CN105250226B - A kind of preparation method of pig thrombiase freeze-dried powder - Google Patents
A kind of preparation method of pig thrombiase freeze-dried powder Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of pig thrombiase freeze-dried powder, comprise the following steps：Anti-freezing Swine plasma is mixed with gel and adsorbs, and crosses column elution and obtains prothrombin solution；Physiological saline is added into rabbit brain powder, adds prothrombin solution and CaCl2Activation of zymogen is carried out, obtains fibrin ferment crude enzyme liquid, ultrafiltration desalination simultaneously concentrates, and carries out inactivation of virus, and gained crude enzyme liquid crosses DEAE Sepharose Fast Flow chromatographic columns, and purpose peak is collected in elution, up to pig thrombiase；Pig thrombiase adds mannitol or Dextran 40, and filtration sterilization, dispenses, and freezes, vacuum tamponade, after rolling aluminium-plastic combined cover, obtains pig thrombiase dried frozen aquatic products；Dried frozen aquatic products carries out dry heat treatment with inactivation of viruses again at 100 DEG C, packs up to pig thrombiase freeze-dried powder.Pig thrombiase specific activity is not less than 130U/mg in product, and whole processing step is simple, easily implements, and improves products safety, is suitable for industrialized production.
Description
Technical field
The present invention relates to the preparation process of pig thrombiase freeze-dried powder, and in particular to a kind of preparation side of pig thrombiase freeze-dried powder
Method.
Background technology
Fibrin ferment is the serine protein hydrolase to play an important role in blood clotting system, it can hydrolyze fibrin egg
White 4 former Arg-Gly peptide bonds, make the fibrinogen of dissolved colloidal state be changed into the fibrin of gel state, promote blood clotting,
Platelet aggregation can be promoted at the same time, blood clotting is further speeded up, achieve the purpose that to stop blooding rapidly.
The thrombin preparation clinically used at present is largely the factor extracted from ox blood or pig blood, through blood coagulation
Activation of zymogen thing activates and obtains the aseptic freeze-dried product of fibrin ferment, has higher specificity, is a kind of quick-acting local hemostatics, extensively
It is general to be applied to hemorrhage of digestive tract and surgical operation hemostasis.(Song Hongxin, Ma Yongzheng, Li Minkang separating and purifying method for thrombase is ground
Study carefully progress [J] food research and developments, 2004,25 (6)：65-67.).
Pig thrombiase molecular weight is about 35~39kDa, is formed by connecting by two polypeptide chains by a disulfide bond, a chain
Molecular weight is about 34kDa, and another chain molecular weight is about 5kDa；Isoelectric point (pI) is that 5.0~5.5, IEF electrophoresis showeds are two
Band, pI are respectively 5.0,5.4 (Xu Changfa, Wang Yuxian, Liu Defu, wait isolating and purifying for pig thrombiases with identification [J] Beijing to join
Close college journal, 1994,8 (1)：44-49.).The process that isolates and purifies of pig thrombiase generally comprises pre-processing, coagulating for Swine plasma
The extraction of hemase original, the activation of factor and the preparation of thick fibrin ferment, the purifying of fibrin ferment.Due to blood coagulation enzyme preparation
It is that the multicomponent biochemical drug being prepared is extracted from pig blood, the potential hidden danger for thering is pig borne virus to pollute, to improve product
Security, it is necessary to carry out the research of fibrin ferment virus inactivation technology.
The Chinese invention patent of Publication No. CN104328101A discloses a kind of preparation method of fibrin ferment, and this method is adopted
The mode being combined with virus-inactivating agent inactivation treatment and nanofiltration membrane filtering, to inactivate and remove potential pig source in product
Venereal disease poison pollution, obtains certain beneficial effect.But this method is more, complicated in the presence of 1. processing step, 2. PROTHROMBIN ACTIVATOR side
Method is complicated, cumbersome, is not 3. avoided that during thrombin preparation that there are the shortcomings that exogenous virus secondary pollution risk.
The content of the invention
In order to solve the above problem of the prior art, the present invention provides a kind of preparation side of pig thrombiase freeze-dried powder
Method.
The preparation method of pig thrombiase freeze-dried powder provided by the invention, comprises the following steps：
(1) anti-freezing Swine plasma contains Na with passing through2B4O73.75mmol/L and H3BO385mmol/L and pH value are 8.0
The QAE-SephadexA50 gels of boric acid-borate buffer solution balance are according to 1L:The dosage mixing of 1~1.5g, is stirred at room temperature suction
It is attached, the gel for being adsorbed with factor is collected by filtration with 400 mesh silks of bilayer, loads chromatographic column, is first washed till base with purifying current
Line walk it is flat, then with the NaCl streams of 0.1mol/L be washed till baseline walk it is flat, finally with the NaCl's containing 0.4mol/L and 2mmol/L
BaCl2Elution, collect purpose peak, obtain prothrombin solution；
(2) it is 1g by rabbit brain powder and physiological saline:The ratio of 20mL, 45 DEG C of physiological saline is added into rabbit brain powder, is protected
Hold 45~50 DEG C stirring extraction 15~after twenty minutes, 2000rpm centrifuge 5 minutes, collect supernatant；According to supernatant:Fibrin ferment
Original solution:CaCl2=4.7mL:1L:The dosage of 1.4g after proportioning is mixed evenly, stood in 25~31 DEG C of water-baths
Activate 1~2hr and carry out activation of zymogen, then centrifuge 10min in 4 DEG C of 5000rpm, filtered with double-deck 400 mesh silks, collect supernatant
Obtain fibrin ferment crude enzyme liquid；
(3) ultrafiltration membrane ultrafiltration desalination and concentration 10~20 times of the fibrin ferment crude enzyme liquid with 8000 dalton of molecular cut off,
Into hydraulic control within 0.15M Pa, reflux pressure is controlled within 0.1MPa, and inactivation of virus is added in the crude enzyme liquid after concentration
After agent stirs evenly, 6~8hr is kept the temperature at 24~26 DEG C, carries out inactivation of virus；The virus inactivator includes final concentration 3g/L
Tributyl phosphate and final concentration 10g/L Tween-80；Or the virus inactivator includes the tricresyl phosphate fourth of final concentration 3g/L
The Triton X-100 of ester and final concentration 10g/L.
(4) crude enzyme liquid after inactivation of viruses crosses DEAE-Sepharose Fast Flow chromatographic columns, is first washed with balance liquid stream
Walked to baseline after putting down, purpose peak is collected with elution, up to pig thrombiase；
(5) pig thrombiase obtained by step (4) adds the mannitol or Dextran 40 of final concentration of 15~35g/L, filtering
It is degerming, dispense, freeze, vacuum tamponade, after rolling aluminium-plastic combined cover, obtains pig thrombiase dried frozen aquatic products；
(6) pig thrombiase dried frozen aquatic products carries out 30~120min of dry heat treatment with inactivation of viruses again, Ran Houjin at 100 DEG C
Row packaging, up to pig thrombiase freeze-dried powder.
Preferably, the PB buffer solutions that equilibrium liquid is pH value 7.9 and concentration is 0.02mol/L described in step (4), eluent
For pH value 7.9, and the PB buffer solutions that the concentration of the NaCl containing 0.2mol/L is 0.02mol/L.
Preferably, in step (4), equilibrium liquid is pH value 8.0 and concentration is 0.05mol/L Tris-HCl buffer solutions are washed
De- liquid is pH value 8.0, and the Tris-HCl buffer solutions that the concentration of the NaCl containing 0.2mol/L is 0.05mol/L.
Preferably, in step (5), the final concentration of 25g/L of mannitol or Dextran 40.
Preferably, in step (6), the dry heat treatment time is 60min.
The present invention also provides the product that the preparation method of the pig thrombiase freeze-dried powder described in any of the above obtains.
Compared with prior art, the invention has the advantages that：
The preparation method of pig thrombiase freeze-dried powder of the present invention, chromatographs post separation by two steps, obtains pig thrombiase, specific activity
Not less than 130U/mg；The mode being combined at the same time using the inactivation of S/D methods and xeothermic inactivation, has effectively been inactivated potential in product
Exogenous virus, improves products safety.Whole processing step is simple, easily implements, and is suitable for industrialized production.Work of the present invention
The pig thrombiase freeze-dried powder preparation that skill is prepared, potential pig borne virus pollution substantially reduce, and improve Product Safety.
Brief description of the drawings
Fig. 1 is the process flow diagram of present invention process.
Embodiment
With reference to specific embodiment, the invention will be further described, so that those skilled in the art can be more preferable
Understand the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
Experiment material：Borax, boric acid, sodium chloride, calcium chloride, barium chloride, rabbit brain powder, QAE-Sephadex A50 gels,
DEAE-Sepharose Fast Flow gels, disodium hydrogen phosphate, sodium dihydrogen phosphate, trishydroxymethylaminomethane, hydrochloric acid, phosphoric acid
Tributyl, Tween-80, Triton X-100, mannitol, Dextran 40 are commercial product.
The preparation of the pig thrombiase freeze-dried powder preparation of the present invention of embodiment 1
1st, the preparation of pig thrombiase
(1) preparation of factor
Take anti-freezing Swine plasma 40L；QAE-Sephadex A50 gel dry powder 40g separately are taken, with Na containing 3.75mmol/L2B4O7
With 85mmol/L H3BO3, after 8.0 buffer solutions of pH are swollen and balance, add into the blood plasma, absorption 2hr be stirred at room temperature；With
The above-mentioned gel for being adsorbed with factor is collected by filtration in double-deck 400 mesh silks, loads chromatographic column；First baseline is washed till with purifying current
Walk it is flat, then with 0.1mol/L NaCl streams be washed till baseline walk it is flat, finally use NaCl containing 0.4mol/L and 2mmol/L BaCl2Wash
De- liquid elution, collects eluting peak, obtains prothrombin solution 8570mL.
(2) preparation of thromboplastin solution
Rabbit brain powder 5g is taken, the physiological saline that 100mL is preheated to 45 DEG C is added, is extracted 20 minutes in 45 DEG C of stirred in water bath
Afterwards, centrifuge 5 minutes for 2000 revs/min, collect supernatant up to thromboplastin solution.
(3) PROTHROMBIN ACTIVATOR
The thromboplastin solution peace treaty of about 40mL steps (2) preparation is added in prothrombin solution prepared by step (1)
12g anhydrous calcium chlorides, after stirring evenly, stand activation 2hr in 25 DEG C of water-baths；By the crude enzyme liquid after activation in 4 DEG C of 5000rpm
10min is centrifuged, supernatant is collected by filtration with 400 mesh silks of bilayer, up to fibrin ferment crude enzyme liquid.
(4) ultrafiltration desalination and crude enzyme liquid is concentrated
It is the ultrafiltration membrane ultrafiltration desalination of 8000 dalton and concentration 10 by crude enzyme liquid molecular cut off made from step (3)
Times, into hydraulic control within 0.15MPa, reflux pressure is controlled within 0.1M Pa.
(5) S/D methods inactivation of virus
In crude enzyme liquid after being concentrated by ultrafiltration to step (4), the phosphoric acid of final concentration of 0.3% (w/v, i.e. 3g/L) is separately added into
Tributyl, the Triton X-100 of final concentration of 1% (w/v, i.e. 10g/L), after stirring evenly, keeps the temperature 8hr at 24 DEG C, carries out
Inactivation of virus.
(6) thrombin purification
DEAE-Sepharose Fast Flow chromatographic columns, chromatographic column on crude enzyme liquid after step (5) inactivation of virus is pre-
First balanced with 7.9 buffer solution of 0.02mol/L PB, pH, after loading, with the balance liquid stream be washed till baseline walk it is flat, then with containing
7.9 buffer solution of 0.02mol/L PB, the pH elution of 0.2mol/L NaCl, collects purpose peak, up to pig thrombiase.
2nd, the preparation of pig thrombiase freeze-dried powder
In pig thrombiase prepared by step 1, the mannitol of final concentration of 1.5% (w/v, i.e. 15g/L) is added, through degerming
After filtering, dispense, freeze, vacuum tamponade, after rolling aluminium-plastic combined cover, obtains pig thrombiase dried frozen aquatic products；It is placed in 100 DEG C of dry heat treatments
After 120min, packaging, up to pig thrombiase freeze-dried powder preparation finished product.
The preparation of the pig thrombiase freeze-dried powder preparation of the present invention of embodiment 2
Schematic diagram as shown in Figure 1, prepares pig thrombiase freeze-dried powder preparation of the present invention.
