CN102416171B - 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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- CN102416171B CN102416171B CN2011104004991A CN201110400499A CN102416171B CN 102416171 B CN102416171 B CN 102416171B CN 2011104004991 A CN2011104004991 A CN 2011104004991A CN 201110400499 A CN201110400499 A CN 201110400499A CN 102416171 B CN102416171 B CN 102416171B
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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; several and several groups of former composite articles of high-purity thrombase (four kinds of prothrombins, VII, IX, X specific activity all >=3.5IU/mg) 100 ℃, the xeothermic inactivation of virus process of 30min, can effectively prevent the protective agent of Main Function plasma thromboplastin component and prothrombin in 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 xeothermic inactivation of virus process.
Background technology
Human Factor Ⅸ Complex (Prothrombin Complex Concentrate, PCC), claim again the plasma thromboplastin component complex, it is the frozen dry blood plasma protein product be prepared into by the Healthy People pooled plasma, except containing plasma thromboplastin component, also contain the synthetic prothrombin of other vitamin K dependents, VII, X.The 1950's PCC start the preparation and for clinical, at present these goods except for hemophilia B, have the hemophilia A of anticoagulin VII, also be widely used in and the hemorrhage that cause low by hepatic disease, the caused thrombin level of vitamin K deficiency and substitute fresh frozen plasma and reverse the excessive and various hemorrhages that cause of coumarin anticoagulant, along with the expansion of PCC clinical indication, its use amount is also increasing year by year.
For since clinical treatment, the report that causes the thrombosis complication has constantly been arranged after clinical infusion from PCC, mainly comprise disseminated inravascular coagulation, phlebothrombosis, pulmonary edema and lethal myocardial infarction etc.Although PCC causes thrombotic exact cause also not illustrated fully at present, but animal model experiment discloses, contain activation kallikreinogen, high molecular weight kininogen, phospholipid and contact factor etc. in goods and all may impel thrombosis, therefore, high-purity PCC goods, other impurity albumen contain less, can effectively reduce thrombotic risk.Present Domestic molding PCC goods, the specific activity of prothrombin, VII, IX, X is mostly between 0.3-1.0IU/mg, and the contained ratio of foreign protein is relatively large, has increased to a certain extent the probability that the thrombosis side reaction occurs.
PCC is prepared from by human normal plasma, have a liking for but can not get rid of hepatitis B virus (HBV), hepatitis C virus (HCV), HIV (human immunodeficiency virus) (HIV), people possibility that T cell virus (HTLV) etc. infects, so in preparation process, the method for inactivation of virus or removal must be arranged.In May, 2002, clear in National Drug Administration's " blood products removal/inactivation of viruses technical method and verification guide principle ": the method for specific energy removal/deactivation fat peplos and non-lipid-coated virus should be arranged in 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; this virus inactivating method carries out usually after the goods lyophilizing; easy and simple to handle; controllability is strong; adopted by a lot of manufacturers, but goods process the easy inactivation degeneration of effective active albumen 100 ℃ of hot conditionss; 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 relatively less; more harsh to protectant requirement; so far there are no to the protectant research of the xeothermic inactivation of virus process of high-purity PCC; in addition, also have no trehalose or/and histidine for the protectant report of the xeothermic inactivation of virus process of PCC.
Summary of the invention
The technical problem solved
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 invention provides protective agents several and several groups of xeothermic inactivation of virus processes of high-purity PCC; 100 ℃, the xeothermic viral inactivation treatment of 30min have and protect preferably effect prothrombin, VII, IX, X, and wherein the plasma thromboplastin component activity yield all>74%.
Technical scheme
The present invention be take high-purity PCC as experiment material, by 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 as follows:
A) high-purity PCC preparation
2-4 ℃ of centrifugal removal cryoprecipitate of Healthy People pooled plasma, take the blood plasma supernatant as raw material, utilizes the expansion bed technology to prepare high-purity PCC.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 for eluent by collecting, obtain high-purity PCC experiment material.
The high-purity PCC composition characteristic of above-mentioned preparation is:
| The thrombin kind | Specific activity (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 adjust plasma thromboplastin component units activity in concentrated solution, add trehalose by a certain percentage or/and the histidine protective agent is adjusted pH to 7.0, aseptic filtration, packing.
C) lyophilization
After above-mentioned packing goods-40 ℃ pre-freeze 8hr, freeze dryer vacuum lyophilization.
D) xeothermic inactivation of virus
After lyophilizing, goods are placed in water-bath, are warming up to 100 ℃ of timing, after 30min, take out.
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 is all higher than 74%, and optimum protective agent combines II, VII after xeothermic viral inactivation treatment, IX, tetra-kinds of coagulation factor activity response rate of X all can reach more than 98%.
The specific embodiment
To protective agent of the present invention, in xeothermic inactivation of virus process, the protective effect to high-purity PCC elaborates in conjunction with the embodiments, but embodiments of the present invention are not limited to this.
Embodiment 1: the protection test to the former complex Main Function of high-purity thrombase plasma thromboplastin component of trehalose and histidine
Do not add protectant blank goods after 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, in (in Table 1) more than 74%, illustrates that the present invention protects agent and can effectively protect high-purity PCC Main Function plasma thromboplastin component under single factor condition.
