CN102416171B - Protective agent in process for performing dry heat virus inactivation on high-purity prothrombin complex concentrate products - Google Patents

Abstract

The invention relates to the field of medical biotechnology, which is a high-purity prothrombin complex product (four coagulation factors II, VII, IX, and X specific activities are all ≥ 3.5IU/mg protein) at 100°C, 30min dry heat virus inactivation process , which can effectively prevent the inactivation of coagulation factor IX and other coagulation factors II, VII, and X in the product. The protective agent of the present invention refers to trehalose or/and histidine, and also contains commonly used glycine. Tests have shown that as long as the high-purity prothrombin complex contains 0.1%-8% trehalose or/and 0.1%-8% histidine, it can effectively protect the coagulation factor during the virus inactivation process at 100°C for 30 minutes IX and coagulation factor II, VII, X activity. Therefore, the invention can be used as a protective agent in the process of inactivating high-purity prothrombin complex dry heat virus.

Description

Protective agent in the process of dry heat virus inactivation of high-purity prothrombin complex products

technical field

The invention relates to the field of medical biotechnology, which is several or several groups of high-purity prothrombin complex products (four blood coagulation factors II, VII, IX, and X specific activities are all ≥ 3.5IU/mg) at 100°C, 30min dry heat The virus inactivation process can effectively prevent the protective agent that mainly acts on blood coagulation factor IX and blood coagulation factors II, VII, X inactivation in the product. Specifically, adding 0.1%-8% trehalose or/and 0.1%-8% histidine to the high-purity prothrombin complex can effectively protect coagulation factor IX, coagulation factor II, Activity of VII, X.

Background technique

Human prothrombin complex (Prothrombin Complex Concentrate, PCC), also known as coagulation factor IX complex, is a freeze-dried plasma protein product prepared from the mixed plasma of healthy people. In addition to coagulation factor IX, it also contains other vitamin K-dependent Synthetic coagulation factors II, VII, X. In the 1950s, PCC began to be prepared and used clinically. At present, this product is not only used for hemophilia B and hemophilia A with anticoagulant factor VII, but also widely used for liver disease and vitamin K deficiency. With the expansion of clinical indications of PCC, its use is also increasing year by year.

Since PCC was used in clinical treatment, there have been reports of thrombotic complications after clinical infusion, mainly including disseminated intravascular coagulation, venous thrombosis, pulmonary edema, and fatal myocardial infarction. Although the exact cause of PCC leading to thrombosis has not yet been fully elucidated, animal model experiments have revealed that products containing activated kallikrein, high-molecular-weight kininogen, phospholipids, and contact factors may all promote thrombosis. Therefore, high Purity PCC products contain less other impurity proteins, which can effectively reduce the risk of thrombus formation. At present, the specific activities of coagulation factors II, VII, IX, and X in domestic molded PCC products are mostly between 0.3-1.0IU/mg, and the proportion of miscellaneous proteins is relatively large, which increases the probability of thrombotic side effects to a certain extent. .

PCC is prepared from healthy human plasma, but the possibility of infection with hepatitis B virus (HBV), hepatitis C virus (HCV), HIV (HIV), and human T-cell-tropic virus (HTLV) cannot be ruled out. There must be a method for virus inactivation or removal during the process. In May 2002, the National Medical Products Administration's "Blood Products Removal/Inactivation Virus Technical Methods and Validation Guidelines" clearly stipulated that in the production process of blood coagulation factor products, there should be specific methods that can remove/inactivate lipid envelopes and non-viral substances. For lipid-enveloped viruses, one or more methods can be used in combination to remove/inactivate the virus. 100℃, 30min dry heat virus inactivation method can inactivate both lipid-enveloped and non-lipid-enveloped viruses. This virus inactivation method is usually carried out after the product is freeze-dried. It is easy to operate and highly controllable. It is used by many manufacturers. The manufacturer adopts it, but the product is processed at a high temperature of 100°C, and the effective active protein is easily inactivated and denatured. Therefore, during the dry heat virus inactivation process, the product needs to add a certain amount of protective agent to protect the active protein.