1st, the preparation of pig thrombiase
(1) preparation of factor
Take anti-freezing Swine plasma 35L；QAE-SephadexA50 gel dry powder 52.5g separately are taken, with 3.75mmol/L Na2B4O7-
85mmol/L H3BO3, the swelling of pH8.0 buffer solutions, and balance it is good after, add into the blood plasma, absorption 2hr be stirred at room temperature；With double
The above-mentioned gel for being adsorbed with factor is collected by filtration in 400 mesh silk of layer, loads chromatographic column；First baseline is washed till with purifying current to walk
It is flat, then with 0.1mol/L NaCl streams be washed till baseline walk it is flat, finally with 0.4mol/L NaCl-2mmol/L BaCl2Elution, is collected
Eluting peak, obtains prothrombin solution 7500mL.
(2) preparation of thromboplastin solution
Rabbit brain powder 4g is taken, adds the physiological saline that 80mL is preheated to 45 DEG C, after 50 DEG C of stirred in water bath are extracted 15 minutes,
2000 revs/min centrifuge 5 minutes, collect supernatant up to thromboplastin solution.
(3) PROTHROMBIN ACTIVATOR
Thromboplastin solution and 10.5g prepared by 35mL steps (2) is added in prothrombin solution prepared by step (1)
Anhydrous calcium chloride, after stirring evenly, stands activation 1hr in 31 DEG C of water-baths；By the crude enzyme liquid after activation in 4 DEG C of 5000rpm from
Heart 10min, is collected by filtration supernatant, up to fibrin ferment crude enzyme liquid with 400 mesh silks of bilayer.
(4) ultrafiltration desalination and crude enzyme liquid is concentrated
It is the ultrafiltration membrane ultrafiltration desalination of 8000 dalton and concentration 20 by crude enzyme liquid molecular cut off made from step (3)
Times, into hydraulic control within 0.15MPa, reflux pressure is controlled within 0.1M Pa.
(5) S/D methods inactivation of virus
In crude enzyme liquid after being concentrated by ultrafiltration to step (4), the phosphoric acid of final concentration of 0.3% (w/v, i.e. 3g/L) is separately added into
Tributyl, the Tween-80 of final concentration of 1% (w/v, i.e. 10g/L), after stirring evenly, keeps the temperature 6hr at 26 DEG C, carries out virus
Inactivation.
(6) thrombin purification
DEAE-Sepharose Fast Flow chromatographic columns, chromatographic column on crude enzyme liquid after step (5) inactivation of virus is pre-
First 0.05mol/L Tris-HCl, pH8.0 buffer solutions balance, after loading, is washed till baseline with the balance liquid stream and walks flat, then use
The 0.05mol/L Tris-HCl of the NaCl containing 0.2mol/L, pH8.0 buffer solutions elution, collect purpose peak, up to pig thrombiase.
2nd, the preparation of pig thrombiase freeze-dried powder
In pig thrombiase prepared by step 1, the Dextran 40 of final concentration of 3.5% (w/v, i.e. 35g/L), warp are added
After aseptic filtration, dispense, freeze, vacuum tamponade, after rolling aluminium-plastic combined cover, obtains pig thrombiase dried frozen aquatic products；It is placed in 100 DEG C of xeothermic places
After managing 30min, packaging, up to pig thrombiase freeze-dried powder preparation finished product.
3 pig thrombiase purification effect of embodiment is evaluated
Anti-freezing Swine plasma 35L is taken, pig thrombiase is purified using 2 process extracting and developing of embodiment, continuously repeats three
It is secondary, technic index and the quality index such as overall merit thrombin purification effect, yield and S/D residual quantities.Experimental result is shown in Table 1.
1 pig thrombiase purification effect evaluation experimental result of table
According to 1 result of table, fibrin ferment is after two steps chromatography, and specific activity is up to more than 130U/mg, higher than National Pharmacopeia
1 order of magnitude of standard (10U/mg)；Every liter of Swine plasma can produce more than 80000U fibrin ferment sterling, and Tween-80 therein
(Tween-80) and tributyl phosphate (TNBP) residual quantity meets national standard (100 μ g/mL, TNBP ＜ of Tween-80 ＜, 10 μ
g/mL).And the buffer solution that whole process of purification uses is simple, common, inexpensive inorganic reagent, production amplification is held
Easily, cost is low, is suitable for industrialized production.