The impact on high-purity PCC plasma thromboplastin component activity recovery of xeothermic viral inactivation treatment of table 1 trehalose and histidine
Embodiment 2: trehalose and the histidine protection effect to 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%, and add 2.0% trehalose, make protectant goods, 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 are all in (in Table 2) more than 76%, illustrate that the present invention protects the four kind thrombins of agent can effectively protect high-purity PCC under single factor condition in.
The impact on tetra-kinds of coagulation factor activity response rate of high-purity PCC of xeothermic viral inactivation treatment of table 2 trehalose and histidine
| 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 or/and histidine+glycine to the protection test of the former complex Main Function of high-purity thrombase plasma thromboplastin component
On 2.0% glycine basis, add variable concentrations (0.5%, 1.5%) trehalose and make protective agent, the plasma thromboplastin component activity yield is in (in 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 in (in Table 3) more than 76%, all higher than the yield of 2.0% glycine matched group 67.4%.On 2.0% glycine basis; add respectively 0.5% histidine+0.5% trehalose, 1.0% histidine+0.5% trehalose, 0.5% histidine+1.0% trehalose, 1.0% histidine+1.0% trehalose and make protective agent; plasma thromboplastin component lowest activity yield is 92.8% (in Table 4); all higher than 2.0% glycine matched group; can obviously find out, protective agent trehalose of the present invention is or/and histidine has good protective effect to high-purity PCC Main Function plasma thromboplastin component activity in xeothermic inactivation process.
The impact on high-purity PCC plasma thromboplastin component activity recovery of xeothermic viral inactivation treatment of table 3 trehalose+glycine and histidine+glycine
The impact of table 4 histidine+trehalose+glycine on high-purity PCC plasma thromboplastin component activity recovery of xeothermic viral inactivation treatment
| The thrombin type | Plasma thromboplastin component |
| 2.0% glycine contrast | 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 protection effects of high-purity prothrombin complex
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 is 98.4% (in 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 is 95.3% (in Table 6); and 2.0% glycine matched group; four kinds of coagulation factor activity yields be up to 70.1% (in Table 5 and table 6); as can be seen here, protective agent trehalose of the present invention and histidine all have active protection effect preferably to tetra-kinds of thrombins of high-purity PCC in xeothermic inactivation process.
The impact of table 5 trehalose+histidine+glycine on tetra-kinds of coagulation factor activity response rate of high-purity PCC of xeothermic viral inactivation treatment
| The thrombin type | 2.0% glycine contrast | 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％ |
The impact of table 6 trehalose+histidine+glycine on tetra-kinds of coagulation factor activity response rate of high-purity PCC of xeothermic viral inactivation treatment
| The thrombin type | 2.0% glycine contrast | 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 (3)
1. trehalose is or/and histidine is used as the protectant purposes of the xeothermic inactivation of virus of the former complex of high-purity thrombase, and content of trehalose is 0.1%-8%, and histidine content is 0.1%-8%.
According to claim 1 trehalose or/and histidine as the protectant purposes of the xeothermic inactivation of virus of the former complex of high-purity thrombase, it is characterized in that: four kinds of prothrombins of prothrombin complex, VII, IX, X specific activity all >=3.5IU/mg.
3. trehalose, or/and histidine is used as the protectant purposes of the xeothermic inactivation of virus of the former complex of high-purity thrombase, is characterized in that: also contain the 0-10% glycine in protective agent according to claim 1.
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| CN103341187B (en) * | 2012-06-28 | 2016-06-22 | 四川远大蜀阳药业股份有限公司 | A kind of method of terminal inactivation of pathogenic microorganism |
| 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 |
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| CN1179107A (en) * | 1995-01-19 | 1998-04-15 | 廓德伦特控股剑桥有限公司 | Dried blood factor composition comprising trehalose |
| CN1524578A (en) * | 2003-09-18 | 2004-09-01 | 上海新兴医药股份有限公司 | Dry heat processing stabilizer for prothrombin complex or factor v a IX preparation |
| CN102105141A (en) * | 2008-04-16 | 2011-06-22 | 一般财团法人化学及血清疗法研究所 | Method of producing thrombin-immobilized bioabsorbable sheet preparation |
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| DE10022092A1 (en) * | 2000-05-08 | 2001-11-15 | Aventis Behring Gmbh | Stabilized protein preparation and process for its preparation |
| CN101544683B (en) * | 2008-03-28 | 2012-11-14 | 上海莱士血液制品股份有限公司 | Method and substance for keeping fibrinogen activity in thermal treatment |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1179107A (en) * | 1995-01-19 | 1998-04-15 | 廓德伦特控股剑桥有限公司 | Dried blood factor composition comprising trehalose |
| CN1524578A (en) * | 2003-09-18 | 2004-09-01 | 上海新兴医药股份有限公司 | Dry heat processing stabilizer for prothrombin complex or factor v a IX preparation |
| CN102105141A (en) * | 2008-04-16 | 2011-06-22 | 一般财团法人化学及血清疗法研究所 | Method of producing thrombin-immobilized bioabsorbable sheet preparation |
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