CN109182315A - A kind of preparation method of pig thrombiase - Google Patents
A kind of preparation method of pig thrombiase Download PDFInfo
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- CN109182315A CN109182315A CN201811040796.8A CN201811040796A CN109182315A CN 109182315 A CN109182315 A CN 109182315A CN 201811040796 A CN201811040796 A CN 201811040796A CN 109182315 A CN109182315 A CN 109182315A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6429—Thrombin (3.4.21.5)
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- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21005—Thrombin (3.4.21.5)
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Abstract
The invention discloses a kind of preparation methods of pig thrombiase, comprising the following steps: 1) preparation of blood plasma, 2) S/D method inactivation of virus, 3) factor crude product preparation, 4) thrombin activation liquid preparation, 5) thrombin purification, 6) ultrafiltration desalination, 7) nanometer film except virus；The present invention combines viral inaction steps by two steps into technical process, and the viral level being effectively controlled in finished product improves the safety of fibrin ferment；(Fig. 2) is confirmed by Marker electrophoresis, and two-step purifying significantly improves the purity of fibrin ferment, while controlling the residual of S/D, improves nanofiltration membrane except the efficiency of virus.
Description
Technical field
The present invention relates to a kind of preparation methods of pig thrombiase.
Background technique
Fibrin ferment (Thombin, EC3.4.21.5) is the serine stretch protein water to play an important role in blood clotting system
Enzyme is solved, is activated by thrombokinase, specificity with higher, is that one kind of domestic-developed in recent years is novel quick-acting
Local hemostatic is clinically widely used in hemorrhage of digestive tract and surgical operation hemostasis.The fibrin ferment of high-purity is in genetic engineering
There is special role in postorder working process.Fibrin ferment hemostatic mechanism is that the fibrinogen in blood is promoted to be converted into fiber egg
It is white, while promoting platelet aggregation, accelerate blood clotting, achievees the purpose that stop blooding rapidly.
It mainly prepares factor from several human plasmas of animal blood plasma both at home and abroad at present, then is activated through activator and become solidifying
Hemase.
Fibrin ferment is a kind of protein, and preparation method generally comprises three basic steps: it extracts, purify and is lyophilized,
The production of Chinese patent CN201010230028.6 is centrifugated to obtain blood plasma the following steps are included: anti-coagulants 1) is added in blood；
2) BaCL2 is added in blood plasma, obtains factor precipitating；3) factor precipitating EDTA dissolves washing centrifugation purification；4) it dissolves
Precipitating, is activated with CaCL2, obtains fibrin ferment；Patent No. CN200510010518.4 production stage is anti-including being added in 1) blood
Solidifying agent, is centrifugated to obtain blood plasma；2) Pig Prothrombin multienzyme complex 3 living is prepared) by isoelectric point precipitation, centrifugation obtains blood coagulation
Proenzyme solution 4) with Pig Prothrombin multienzyme complex living prothrombin solution is activated, obtain fibrin ferment.Obtained by two above patent
Fibrin ferment foreign protein it is more, and virus that may be present in untreated thrombin preparation, there are security risk, fibrin ferment is made
For blood extract, belong to containing the high-risk product of virus, but there is no viral inaction steps in technique, seriously affects the safety of product
Property.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the impurity of the fibrin ferment prepared in the prior art is more, and to solidifying
Virus that may be present does not process in hemase preparation, haves the defects that security risk, provides a kind of preparation side of pig thrombiase
Method.
In order to solve the above-mentioned technical problems, the present invention provides the following technical solutions:
A kind of preparation method of pig thrombiase, comprising the following steps:
1) preparation of blood plasma
Sodium citrate solution is added into the fresh pig blood of collection, 15 DEG C hereinafter, centrifuge separation erythrocyte and blood plasma, institute
Blood plasma is placed in -20 DEG C and freezes；
2) S/D method inactivation of virus
It will be filtered by the blood plasma of thaw at RT with qualitative filter paper, triton x-100 and tricresyl phosphate fourth is added in gained filtrate
Ester stirs 3~6 hours；
3) prepared by factor crude product
Blood plasma Jing Guo S/D inactivation treatment is filtered with qualitative filter paper, DEAE Sephadex A- is added in gained filtrate
50 gels, stirring；Filtering, discards filtrate；Sodium citrate, the 0.05~0.8moL/L sodium chloride for configuring 0.01~0.1moL/L are molten
The high salt wash liquid of liquid first removes foreigh protein removing with high level salt solution elution gel；Again with high level salt solution eluent impregnate gel 10~
20 minutes, filtering collected filtrate in triplicate or more than three times；
4) prepared by thrombin activation liquid
Calcium chloride solution is added into above-mentioned filtrate, adjusts Ca in filtrate+Concentration be finally adjusted to 0.02~
0.15moL/L, 25 DEG C~32 DEG C stirrings activate 0.5 hour；
5) thrombin purification
With the pillar that Sp-Sepharose FF is filled, by above-mentioned thrombin activation liquid in the ratio citric acid of 1:3~6
Sodium solution dilution, loading；>=3 column volumes are eluted with high salt wash liquid after loading, wash foreign protein；High salt wash is used again
Liquid elutes 2 column volumes, collects fibrin ferment sterling；
6) ultrafiltration desalination
Above-mentioned collection liquid is subjected to ultrafiltration desalination；
7) nanometer film is except virus
It after DV20 NI0552 nanofiltration membrane is correctly installed, pressurizes to the ultrafiltration concentration liquid in fluid reservoir, maintains filter membrane front and back
Pressure difference in 2~5bar, filtration Ultrafiltration concentrate obtains thrombin solution.
