CN104181313A - Preparation method of blood coagulation factor IX quality control product - Google Patents

Abstract

The invention relates to a preparation method of a clinical blood coagulation test preparation, specifically a preparation method of a blood coagulation factor IX quality control product, which uses an anti-human blood coagulation factor IX monoclonal antibody immunoaffinity chromatography column to perform affinity chromatography on the mixed plasma of multiple parts to remove multiple blood coagulation factors. The blood coagulation factor IX in the mixed plasma of one person is the blood coagulation factor IX-deficient plasma; then the multiple people’s mixed plasma is mixed with the blood coagulation factor IX-deficient plasma according to a certain ratio to prepare blood coagulation factor IX substances with different concentrations of blood coagulation factor IX For the control product, a lyoprotectant was added, subpackaged, and freeze-dried to obtain a blood coagulation factor IX quality control product. The blood coagulation factor IX quality control product prepared by the present invention has good product uniformity, stability and stability after rethawing of the freeze-dried product, and can replace imported products for quality control of blood coagulation factor IX detection, which is beneficial to reduce detection costs and improve my country's ability to detect coagulation factor IX.

Description

Coagulation factor IX quality control substance preparation method

technical field

The invention relates to a preparation method of a clinical blood coagulation test preparation, in particular a preparation method of a blood coagulation factor IX quality control product.

Background technique

Determination of coagulation factor IX has important clinical significance. As the blood coagulation factor IX activity test project continues to expand to various fields in disease diagnosis and treatment, it is particularly important to use blood coagulation factor IX quality control products to conduct internal quality control and external quality assessment for blood coagulation factor IX activity detection. At present, there are many reagents and instruments for the detection of blood coagulation factor IX. Different reagents, detection instruments, and detection principles are different, resulting in great differences in the results. Regardless of the method used, internal quality control and external quality assessment are necessary means to ensure good repeatability and comparability of test results, and these quality assurance measures are inseparable from coagulation factor IX quality control products.

At present, the blood coagulation quality control substances used in clinical laboratories are usually multi-parameter blood coagulation quality control substances, and the abnormal values of the blood coagulation quality control substances are also abnormal except for the abnormal level of coagulation factor IX. At the same time, it is difficult to guarantee the effectiveness of the coagulation factor IX internal quality control and external quality assessment.

Domestic coagulation quality control products are almost monopolized by foreign reagent manufacturers. The products are expensive and have a single level, which cannot meet the needs of clinical testing with multiple activity levels.

Contents of the invention

In view of the above technical problems, the present invention provides a method for preparing a blood coagulation factor IX quality control product and obtaining a blood coagulation factor IX quality control product. The blood coagulation factor IX quality control product prepared by the method of the present invention has multiple concentration levels of blood coagulation factor IX, and the content of other blood coagulation factors is in the normal range; the product has good uniformity and stability, and meets the clinical requirements for the blood coagulation factor IX quality control product inspection. Quality control requirements.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions: a method for preparing a blood coagulation factor IX quality control product, comprising the following steps:

(1) Add and mix the blood collected from human veins and anticoagulant at a volume ratio of 9:1, centrifuge at 2°C to 8°C for 15 minutes, and the relative centrifugal force is 2500g, separate and absorb the upper layer of plasma, and separate the plasma from several people Mixing, that is, multiple servings of mixed plasma; the anticoagulant is trisodium citrate solution, and its concentration is 0.109mol/L to 0.129mol/L.

(2) The immunoaffinity chromatography column is pre-balanced with a buffer solution at 10°C to 18°C. The buffer solution includes 0.02mol/L Tris, 0.15mol/L NaCl, 8mmol/L MgCl ₂ , pH7.25.

(3) After adding MgCl ₂ to the raw plasma, the concentration of MgCl ₂ in the raw plasma is 4.5-5.5 mmol/L. Put on the immunoaffinity chromatography column, and collect the plasma flowing out from the bottom of the immunoaffinity chromatography column, which is plasma lacking coagulation factor IX.

(4) By mixing the blood coagulation factor IX-deficient plasma and the mixed blood plasma of multiple parts according to the required ratio, the blood coagulation factor IX quality control products with different concentration levels can be prepared.

(5) The prepared blood coagulation factor IX quality control substance was added into a lyoprotectant, subpackaged, and freeze-dried. The added lyoprotectant includes 0.5-1.5% Hepes, 1-5% glycine, and 0.5-5% mannitol whose mass proportion accounts for the coagulation factor IX quality control product.

The preparation method of the blood coagulation factor IX quality control product provided by the present invention uses an anti-human blood coagulation factor IX monoclonal antibody immunoaffinity chromatography column to perform affinity chromatography on the mixed plasma of multiple parts to remove the coagulation factor in the mixed plasma of multiple parts IX is blood coagulation factor IX-deficient plasma; then multiple parts of mixed plasma and coagulation factor IX-deficient plasma are mixed according to a certain ratio to prepare coagulation factor IX quality control products with different concentrations of coagulation factor IX, and add lyoprotectant , aliquoted, and freeze-dried to obtain a blood coagulation factor IX quality control substance.

In the preparation method provided by the present invention, the preparation method of the anti-human coagulation factor IX monoclonal antibody immunoaffinity chromatography column is to couple a highly specific monoclonal antibody linked to coagulation factor IX to CNBr-activated Sepharose4B Immunoaffinity chromatography column on agarose gel. In the presence of Mg ²⁺ , when plasma passes through the immunoaffinity chromatography column, only coagulation factor IX is adsorbed, and the activities of other coagulation factors remain basically unchanged.

The blood coagulation factor IX quality control product prepared by the present invention has good product uniformity, stability and stability after rethawing of the freeze-dried product, and can replace imported products for quality control of blood coagulation factor IX detection, which is beneficial to reduce detection costs and improve my country's ability to detect coagulation factor IX.

Detailed ways

In conjunction with embodiment, further set forth the present invention:

The embodiment takes the preparation of a blood coagulation factor IX quality control product with an activity of 30% as an example.

(1) Add and mix the blood collected from human veins and anticoagulant at a volume ratio of 9:1, centrifuge at 2°C to 8°C for 15 minutes, and the relative centrifugal force is 2500g, separate and absorb the upper layer of plasma, and separate the plasma from several people Mixing, that is, multiple servings of mixed plasma; the anticoagulant is trisodium citrate solution, and its concentration is 0.109mol/L to 0.129mol/L.

(2) The immunoaffinity chromatography column is pre-balanced with a buffer solution at 10°C to 18°C. The buffer solution includes 0.02mol/L Tris, 0.15mol/L NaCl, 8mmol/L MgCl ₂ , pH7.25.

(3) After adding MgCl ₂ to the raw plasma, the concentration of MgCl ₂ in the raw plasma is 4.5-5.5 mmol/L. Put on the immunoaffinity chromatography column, and collect the plasma flowing out from the bottom of the immunoaffinity chromatography column, which is plasma lacking coagulation factor IX.

(4) Mixing the blood coagulation factor IX-deficient plasma and the mixed blood plasma of multiple parts according to the required ratio, and preparing the blood coagulation factor IX quality control product with the activity of blood coagulation factor IX at a concentration level of about 30%.

(5) Add the prepared coagulation factor IX quality control substance into the lyoprotectant. Protectants include 1% Hepes, 2% Glycine, 2% Mannitol. Aliquot and freeze-dry.

Product performance inspection test:

(1) