CN102416171A - Protective agent in process for performing dry heat virus inactivation on high-purity prothrombin complex concentrate products - Google Patents
Protective agent in process for performing dry heat virus inactivation on high-purity prothrombin complex concentrate products Download PDFInfo
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- CN102416171A CN102416171A CN2011104004991A CN201110400499A CN102416171A CN 102416171 A CN102416171 A CN 102416171A CN 2011104004991 A CN2011104004991 A CN 2011104004991A CN 201110400499 A CN201110400499 A CN 201110400499A CN 102416171 A CN102416171 A CN 102416171A
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Abstract
The invention relates to the field of medicinal biotechnologies, and discloses a protective agent which can effectively prevent a mainly acting blood coagulation factor IX and other blood coagulation factors II, VII and X in high-purity prothrombin complex concentrate products (the specific activities of the blood coagulation factors II, VII, IX, and X are more than and equal to 3.5IU/mg protein) from being inactivated in the process of performing dry heat virus inactivation at the temperature of 100 DEG C for 30 minutes. The protective agent is trehalose or/and histidine and also contains two or three of common glycine, sodium citrate and NaCl. Experiments prove that if only high-purity prothrombin complex concentrate products contain 0.1 to 8 percent of trehalose or/and 0.1 to 8 percent of histidine, the activities of the blood coagulation factors IX, II, VII and X can be effectively protected in the process of performing dry heat virus inactivation at the temperature of 100 DEG C for 30 minutes. Therefore, the invention can be used as the protective agent in the process for performing dry heat virus inactivation on the high-purity prothrombin complex concentrate products.
Description
Technical field
The present invention relates to the medical biotechnology field; Be several kinds and several groups of former composite articles of high-purity thrombase (four kinds of prothrombins, VII, IX, X be more equal than living >=3.5IU/mg) 100 ℃, the xeothermic inactivation of virus process of 30min, can effectively prevent the protective agent of main effect plasma thromboplastin component and prothrombin in the goods, VII, X inactivation.Particularly, add the 0.1%-8% trehalose in the former complex of high-purity thrombase or/and the 0.1%-8% histidine, can effectively protect the activity of plasma thromboplastin component and prothrombin, VII, X in the xeothermic inactivation of virus process.
Background technology
Human Factor (Prothrombin Complex Concentrate; PCC); Claim the plasma thromboplastin component complex again; Be the frozen dry blood plasma protein product that is prepared into by the healthy subjects pooled plasma, except that containing plasma thromboplastin component, also contain the synthetic prothrombin of other vitamin K dependents, VII, X.PCC began preparation and was used for clinical the 1950's; These goods are except that being used for hemophilia B, having the hemophilia A of anticoagulin VII at present; Also be widely used in and the hemorrhage that cause low and substitute FFP and reverse the excessive and various hemorrhages that cause of coumarin anticoagulant by hepatic disease, the caused thrombin level of vitamin K deficiency; Along with the expansion of PCC clinical indication, its use amount is also increasing year by year.
Since PCC is used for clinical treatment, the report that causes the thrombosis complication behind the clinical infusion is constantly arranged, mainly comprise disseminated inravascular coagulation, phlebothrombosis, pulmonary edema and lethal myocardial infarction etc.Though PCC causes thrombotic definite reason also not illustrated fully at present; But animal model experiment discloses; Contain activation kallikreinogen, high molecular weight kininogen, phospholipid and contact factor etc. in the goods and all possibly impel thrombosis, therefore, high-purity PCC goods; Other impurity albumen contain less, can effectively reduce thrombotic risk.Current domestic molding PCC goods, between 0.3-1.0IU/mg, the contained ratio of foreign protein is relatively large mostly for the specific activity of prothrombin, VII, IX, X, has increased the probability that the thrombosis side reaction takes place to a certain extent.