High-purity PCC has higher purity of coagulation factors, relatively less protein content, and more stringent requirements for protective agents. So far, there has been no research on protective agents for high-purity PCC dry heat virus inactivation process. In addition, no seaweed The report of sugar and/or histidine used as protective agent in PCC dry heat virus inactivation process.

Contents of the invention

technical problem to be solved

In order to reduce the activity loss of coagulation factors in the dry heat virus inactivation process of high-purity PCC and improve the activity recovery rate of the product, the present invention provides several and several groups of protective agents for the dry heat virus inactivation process of high-purity PCC, 100°C, 30min Dry heat virus inactivation treatment has a good protective effect on blood coagulation factors II, VII, IX, and X, and the activity yield of blood coagulation factor IX is all>74%.

Technical solutions

The present invention uses high-purity PCC as the experimental material, through freeze-drying, dry-heat virus inactivation (100°C, 30min), activity measurement, and comparative test, to obtain 2 kinds and 8 groups of protective agents for high-purity PCC dry-heat virus inactivation treatment ,Specific steps are as follows:

a) Preparation of high-purity PCC

The mixed plasma of healthy people was centrifuged at 2-4°C to remove cryoprecipitate, and the plasma supernatant was used as raw material to prepare high-purity PCC by using expanded bed technology. The collected eluate was ultrafiltered, desalted, concentrated and tested for activity with 0.01M sodium citrate, 0.5% NaCl, pH7.0 ultrafiltration buffer in an ultrafiltration concentration system to obtain high-purity PCC experimental materials.

The high-purity PCC composition feature of above-mentioned preparation is:

Types of coagulation factors Specific activity (IU/mg) coagulation factor II 3.5-6.0 coagulation factor VII 3.5-6.0 coagulation factor IX 3.5-6.0 coagulation factor X 3.5-6.0

b) Add protective agent

Properly adjust the activity of blood coagulation factor IX unit in the concentrated solution, add trehalose or/and histidine protective agent in a certain proportion, adjust the pH to 7.0, sterilize, filter and pack.

c) freeze drying

After pre-freezing at -40°C for 8 hours, the above subpackaged products were vacuum freeze-dried in a freeze dryer.

d) Dry heat virus inactivation

After lyophilization, the product was placed in a water bath, and the temperature was raised to 100°C for timing, and it was taken out after 30 minutes.

e) Activity assay

The products before and after dry heat virus inactivation were dissolved in water for injection, and the activity of blood coagulation factors was determined by a one-stage method for comparative analysis.

Beneficial effect

The beneficial effect of the present invention is to provide 2 kinds and 8 groups of protective agents for dry heat virus inactivation treatment of high-purity PCC, the activity yield of blood coagulation factor IX is higher than 74%, and the optimal protective agent combination dry heat virus inactivation After treatment, the activity recovery rates of the four coagulation factors II, VII, IX, and X can all reach more than 98%.

Detailed ways

The protective effect of the protective agent of the present invention on high-purity PCC during the dry heat virus inactivation process will be described in detail with reference to the examples, but the embodiments of the present invention are not limited thereto.

Example 1: Protection test of trehalose and histidine on the main effect of high-purity prothrombin complex coagulation factor IX

After the blank control product without protective agent was inactivated by dry heat at 100°C for 30 minutes, the recovery rate of coagulation factor IX was lower than 58%, while the products with different concentrations (1.5%, 2.5%) of trehalose as protective agent, Blood coagulation factor IX activity recovery rate is more than 75%; Add different concentrations (1.5%, 2.5%) histidine as the goods of protective agent, blood coagulation factor IX activity recovery rate is more than 74% (see table 1), illustrates that the present invention protects The agent can effectively protect the coagulation factor IX, which is the main role of high-purity PCC, under the condition of single factor.

Table 1 Effect of trehalose and histidine on recovery rate of high-purity PCC coagulation factor IX activity treated by dry heat virus inactivation

Example 2: Protective effect of trehalose and histidine on four coagulation factors of high-purity prothrombin complex

After the blank control product without protective agent was inactivated by dry heat, the recovery rate of the four coagulation factors was not more than 59%, while the product added with 2.0% trehalose as the protective agent had the activity recovery rate of the four coagulation factors at 81%. More than %; add 2.0% histidine to make the goods of protecting agent, four kinds of blood coagulation factor activity recoveries are all more than 76% (see table 2), illustrate that protecting agent of the present invention can effectively protect high-purity PCC under single factor condition four coagulation factors.

