CN101757617A - Stable animal source thrombase preparation and preparation method thereof - Google Patents
Stable animal source thrombase preparation and preparation method thereof Download PDFInfo
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- CN101757617A CN101757617A CN200810202980A CN200810202980A CN101757617A CN 101757617 A CN101757617 A CN 101757617A CN 200810202980 A CN200810202980 A CN 200810202980A CN 200810202980 A CN200810202980 A CN 200810202980A CN 101757617 A CN101757617 A CN 101757617A
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- thrombase
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Abstract
The invention relates to a stable animal source thrombase preparation, which comprises thrombase and stabilizing agents, wherein the thrombase preparation bears 30 minutes of virus inactivation at 100 DEG C, and the functions and the structure of the thrombase are maintained at the same time of ensuring that possibly polluted pathogenic microorganisms are effectively killed. Compared with the prior art, the animal thrombase of the invention has wide sources and low price, the addition of the stabilizing agents ensures that the animal source thrombase preparation has high stability and can bear 30 minutes of virus inactivation at 100 DEG C, the functions and the structure of the thrombase are maintained at the same time of effectively killing possibly polluted pathogenic microorganisms (including viruses expect for prion viruses, bacteria, mycoplasma, mucedine and the like), and the use safety and the effectiveness are ensured. In addition, the preparation method adopted by the invention has the advantages of reasonable process and simple operation, and has good application performance.
Description
Technical field
The present invention relates to thrombin, relate in particular to a kind of stable animal source thrombase preparation and preparation method thereof.
Background technology
Inactivation of virus/removal the method for existing protein product has its limitation, and is only effective to enveloped virus as S/D method (organic solvent/surfactant method), thereby causes the application of protein product to still have certain risk.Wet heating (60 ℃, 10 hours), dry heating method (80 ℃, 72 hours or 100 ℃, 30 minutes) can kill human no lipid-coated virus effectively, as B19 virus and hepatitis A virus (HAV).But, for zoogenous virus, as bovine parvovirus, pig parvoviral with porcine circovirus, because it is no lipid-coated virus, S/D can not kill it effectively, simultaneously, because this viroid is to the opposing of heat, damp and hot and dry heating method commonly used is invalid substantially to it.And in numerous biomaterial for medical purpose of at home and abroad ratifying, the product proportion of animal origin is not little.
Therefore seek a kind of all viruses of energy deactivation, and the method less, easy to operate to the protein product activity influence, will help to improve the safety that protein product is used.
Summary of the invention
Purpose of the present invention is exactly that a kind of with low cost, stable performance, radiotolerant stable animal source thrombase preparation and preparation method thereof are provided in order to overcome the defective that above-mentioned prior art exists.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of stable animal source thrombase preparation, it is characterized in that this thrombin preparation comprises thrombin, stabilizing agent, described thrombin preparation bore 100 ℃ of inactivation of virus 30 minutes, when guaranteeing to kill the pathogenic microorganism that may pollute effectively, keep the function and the structure of thrombin.
Described thrombin is a protein, and described stabilizing agent comprises L-arginine monohydrochloride, sodium chloride and citric acid trisodium;
The content of described protein, L-arginine monohydrochloride, sodium chloride, citric acid trisodium following (g/100ml):
Protein 0.1-10;
L-arginine monohydrochloride 0.1-10;
Sodium chloride 0.01-10;
Citric acid trisodium 0.01-5.
The content of described protein, L-arginine monohydrochloride, sodium chloride, citric acid trisodium is preferably as follows (g/100ml):
Protein 0.5-5;
L-arginine monohydrochloride 0.1-2;
Sodium chloride 0.3-1;
Citric acid trisodium 1-3.
Described thrombin preparation also comprises residual moisture, and the content of this residual moisture is 0.5-15%.
The preferred 0.5-5% of the content of described residual moisture.
Described thrombin comprises the thrombin in mammal source.
Described mammal comprises cattle, horse, pig, sheep.
Described thrombin preparation is a lyophilized powder.
A kind of preparation method of stable animal source thrombase preparation is characterized in that, this method comprises and adopts aseptic technique to obtain mammalian whole blood, carries out the separation of blood plasma in the production site of 100 grades of cleanliness factors, and thrombin is slightly carried, purification; The protein content of thrombin calculates protein wt behind the mensuration purification, adds stabilizing agent, stirring and evenly mixing; Encapsulation, lyophilizing; Under 100 ℃, carry out 30 minutes inactivation of virus, obtain stable animal source thrombase preparation.