PCC is prepared from human normal plasma; But can not get rid of hepatitis B virus (HBV), hepatitis C virus (HCV), HIV (HIV), people have a liking for T cell virus (HTLV) etc. infectious maybe, therefore the method for inactivation of virus or removal must be arranged in preparing process.In May, 2002; Clearly stipulate in National Drug Administration's " blood products removal/inactivation of viruses technical method and verification guide principle ": the method for specific removing/deactivation fat peplos and non-lipid-coated virus should be arranged in the blood clotting factors production of articles process, can adopt one or more method combined removal/inactivation of viruses.100 ℃, the xeothermic inactivation of virus method of 30min all have inactivating efficacy to fat peplos and non-lipid-coated virus, and this virus inactivating method carries out after the goods lyophilizing usually, and is easy and simple to handle; Controllability is strong; Adopted by a lot of manufacturers, but goods handle 100 ℃ of hot conditionss, effective active albumen is prone to the inactivation degeneration; Therefore in xeothermic inactivation of virus process, goods need add a certain amount of protective agent activated protein is protected.
High-purity PCC; Thrombin purity is higher; Protein content is less relatively, and is then more harsh to protectant requirement, do not see so far the protectant research of the xeothermic inactivation of virus process of high-purity PCC; In addition, do not see trehalose or/and histidine is used for the protectant report of the xeothermic inactivation of virus process of PCC yet.
Summary of the invention
The technical problem that solves
In order to reduce the loss of activity of high-purity PCC thrombin in xeothermic inactivation of virus process; Improve the goods activity recovery; The present invention provides the protective agent of several kinds and several groups xeothermic inactivation of virus processes of high-purity PCC; 100 ℃, the xeothermic viral inactivation treatment of 30min have the better protect effect to prothrombin, VII, IX, X, and wherein the plasma thromboplastin component activity yield all＞74%.
Technical scheme
The present invention is an experiment material with high-purity PCC, through lyophilization, xeothermic inactivation of virus (100 ℃, 30min), determination of activity, contrast test, obtains the protective agent of 2 kinds and the 8 groups xeothermic viral inactivation treatment of high-purity PCC, and concrete steps are following:
A) high-purity PCC preparation
2-4 ℃ of centrifugal removal cryoprecipitate of healthy subjects pooled plasma is raw material with the blood plasma supernatant, utilizes expansion bed technology preparation high-purity PCC.The eluent of collecting with contained 0.01M sodium citrate, 0.5%NaCl, the ultrafiltration in the ultrafiltration and concentration system of pH7.0 ultrafiltration buffer, desalination, concentrated and determination of activity, is obtained high-purity PCC experiment material.
The high-purity PCC composition characteristic of above-mentioned preparation is:
| The thrombin kind | Than live (IU/mg) |
| Prothrombin | 3.5-6.0 |
| Proconvertin | 3.5-6.0 |
| Plasma thromboplastin component | 3.5-6.0 |
| Stuart factor | 3.5-6.0 |
B) add protective agent
Suitably plasma thromboplastin component units activity in the adjustment concentrated solution adds trehalose by a certain percentage or/and the histidine protective agent is adjusted pH to 7.0, aseptic filtration, packing.
C) lyophilization
Behind the above-mentioned packing goods-40 ℃ pre-freeze 8hr, freeze dryer vacuum lyophilization.
D) xeothermic inactivation of virus
Goods place water-bath after the lyophilizing, are warming up to 100 ℃ of timing, take out behind the 30min.
E) determination of activity
Water for injection dissolves xeothermic inactivation of virus front and back goods, and the first phase method is measured coagulation factor activity, relative analysis.
Beneficial effect
The invention has the beneficial effects as follows; The protective agent of 2 kinds and the 8 groups xeothermic viral inactivation treatment of high-purity PCC is provided; The activity yield of plasma thromboplastin component all is higher than 74%, and optimum protective agent makes up II after the xeothermic viral inactivation treatment, VII, IX, four kinds of coagulation factor activity response rate of X and all can reach more than 98%.
The specific embodiment
In conjunction with embodiment protective agent of the present invention is elaborated in the protective effect of xeothermic inactivation of virus process to high-purity PCC, but embodiment of the present invention is not limited thereto.