Table 2 Effects of trehalose and histidine on the activity recovery rate of four coagulation factors of high-purity PCC treated with dry heat virus inactivation

coagulation factor type blank control 2.0% trehalose 2.0% Histidine coagulation factor IX 57.9% 82.9% 79.5% coagulation factor II 58.6% 81.4% 84.4% coagulation factor VII 53.1% 85.2% 76.2% coagulation factor X 49.5% 91.8% 80.6%

Example 3: Protection test of trehalose or/and histidine+glycine on high-purity prothrombin complex mainly acting on blood coagulation factor IX

On the basis of 2.0% glycine, different concentrations (0.5%, 1.5%) of trehalose were added as protective agents, and the activity yield of blood coagulation factor IX was above 97% (see Table 3); on the basis of 2.0% glycine, different concentrations ( 0.5%, 1.5%) histidine as protective agent, the activity yield of blood coagulation factor IX was more than 76% (see Table 3), all higher than the 67.4% yield of 2.0% glycine control group. On the basis of 2.0% glycine, 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 is used as a protective agent, and the minimum active yield of blood coagulation factor IX is 92.8% (see Table 4), which is higher than the 2.0% glycine control group. During the heat inactivation process, the activity of coagulation factor IX, which is the main role of high-purity PCC, has a good protective effect.

Table 3 Effects of trehalose+glycine and histidine+glycine on the recovery rate of high-purity PCC blood coagulation factor IX activity treated by dry heat virus inactivation

Table 4 Effect of histidine+trehalose+glycine on the recovery rate of high-purity PCC coagulation factor IX activity treated by dry heat virus inactivation

coagulation factor type coagulation factor IX 2.0% glycine control 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%

Example 4: Protective effect of trehalose + histidine + glycine on four coagulation factors of high-purity prothrombin complex

On the basis of 2.0% glycine, add 0.5% histidine + 1.0% trehalose as protective agent, and the activity yield of the four coagulation factors is the lowest 98.4% (see Table 5); on the basis of 2.0% glycine, add 1.0% trehalose Glycine+0.5% trehalose is used as protective agent, and the activity yield of four kinds of blood coagulation factors is the lowest 95.3% (seeing table 6), and 2.0% glycine control group, the highest activity yield of four kinds of blood coagulation factors is 70.1% (seeing table 5 and Table 6), it can be seen that the protective agent trehalose and histidine of the present invention have better active protective effects on the four coagulation factors of high-purity PCC in the dry heat inactivation process.

Table 5 The influence of trehalose+histidine+glycine on the activity recovery rate of four coagulation factors of high-purity PCC treated with dry heat virus inactivation

coagulation factor type 2.0% glycine control 2.0% Glycine + 0.5% Histidine + 1.0% Trehalose coagulation factor IX 67.4% 113.5% coagulation factor II 70.1% 110.2% coagulation factor VII 57.6% 105.4% coagulation factor X 68.5% 98.4%

Table 6 The effect of trehalose+histidine+glycine on the activity recovery rate of four coagulation factors of high-purity PCC treated with dry heat virus inactivation

coagulation factor type 2.0% glycine control 2.0% Glycine + 1.0% Histidine + 0.5% Trehalose coagulation factor IX 67.4% 112.7% coagulation factor II 70.1% 108.6% coagulation factor VII 57.6% 95.3% coagulation factor X 68.5% 99.5%

Claims (3)

1. The use of trehalose or/and histidine as a high-purity prothrombin complex dry heat virus inactivation protection agent, the content of trehalose is 0.1%-8%, and the content of histidine is 0.1%-8%. 2. According to claim 1, the use of trehalose or/and histidine as a high-purity prothrombin complex dry heat virus inactivation protection agent is characterized in that: the prothrombin complex has four coagulation factors II, The specific activities of VII, IX, and X were all ≥3.5IU/mg. 3. The use of trehalose or/and histidine as a protective agent for dry heat virus inactivation of high-purity prothrombin complex according to claim 1, characterized in that: the protective agent also contains 0-10% glycine.