4 pig thrombiase freeze-dried powder preparation prescription of embodiment and xeothermic inactivation technology parameter influence
Pig thrombiase is purified by 2 process extracting and developing of embodiment, freeze-dried powder is made by different preparation prescriptions respectively
(filling amount is 2mL/ branch, and preparation specification is 1000U/ branch), in 100 DEG C of xeothermic inactivation 2hr, and in 0min (untreated),
30min, 60min, 120min are sampled, and are pressed《Chinese Pharmacopoeia》Three methods detect product potency.Experimental result is shown in Table 2.
2 pig thrombiase freeze-dried powder dry heating method of table processing sample activity change (100 DEG C, 2hr)
As can be seen from Table 2, as Dextran 40 or mannitol concentration raise, pig thrombiase freeze-dried powder preparation heat resistance
It is stronger.Considering cost factor, Dextran 40 or mannitol concentration are with 2.5% (w/v, i.e. 25g/L) in preparation prescription
Most preferably.
5 pig thrombiase freeze-dried powder dry heating method of embodiment verifies the inactivating efficacy of pig parvoviral
Pig parvoviral (PPV, substantial titer 7.5LgTCID50/ 0.1mL), viral diagnosis cell：IBRS2 cells, with
And the detection of sample virus titre, provided by animal medicine institute of Hua Zhong Agriculture University.
Pig thrombiase is prepared by 2 process of embodiment, by " 500U/mL fibrin ferments, 2.5% (w/v, i.e. 25g/L) sweet dew
The preparation prescription of alcohol " prepares semi-finished product.Semi-finished product 36mL is taken, adds 4mL pig parvoviral liquid, is mixed, by 2.22mL/ branch point
Loaded in cillin bottle, partly jump a queue；After lyophilized, jump a queue entirely, roll aluminium-plastic combined cover；In 100 DEG C of xeothermic inactivation 2hr, and in 0min (not
Processing), 30min, 60min, 120min sampling, detect virus titer.Repeat experiment three times.Experimental result is shown in Table 3.
3 dry heating method of table is to PPV inactivating efficacies in pig thrombiase freeze-dried powder (100 DEG C of 2hr, unit：LgTCID50/0.1mL)
Note：Experiment lowest detection is limited to 0.50LogTCID50/0.1mL。
As can be seen from Table 3, through 100 DEG C of xeothermic 30min, you can decline the pig parvoviral in pig thrombiase freeze-dried powder
More than 4logs；After xeothermic 60min, virus titer is down to below test limit.Therefore, present invention process does final finished
Hot inactivation treatment, can potential pig borne virus pollution effectively in inactivated preparation, further increase products safety.While because
It is that inactivation of virus is carried out to finished product, recontamination after heat inactivation can be prevented, method is simple, easy to implement.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention
Enclose not limited to this.The equivalent substitute or conversion that those skilled in the art are made on the basis of the present invention, in the present invention
Protection domain within.Protection scope of the present invention is subject to claims.