Described mammalian whole blood is the mammalian whole blood through producing with the fixed point plant of butchering the back quarantine before strict the butchering; Described stabilizing agent comprises L-arginine monohydrochloride, sodium chloride and citric acid trisodium; The condition of described deactivation is 100 ℃ of following inactivation of virus 30 minutes.
The present invention adopts the source of extensive, the cheap animal blood source of resource as thrombin, adds stabilizing agent and improves its stability, adopts xeothermicly to carry out inactivation of virus, and keeps excellent function and structure.
Compared with prior art, animal source thrombase wide material sources of the present invention, cheap, add stabilizing agent and guarantee to have advantages of excellent stability, 100 ℃ of 30 minutes inactivation of virus, kill effectively may pollute film virus is arranged in, keep the function and the structure of thrombin, guarantee the safety and the effectiveness that use; In addition, the preparation method technology that the present invention adopts is reasonable, simple to operate, has excellent application performance.
The specific embodiment
The invention will be further described for the contrast specific embodiment below.
Embodiment 1
A kind of preparation method of safe lyophilizing Sanguis sus domestica source thrombase preparation may further comprise the steps:
According to the production standard operating process, adopt aseptic technique to obtain the porker whole blood of producing with the fixed point plant of butchering the back quarantine through before strict the butchering; Carry out the separation of blood plasma in the production site of 100 grades of cleanliness factors, thrombin is slightly carried, purification; The protein content of thrombin calculates albumen weight behind the mensuration purification, and stabilizing agent is added, stirring and evenly mixing, final solution contains protein: 1.5g/100ml, sodium chloride: 1g/100ml, L-arginine monohydrochloride: 0.5g/100ml, citric acid trisodium: 2.4g/100ml; Encapsulation, lyophilizing, aseptic filtration; 100 ℃ of 30 minutes inactivation of virus make safe lyophilizing Sanguis sus domestica source thrombase preparation.
Embodiment 2
The inactivation of virus experiment:
According to the production standard operating process, adopt aseptic technique to obtain the porker whole blood of producing with the fixed point plant of butchering the back quarantine through before strict the butchering; Carry out the separation of blood plasma in the production site of 100 grades of cleanliness factors, thrombin is slightly carried, purification; The protein content of thrombin calculates albumen weight behind the mensuration purification, and refined solution is divided into two parts:
First part, the constituent of per 100 ml solns is as follows: protein: 1.5 grams, sodium chloride: 1 gram, L-arginine monohydrochloride: 0.5 gram, citric acid trisodium: 2.4 grams; Packing, lyophilizing, aseptic filtration; Second part, the constituent of per 100 ml solns is as follows: protein: 1.5 grams, vitamin C: 200mM, sodium chloride: 1 gram, citric acid trisodium: 2.4 grams, aseptic filtration.
With above-mentioned two parts liquid branch that classifies in three categories separately, every five equilibrium adds respectively approximately～10
7The pig parvoviral of qfu/ml (PPV) divides to install to cillin bottle, the 1ml/ bottle, and after the pulverizing, lyophilization once more makes moisture≤1% of lyophilized powder.100 ℃ of 30 minutes inactivation of virus. adopt Reed-muench TCID
50Virus titer reduced (Total Reduction Factor, TRF) situation after method was measured predose.
The result shows; compare with the second assembly side; adopt the first assembly side as protective agent; 100 ℃ of 30 minutes inactivation of virus; the TRF of PPV has reduced nearly 4.5-5.5; and the ascorbic acid group has only reduced 2-3.5, illustrates that stabilizing agent of the present invention has strengthened the inactivation of virus effect more effectively than the ascorbic acid group.
Embodiment 3
Thrombin stability test of the present invention and titration:
According to the technological process of production, prepare thrombin according to embodiment 1 method.
Randomly draw two groups of lot numbers and carry out stability test, testing index is a thrombin titer.From each lot number: get 100 respectively and be placed on (3 years by a definite date), 80 in the household freezer and be placed in the baking oven of 37 ℃ and 75% relative humidity (6 months by a definite date), 80 and be placed in 25 ℃ of air-conditioned rooms (6 months by a definite date) as test sample book.Before this, carried out the baseline time test, and test result is all qualified.In the real-time testing process in 3 years by a definite date, chose interior every month of the first half 10 cover samples and detect, after this, to choose 10 cover samples every half a year and detect, testing result is recorded in the table 1.