Further, the additive amount of triton x-100 and tributyl phosphate is 0.3%-2% in the step 2).
Further, the pH of the high salt wash liquid is 6~9.
Further, the sodium citrate in the step 5) in high salt wash liquid is replaced with into trisodium citrate or three (hydroxyls
Methyl) aminomethane.
Preferably, a kind of preparation method of pig thrombiase, comprising the following steps:
1) preparation of blood plasma
Into the fresh pig blood of collection in the sodium citrate solution of the ratio addition 10% of 20:1；15 DEG C hereinafter, 3500r/
Min centrifuge separation erythrocyte and blood plasma, gained blood plasma are placed in -20 DEG C and freeze.
2) S/D method inactivation of virus
It will be filtered by the blood plasma of thaw at RT with qualitative filter paper, triton x-100 and tricresyl phosphate fourth is added in gained filtrate
Ester stirs 3~6 hours at room temperature.
3) prepared by factor crude product
Blood plasma Jing Guo viral inactivation treatment is filtered with qualitative filter paper, DEAE is added by 10:1 in gained filtrate
Sephadex A-50 gel stirs 30 minutes；Filtering, discards filtrate；With 3 times and the 0.02moL/L sodium citrate of gel volume
+ 0.1moL/L NaCl liquid elutes gel and removes foreigh protein removing；Again with isometric 0.02moL/L sodium citrate+0.5moL/
L sodium chloride eluent impregnates gel 15 minutes, filtering, in triplicate, collects filtrate.
4) prepared by thrombin activation liquid
Calcium chloride solution is added into above-mentioned filtrate, the concentration of Ca+ in filtrate is made finally to be adjusted to 0.05moL/L, 30 DEG C
Stirring activation 0.5 hour
5) thrombin purification
Prepare the pillar filled with Sp-Sepharose FF, above-mentioned thrombin activation liquid is used in the ratio of 1:5
The dilution of 0.02moL/L sodium citrate solution, loading；It is washed after loading with 0.02moL/L sodium citrate+0.1moL/L sodium chloride
It washs liquid and elutes 3 column volumes, wash foreign protein；0.02moL/L sodium citrate+0.3moL/L sodium chloride elution 2 is used again
A column volume collects fibrin ferment sterling.
6) ultrafiltration desalination
Above-mentioned collection liquid is subjected to ultrafiltration desalination, chlorine ion concentration detection effect is equal to tap water detection effect containing chlorine,
I.e. desalination finishes.
7) nanometer film is except virus
It after DV20 NI0552 nanofiltration membrane is correctly installed, pressurizes to the ultrafiltration concentration liquid in fluid reservoir, maintains filter membrane front and back
Pressure difference in 3bar or so, filtration Ultrafiltration concentrate obtains thrombin solution.
The beneficial effects obtained by the present invention are as follows being:
The present invention combines viral inaction steps, the disease being effectively controlled in finished product by two steps into technical process
Malicious content improves the safety of fibrin ferment；After artificially addition virus BVDV, PRV, VSV, EMCV, PPV, by third party's disease
Malicious inactivation technology verifying, viral reduction amount (Log10) >=4Logs (table 1), it is effectively to grasp that two steps joint, which inactivates/remove viral technique,
Make, meets national standard.
Detailed description of the invention
Attached drawing is used to provide further understanding of the present invention, and constitutes part of specification, with reality of the invention
It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the process flow chart of Example 1 and Example 2 of the present invention；
Fig. 2 is Marker electrophorogram.
Specific embodiment
Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings, it should be understood that preferred reality described herein
Apply example only for the purpose of illustrating and explaining the present invention and is not intended to limit the present invention.
