin is from snake venom, to extract purification and obtain through DEAE-Sepharose Fast Flow chromatography, affinity chromatograph, gel permeation chromatography, lyophilization.
3. batroxobin freeze-dried powder according to claim 1 is characterized in that, said freeze drying protectant is to be selected from aminoacid, saccharide, polyhydroxy-alcohol, protein and the perfluorooctane sulfonate one or more.
4. batroxobin freeze-dried powder according to claim 3 is characterized in that,
Said aminoacid is to be selected from glycine, proline, L-tryptophan, sodium glutamate, alanine, lysine hydrochloride, sarcosine, L-tyrosine, phenylalanine, the arginine one or more;
Said saccharide is to be selected from glucose, fructose, lactose, mannose, sucrose, trehalose, maltose, cyclodextrin and derivant thereof, the dextran one or more;
Said polyhydroxy-alcohol is to be selected from glycerol, mannitol, sorbitol, inositol and the Polyethylene Glycol one or more;
Said protein is to be selected among bovine serum albumin and the human serum albumin one or both.
5. batroxobin freeze-dried powder according to claim 1 is characterized in that, said excipient is to be selected from mannitol, dextran, glycine, alanine, arginine and the lysine one or more.
6. the method for preparing of the said batroxobin freeze-dried powder of claim 1 comprises the steps:
1) gets batroxobin, it is joined in the buffer solution dissolve, get lysate;
2) in lysate, add freeze drying protectant and excipient;
3) packing, lyophilization promptly gets the batroxobin freeze-dried powder;
The pH value of wherein said buffer is 4.8～6.0; Said batroxobin is 10BU: 0.01～10mg with the ratio of freeze drying protectant, and batroxobin is 10BU: 2～200mg with the ratio of excipient.
7. method for preparing according to claim 6 is characterized in that, said buffer solution is acetate buffer and/or citrate buffer.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101200842A CN101361717B (en) | 2007-08-08 | 2007-08-08 | Batroxobin freeze-dry powder injection and preparation method thereof |
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| CN2007101200842A CN101361717B (en) | 2007-08-08 | 2007-08-08 | Batroxobin freeze-dry powder injection and preparation method thereof |
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| CN101361717B true CN101361717B (en) | 2012-04-25 |
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| CN112924701A (en) * | 2021-01-29 | 2021-06-08 | 郑州普湾医疗技术有限公司 | Batroxobin cup detection reagent with platelet aggregation function and preparation method thereof |
| CN112730769A (en) * | 2021-01-29 | 2021-04-30 | 郑州普湾医疗技术有限公司 | Platelet aggregation functional adenosine diphosphate cup detection reagent and preparation method thereof |
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