Table 1 thrombin temperature experiment result
| ??2-10℃ | ??25℃ | ??37℃ | |
| Time (moon) | (IU/ml) tires | (IU/ml) tires | (IU/ml) tires |
| January | ??565 | ??570 | ??585 |
| February | ??/ | ??565 | ??580 |
| March | ??555 | ??560 | ??576 |
| April | ??/ | ??556 | ??563 |
| May | ??/ | ??545 | ??543 |
| June | ??550 | ??540 | ??521 |
| December | ??545 | ??/ | ??/ |
| The 18th month | ??533 | ??/ | ??/ |
| The 24th month | ??526 | ??/ | ??/ |
| The 30th month | ??520 | ??/ | ??/ |
| The 36th month | ??516 | ??/ | ??/ |
Thrombin titer is measured:
Fibrinogen is made into the Fibrinogen normal saline solution that is equivalent to 0.2% coagulated substance, regulating pH value with disodium phosphate soln (0.05mol/L) or sodium dihydrogen phosphate (0.05mol/L) is 7.0～7.4, the reuse normal saline is diluted to 0.1% solution, and is standby.
The preparation of standard curve: get the thrombin standard substance, add physiological saline solution, be made into every ml respectively and contain 5.0,6.4, the standard solution of 8.0,10.0 units.Other gets internal diameter 1cm, 4 in the test tube of long 10cm, and each accurate each 0.1ml of Fibrinogen normal saline solution that adds puts in 37 ℃ of water-baths, observes the fibrinous presetting period, and every kind of concentration is surveyed 5 times, averages.Concentration (U) with standard solution in every pipe is abscissa, and be vertical coordinate setting time (second), the drawing standard curve.
Algoscopy: 3 bottles of sample thiefs, the accurate respectively weight that takes by weighing its content, add the solution that normal saline is mixed with standard curve concentration respectively by labelled amount, the accurate 0.1ml that draws presses the meansigma methodss (the same standard curve of error requirements) of 5 setting times of preparation parallel assay of standard curve.On standard curve or with linear regression equation, try to achieve units, be calculated as follows.
Thrombin (unit/bottle)=Ux10xV
In the following formula:
The U:0.1ml sample on standard curve, read the effective unit number
V: dissolve every bottle of milliliter number that thrombin is used
W: the weight of sample (mg)
10:0.1ml be converted into the number of 1.0ml
Embodiment 4
A kind of preparation method of safe lyophilizing Sanguis sus domestica source thrombase preparation may further comprise the steps:
According to the production standard operating process, adopt aseptic technique to obtain the porker whole blood of producing with the fixed point plant of butchering the back quarantine through before strict the butchering; Carry out the separation of blood plasma in the production site of 100 grades of cleanliness factors, thrombin is slightly carried, purification; The protein content of thrombin calculates albumen weight behind the mensuration purification, and stabilizing agent is added, stirring and evenly mixing, final solution contains protein: 0.1g/100ml, L-arginine monohydrochloride: 0.1g/100ml, sodium chloride: 0.01g/100ml, citric acid trisodium: 0.01g/100ml; Encapsulation, lyophilizing, aseptic filtration; 100 ℃ of 30 minutes inactivation of virus make safe lyophilizing Sanguis sus domestica source thrombase preparation.
Embodiment 5
A kind of preparation method of safe lyophilizing Sanguis sus domestica source thrombase preparation may further comprise the steps:
According to the production standard operating process, adopt aseptic technique to obtain the porker whole blood of producing with the fixed point plant of butchering the back quarantine through before strict the butchering; Carry out the separation of blood plasma in the production site of 100 grades of cleanliness factors, thrombin is slightly carried, purification; The protein content of thrombin calculates albumen weight behind the mensuration purification, stabilizing agent is added, and stirring and evenly mixing, final solution contains protein: 10g/100ml, L-arginine monohydrochloride: 10g/100ml, sodium chloride: 10g/100ml, citric acid trisodium: 5g/100ml; Encapsulation, lyophilizing, aseptic filtration; 100 ℃ of 30 minutes inactivation of virus are realized the final sterilization virus of going out, and make safe lyophilizing Sanguis sus domestica source thrombase preparation.
Embodiment 6
A kind of preparation method of safe lyophilizing Sanguis sus domestica source thrombase preparation may further comprise the steps:
According to the production standard operating process, adopt aseptic technique to obtain the porker whole blood of producing with the fixed point plant of butchering the back quarantine through before strict the butchering; Carry out the separation of blood plasma in the production site of 100 grades of cleanliness factors, thrombin is slightly carried, purification; The protein content of thrombin calculates albumen weight behind the mensuration purification, and stabilizing agent is added, stirring and evenly mixing, final solution contains protein: 0.5g/100ml, L-arginine monohydrochloride: 0.1g/100ml, sodium chloride: 0.3g/100ml, citric acid trisodium: 1g/100ml; Encapsulation, lyophilizing; 100 ℃ of 30 minutes inactivation of virus make safe lyophilizing Sanguis sus domestica source thrombase preparation.