Embodiment 1: trehalose and histidine mainly act on the protection test of plasma thromboplastin component to the former complex of high-purity thrombase
Do not add protectant blank goods behind 100 ℃, the xeothermic viral inactivation treatment of 30min; The plasma thromboplastin component response rate is lower than 58%; Make protectant goods and add variable concentrations (1.5%, 2.5%) trehalose, the plasma thromboplastin component activity recovery is more than 75%; Add variable concentrations (1.5%, 2.5%) histidine and make protectant goods, the plasma thromboplastin component activity recovery (is being seen table 1) more than 74%, explains that the present invention protects agent and under single factor condition, can mainly act on plasma thromboplastin component to high-purity PCC and effectively protect.
Table 1 trehalose and histidine are to the influence of high-purity PCC plasma thromboplastin component activity recovery of xeothermic viral inactivation treatment
Embodiment 2: trehalose and histidine are to the protection effect of four kinds of thrombins of the former complex of high-purity thrombase
Do not add protectant blank goods after xeothermic viral inactivation treatment, four kinds of thrombin response rate are no more than 59%, make protectant goods and add 2.0% trehalose, and four kinds of coagulation factor activity response rate are all more than 81%; Add 2.0% histidine and make protectant goods, four kinds of coagulation factor activity response rate all (are being seen table 2) more than 76%, explain that the present invention protects agent and under single factor condition, can effectively protect four kinds of thrombins among the high-purity PCC.
Table 2 trehalose and histidine are to the influence of four kinds of coagulation factor activity response rate of high-purity PCC of xeothermic viral inactivation treatment
| The thrombin type | Blank | 2.0% trehalose | 2.0% histidine |
| Plasma thromboplastin component | 57.9％ | 82.9％ | 79.5％ |
| Prothrombin | 58.6％ | 81.4％ | 84.4％ |
| Proconvertin | 53.1％ | 85.2％ | 76.2％ |
| Stuart factor | 49.5％ | 91.8％ | 80.6％ |
Embodiment 3: trehalose is or/and histidine+glycine mainly acts on the protection test of plasma thromboplastin component to the former complex of high-purity thrombase
On 2.0% glycine basis, add variable concentrations (0.5%, 1.5%) trehalose and make protective agent, the plasma thromboplastin component activity yield (is being seen table 3) more than 97%; On 2.0% glycine basis, add variable concentrations (0.5%, 1.5%) histidine and make protective agent, the plasma thromboplastin component activity yield (is being seen table 3) more than 76%, all is higher than the yield of 2.0% glycine matched group 67.4%.On 2.0% glycine basis; Add 0.5% histidine+0.5% trehalose, 1.0% histidine+0.5% trehalose, 0.5% histidine+1.0% trehalose, 1.0% histidine+1.0% trehalose respectively and make protective agent; Plasma thromboplastin component lowest activity yield is 92.8% (seeing table 4); All be higher than 2.0% glycine matched group; Can find out obviously that protective agent trehalose of the present invention has good protective effect or/and histidine mainly acts on the plasma thromboplastin component activity to high-purity PCC in xeothermic inactivation process.
Table 3 trehalose+glycine and histidine+glycine are to the influence of high-purity PCC plasma thromboplastin component activity recovery of xeothermic viral inactivation treatment
Table 4 histidine+trehalose+glycine is to the influence of high-purity PCC plasma thromboplastin component activity recovery of xeothermic viral inactivation treatment
| The thrombin type | Plasma thromboplastin component |
| The contrast of 2.0% glycine | 67.4％ |
| 2.0% glycine+0.5% histidine+0.5% trehalose | 108.7％ |
| 2.0% glycine+1.0% histidine+0.5% trehalose | 112.7％ |
| 2.0% glycine+0.5% histidine+1.0% trehalose | 113.5％ |
| 2.0% glycine+1.0% histidine+1.0% trehalose | 92.8％ |
Embodiment 4: trehalose+histidine+glycine is to four kinds of thrombin protections of high-purity prothrombin complex effect
On 2.0% glycine basis, add 0.5% histidine+1.0% trehalose and make protective agent, four kinds of coagulation factor activity yields are minimum to be 98.4% (seeing table 5); On 2.0% glycine basis; Add 1.0% histidine+0.5% trehalose and make protective agent; Four kinds of coagulation factor activity yields are minimum to be 95.3% (seeing table 6), and 2.0% glycine matched group, four kinds of coagulation factor activity yields are up to 70.1% (seeing table 5 and table 6); This shows that protective agent trehalose of the present invention and histidine all have active protection effect preferably to four kinds of thrombins of high-purity PCC in xeothermic inactivation process.