Embodiment 1
1 process flow chart configures 10% sodium citrate solution 2.5kg with reference to the accompanying drawings, with the freshly harvested fresh pig blood of 50kg
50kg is stirred evenly, the blood plasma 22kg of centrifugation.220mL triton x-100 and 220mL tributyl phosphate, temperature are added into blood plasma
24 DEG C of degree control, is stirred 4 hours.2.2kg DEAE Sephadex A-50 gel is added after qualitative filter paper filters in blood plasma,
30min, filtering, filter cake 8kg0.03moL/L sodium citrate and the elution of 0.1moL/L sodium chloride solution are stirred, takes out gel again
15min is impregnated with the 0.03moL/L sodium citrate and 0.5moL/L sodium chloride solution of 2.5kg or so, filtering is closed in triplicate
And filtrate about 7.5kg.CaCL is added2, it is Ca in filtrate+Concentration be adjusted to 0.05moL/L, 30 DEG C of stirrings activate 30min.With
0.03moL/L sodium citrate dilutes above-mentioned filtrate to 45kg.Preparation Sp-Sepharose FF pillar, the direct loading of aforesaid liquid,
After loading, 6kg is eluted with 0.03moL/L sodium citrate and 0.1moL/L sodium chloride solution, is discarded, then use 0.03moL/L
Sodium citrate and 0.3moL/L sodium chloride 4kg elute fibrin ferment, obtain thrombin solution.The solution is used after ultrafiltration desalination
DV20 NI0552 nanofiltration membrane removes virus removal, obtains thrombin solution.
Embodiment 2
1 process flow chart configures 10% sodium citrate solution 2.5kg with reference to the accompanying drawings, with the freshly harvested fresh pig blood of 50kg
50kg is stirred evenly, the blood plasma 22kg of centrifugation.330mL triton x-100 and 330mL tributyl phosphate, temperature are added into blood plasma
26 DEG C of degree control, is stirred 4 hours.2.3kg DEAE Sephadex A-50 gel is added after qualitative filter paper filters in blood plasma,
30min, filtering, filter cake 8kg0.05moL/L sodium citrate and the elution of 0.1moL/L sodium chloride solution are stirred, takes out gel again
15min is impregnated with the 0.05moL/L sodium citrate and 0.5moL/L sodium chloride solution of 2.5kg or so, filtering is closed in triplicate
And filtrate about 7.5kg.CaCL is added2, it is Ca in filtrate+Concentration be adjusted to 0.08moL/L, 30 DEG C of stirrings activate 30min.With
(methylol) aminomethane of 0.05moL/L tri- dilutes above-mentioned filtrate to 45kg.Prepare Sp-Sepharose FF pillar, above-mentioned liquid
Body direct loading after loading, is eluted with (methylol) aminomethane of 0.05moL/L tri- and 0.1moL/L sodium chloride solution
6kg is discarded, then elutes fibrin ferment with (methylol) aminomethane of 0.05moL/L tri- and 0.3moL/L sodium chloride 4kg, is coagulated
Hemase solution.The solution removes virus removal after ultrafiltration desalination, with DV20 NI0552 nanofiltration membrane, obtains thrombin solution.
Virus inactivation technology verifying
It after artificially addition virus BVDV, PRV, VSV, EMCV, PPV, is verified by third party's virus inactivation technology, virus drop
Low amounts (Log10) >=4Logs (table 1), the inactivation of two steps joint/except viral technique is effectively operation, meet national standard.