Embodiment 7
A kind of preparation method of safe lyophilizing Sanguis sus domestica source thrombase preparation may further comprise the steps:
According to the production standard operating process, adopt aseptic technique to obtain the porker whole blood of producing with the fixed point plant of butchering the back quarantine through before strict the butchering; Carry out the separation of blood plasma in the production site of 100 grades of cleanliness factors, thrombin is slightly carried, purification; The protein content of thrombin calculates albumen weight behind the mensuration purification, stabilizing agent is added, and stirring and evenly mixing, final solution contains protein: 5g/100ml, L-arginine monohydrochloride: 2g/100ml, sodium chloride: 1g/100ml, citric acid trisodium: 3g/100ml; Encapsulation, lyophilizing, aseptic filtration; 100 ℃ of 30 minutes inactivation of virus make safe lyophilizing Sanguis sus domestica source thrombase preparation.
The various embodiments described above can also adopt the alternative pig whole blood of mammiferous whole bloods such as cattle, horse or sheep to prepare thrombin preparation.
Claims (10)
1. stable animal source thrombase preparation, it is characterized in that this thrombin preparation comprises thrombin, stabilizing agent, described thrombin preparation bore 100 ℃ of inactivation of virus 30 minutes, when guaranteeing to kill the pathogenic microorganism that may pollute effectively, keep the function and the structure of thrombin.
2. stable animal source thrombase preparation according to claim 1 is characterized in that described thrombin is a protein, and described stabilizing agent comprises L-arginine monohydrochloride, sodium chloride and citric acid trisodium;
The content of described protein, L-arginine monohydrochloride, sodium chloride, citric acid trisodium following (g/100ml):
Protein 0.1-10;
L-arginine monohydrochloride 0.1-10;
Sodium chloride 0.01-10;
Citric acid trisodium 0.01-5.
3. stable animal source thrombase preparation according to claim 2 is characterized in that, the content of described protein, L-arginine monohydrochloride, sodium chloride, citric acid trisodium is preferably as follows (g/100ml):
Protein 0.5-5;
L-arginine monohydrochloride 0.1-2;
Sodium chloride 0.3-1;
Citric acid trisodium 1-3.
4. according to claim 2 or 3 described stable animal source thrombase preparations, it is characterized in that described thrombin preparation also comprises residual moisture, the content of this residual moisture is 0.5-15%.
5. stable animal source thrombase preparation according to claim 4 is characterized in that, the preferred 0.5-5% of the content of described residual moisture.
6. stable animal source thrombase preparation according to claim 1 and 2 is characterized in that, described thrombin comprises the thrombin in mammal source.
7. stable animal source thrombase preparation according to claim 6 is characterized in that described mammal comprises cattle, horse, pig, sheep.
8. stable animal source thrombase preparation according to claim 1 is characterized in that described thrombin preparation is a lyophilized powder.
9. the preparation method of a stable animal source thrombase preparation as claimed in claim 1, it is characterized in that this method comprises that the employing aseptic technique obtains mammalian whole blood, carries out the separation of blood plasma in the production site of 100 grades of cleanliness factors, thrombin is slightly carried, purification; The protein content of thrombin calculates protein wt behind the mensuration purification, adds stabilizing agent, stirring and evenly mixing; Encapsulation, lyophilizing; Under 100 ℃, carry out 30 minutes inactivation of virus, obtain stable animal source thrombase preparation.
10. the preparation method of stable animal source thrombase preparation according to claim 9 is characterized in that, described mammalian whole blood is for through before strict the butchering and butcher the mammalian whole blood that the fixed point plant of back quarantine produces; Described stabilizing agent comprises L-arginine monohydrochloride, sodium chloride and citric acid trisodium; The condition of described deactivation is 100 ℃ of following inactivation of virus 30 minutes.
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| CN200810202980A CN101757617A (en) | 2008-11-19 | 2008-11-19 | Stable animal source thrombase preparation and preparation method thereof |
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| CN200810202980A CN101757617A (en) | 2008-11-19 | 2008-11-19 | Stable animal source thrombase preparation and preparation method thereof |
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Open date: 20100630 |