Table 5 trehalose+histidine+glycine is to the influence of four kinds of coagulation factor activity response rate of high-purity PCC of xeothermic viral inactivation treatment
| The thrombin type | The contrast of 2.0% glycine | 2.0% glycine+0.5% histidine+1.0% trehalose |
| Plasma thromboplastin component | 67.4％ | 113.5％ |
| Prothrombin | 70.1％ | 110.2％ |
| Proconvertin | 57.6％ | 105.4％ |
| Stuart factor | 68.5％ | 98.4％ |
Table 6 trehalose+histidine+glycine is to the influence of four kinds of coagulation factor activity response rate of high-purity PCC of xeothermic viral inactivation treatment
| The thrombin type | The contrast of 2.0% glycine | 2.0% glycine+1.0% histidine+0.5% trehalose |
| Plasma thromboplastin component | 67.4％ | 112.7％ |
| Prothrombin | 70.1％ | 108.6％ |
| Proconvertin | 57.6％ | 95.3％ |
| Stuart factor | 68.5％ | 99.5％ |
Claims (6)
- Trehalose or/histidine is as the xeothermic inactivation of virus protective agent of the former complex of high-purity thrombase, content of trehalose is 0.1%-8%, histidine content is 0.1%-8%.
- According to the said trehalose of claim 1 or/histidine is as the xeothermic inactivation of virus protective agent of the former complex of high-purity thrombase, it is characterized in that four kinds of prothrombins of prothrombin complex, VII, IX, X are than the equal >=3.5IU/mg that lives.
- According to the said trehalose of claim 1 or/histidine is as the xeothermic inactivation of virus protective agent of the former complex of high-purity thrombase, it is characterized in that also containing in the protective agent 0-10% glycine.
- According to the said trehalose of claim 1 or/histidine is as the xeothermic inactivation of virus protective agent of the former complex of high-purity thrombase, it is characterized in that also containing in the protective agent sodium citrate, NaCl one or both.
- According to the said trehalose of claim 3 or/histidine is as the xeothermic inactivation of virus protective agent of the former complex of high-purity thrombase, it is characterized in that also containing in the protective agent sodium citrate, NaCl one or both.
- According to the said trehalose of claim 1 or/histidine is as the xeothermic inactivation of virus protective agent of the former complex of high-purity thrombase, is equally applicable to other kinds prothrombin complex goods.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105175486A (en) * | 2015-10-20 | 2015-12-23 | 上海洲跃生物科技有限公司 | Preparation method of high-purity human coagulation factor IX |
| CN105330736A (en) * | 2015-11-06 | 2016-02-17 | 上海洲跃生物科技有限公司 | Method for preparing human blood coagulation factors IX and VII subcutaneously from cold-glue-removed blood plasma |
| RU2629866C1 (en) * | 2012-06-28 | 2017-09-04 | Сычуань Юаньдашуян Фармасьютикал Ко., Лтд. | Method for final inactivation of pathogenic microorganisms |
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Cited By (3)
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| RU2629866C1 (en) * | 2012-06-28 | 2017-09-04 | Сычуань Юаньдашуян Фармасьютикал Ко., Лтд. | Method for final inactivation of pathogenic microorganisms |
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| CN105330736A (en) * | 2015-11-06 | 2016-02-17 | 上海洲跃生物科技有限公司 | Method for preparing human blood coagulation factors IX and VII subcutaneously from cold-glue-removed blood plasma |
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