The verifying of 1 virus inactivation technology of table
Note: BVDV: bovine diarrhea virus；PRV: Pseudorabies virus；VSV: vesicular stomatitis virus；EMCV: encephalomyo-carditis disease
Poison；
PPV: pig parvoviral
The meaning of each band is specific as follows in fig 2:
| Activating fluid | Activating fluid before secondarily purified |
| 0.1M | Sodium chloride concentration is 0.1moL/L |
| Marker | Different molecular weight control |
| 0.3M-1 | Sodium chloride concentration is 0.3moL/L, first column volume of collection |
| 0.3M-2 | Sodium chloride concentration is 0.3moL/L, second column volume of collection |
| 0.3M-3 | Sodium chloride concentration is 0.3moL/L, the third column volume of collection |
| 0.3M-4 | Sodium chloride concentration is 0.3moL/L, the 4th column volume of collection |
From the confirmation of the Marker electrophoresis of Fig. 2 it is found that two-step purifying significantly improves the purity of fibrin ferment, while controlling S/
The residual of D improves nanofiltration membrane except the efficiency of virus.Total titer falls to 90% before purification, and Tot Prot falls to purifying
Preceding 2.9%, as can be seen from Figure 2 a large amount of foreign proteins are removed；
Protein content result before and after 2 two-step purifying of table
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Claims (4)
1. a kind of preparation method of pig thrombiase, which comprises the following steps:
1) preparation of blood plasma
Sodium citrate solution is added into the fresh pig blood of collection, 15 DEG C hereinafter, centrifuge separation erythrocyte and blood plasma, gained blood
Slurry is placed in -20 DEG C and freezes；
2) S/D method inactivation of virus
It will be filtered by the blood plasma of thaw at RT with qualitative filter paper, triton x-100 and tributyl phosphate is added in gained filtrate, stirs
It mixes 3~6 hours；
3) prepared by factor crude product
Blood plasma Jing Guo S/D inactivation treatment is filtered with qualitative filter paper, it is solidifying that DEAE Sephadex A-50 is added in gained filtrate
Glue, stirring；Filtering, discards filtrate；Configure the sodium citrate of 0.01~0.1moL/L, 0.05~0.8moL/L sodium chloride solution
High salt wash liquid first removes foreigh protein removing with high level salt solution elution gel；10~20 points of gel are impregnated with high level salt solution eluent again
Clock, filtering collect filtrate in triplicate or more than three times；
4) prepared by thrombin activation liquid
Calcium chloride solution is added into above-mentioned filtrate, adjusts Ca in filtrate+Concentration be finally adjusted to 0.02~0.15moL/L, 25
DEG C~32 DEG C of stirrings activate 0.5 hour；
5) thrombin purification
It is with the pillar that Sp-Sepharose FF is filled, above-mentioned thrombin activation liquid is molten in the ratio sodium citrate of 1:3~6
Liquid dilution, loading；>=3 column volumes are eluted with high salt wash liquid after loading, wash foreign protein；It is washed again with high salt wash liquid
2 column volumes are taken off, fibrin ferment sterling is collected；
6) ultrafiltration desalination
Above-mentioned collection liquid is subjected to ultrafiltration desalination；
7) nanometer film is except virus
It after DV20NI0552 nanofiltration membrane is correctly installed, pressurizes to the ultrafiltration concentration liquid in fluid reservoir, maintains the pressure before and after filter membrane
Difference obtains thrombin solution in 2~5bar, filtration Ultrafiltration concentrate.
2. the preparation method of pig thrombiase as described in claim 1, which is characterized in that triton x-100 in the step 2)
Additive amount with tributyl phosphate is 0.3%-2%.
3. the preparation method of pig thrombiase as described in claim 1, which is characterized in that the pH of the high salt wash liquid be 6~
9。
4. the preparation method of pig thrombiase as described in claim 1, which is characterized in that by high salt wash liquid in the step 5)
In sodium citrate replace with trisodium citrate or three (methylol) aminomethanes.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110835626A (en) * | 2019-12-04 | 2020-02-25 | 长春雷允上药业有限公司 | Preparation method of thrombin |
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| CN103160486A (en) * | 2013-04-02 | 2013-06-19 | 黑龙江迪龙制药有限公司 | Preparation method of porcine thrombin |
| CN104328101A (en) * | 2014-10-27 | 2015-02-04 | 长春远大国奥制药有限公司 | Preparation method of thrombin |
| CN106011116A (en) * | 2016-07-29 | 2016-10-12 | 上海莱士血液制品股份有限公司 | Preparation method of human thrombin |
| CN107058270A (en) * | 2017-05-08 | 2017-08-18 | 湖南格制药有限公司 | The preparation method and its production system of pig thrombiase |
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2018
- 2018-09-07 CN CN201811040796.8A patent/CN109182315A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160486A (en) * | 2013-04-02 | 2013-06-19 | 黑龙江迪龙制药有限公司 | Preparation method of porcine thrombin |
| CN104328101A (en) * | 2014-10-27 | 2015-02-04 | 长春远大国奥制药有限公司 | Preparation method of thrombin |
| CN106011116A (en) * | 2016-07-29 | 2016-10-12 | 上海莱士血液制品股份有限公司 | Preparation method of human thrombin |
| CN107058270A (en) * | 2017-05-08 | 2017-08-18 | 湖南格制药有限公司 | The preparation method and its production system of pig thrombiase |
Non-Patent Citations (2)
| Title |
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| 招健华等: "凝血酶制备工艺的研究进展", 《广东省药学会.2016年广东省药师周大会论文集》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110835626A (en) * | 2019-12-04 | 2020-02-25 | 长春雷允上药业有限公司 | Preparation method of thrombin |
| 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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Application publication date: 20190111 |