Claims (6)
1. a kind of preparation method of pig thrombiase freeze-dried powder, it is characterised in that comprise the following steps：
(1) anti-freezing Swine plasma contains Na with passing through2B4O73.75mmol/L and H3BO385mmol/L and boric acid-boron that pH value is 8.0
The QAE-SephadexA50 gels of sand buffer solution balance are according to 1L:The dosage mixing of 1~1.5g, is stirred at room temperature absorption, with bilayer
400 mesh silks are collected by filtration the gel for being adsorbed with factor, load chromatographic column, first with purifying current be washed till baseline walk it is flat, then
With the NaCl streams of 0.1mol/L be washed till baseline walk it is flat, finally with NaCl and the BaCl of 2mmol/L containing 0.4mol/L2Eluent
Elution, collects purpose peak, obtains prothrombin solution；
(2) it is 1g: 20mL ratio in rabbit brain powder and physiological saline, the physiological saline of 45 DEG C of addition into rabbit brain powder, keeps 45
~50 DEG C of stirring extractions 15~after twenty minutes, 2000rpm is centrifuged 5 minutes, is collected supernatant；According to supernatant:Factor is molten
Liquid:CaCl2=4.7mL:1L:The dosage of 1.4g carries out that after proportioning is mixed evenly, activation 1 is stood in 25~31 DEG C of water-baths
~2hr carries out activation of zymogen, then centrifuges 10min in 4 DEG C of 5000rpm, is filtered with double-deck 400 mesh silks, and collecting supernatant must coagulate
Hemase crude enzyme liquid；
(3) ultrafiltration membrane ultrafiltration desalination and concentration 10~20 times of the fibrin ferment crude enzyme liquid with 8000 dalton of molecular cut off, feed liquor
Within 0.15M Pa, reflux pressure controls within 0.1MPa voltage-controlled system, and virus inactivator is added in the crude enzyme liquid after concentration and is stirred
After mixing uniformly, 6~8hr is kept the temperature at 24~26 DEG C, carries out inactivation of virus；The virus inactivator includes the phosphorus of final concentration 3g/L
The Tween-80 of sour tributyl and final concentration 10g/L；Or the virus inactivator including final concentration 3g/L tributyl phosphate and
The TritonX-100 of final concentration 10g/L；
(4) crude enzyme liquid after inactivation of viruses crosses DEAE-Sepharose Fast Flow chromatographic columns, is first washed till base with balance liquid stream
Line is walked put down after, purpose peak is collected with elution, up to pig thrombiase；
(5) mannitol or Dextran 40 of the final concentration of 15~35g/L of pig thrombiase addition obtained by step (4), filtration sterilization,
Packing, freezes, vacuum tamponade, after rolling aluminium-plastic combined cover, obtains pig thrombiase dried frozen aquatic products；
(6) pig thrombiase dried frozen aquatic products carries out 30~120min of dry heat treatment with inactivation of viruses again at 100 DEG C, is then wrapped
Dress, up to pig thrombiase freeze-dried powder.
2. the preparation method of pig thrombiase freeze-dried powder according to claim 1, it is characterised in that put down described in step (4)
Weigh the PB buffer solutions that liquid is pH value 7.9 and concentration is 0.02mol/L, and eluent is pH value 7.9, and containing the dense of 0.2mol/LNaCl
Spend the PB buffer solutions for 0.02mol/L.
3. the preparation method of pig thrombiase freeze-dried powder according to claim 1, it is characterised in that in step (4), equilibrium liquid
The Tris-HCl buffer solutions for being 0.05mol/L for pH value 8.0 and concentration, eluent are pH value 8.0, and NaCl containing 0.2mol/L
Concentration be 0.05mol/L Tris-HCl buffer solutions.
4. the preparation method of pig thrombiase freeze-dried powder according to claim 1, it is characterised in that in step (5), mannitol
Or the final concentration of 25g/L of Dextran 40.
5. the preparation method of pig thrombiase freeze-dried powder according to claim 1, it is characterised in that in step (6), xeothermic place
The reason time is 60min.
6. the product that the preparation method of any pig thrombiase freeze-dried powder of Claims 1 to 5 obtains.
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| CN110835626B (en) * | 2019-12-04 | 2021-12-21 | 长春雷允上药业有限公司 | Preparation method of thrombin |
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| US20050265989A1 (en) * | 2004-02-05 | 2005-12-01 | Kald Fisk As | Non-toxic purification and activation of prothrombin and use thereof |
| CN102151289A (en) * | 2011-01-28 | 2011-08-17 | 哈尔滨派斯菲科生物制药股份有限公司 | Method for producing human prothrombin complex |
| CN103160486A (en) * | 2013-04-02 | 2013-06-19 | 黑龙江迪龙制药有限公司 | Preparation method of porcine thrombin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050265989A1 (en) * | 2004-02-05 | 2005-12-01 | Kald Fisk As | Non-toxic purification and activation of prothrombin and use thereof |
| CN102151289A (en) * | 2011-01-28 | 2011-08-17 | 哈尔滨派斯菲科生物制药股份有限公司 | Method for producing human prothrombin complex |
| CN103160486A (en) * | 2013-04-02 | 2013-06-19 | 黑龙江迪龙制药有限公司 | Preparation method of porcine thrombin |
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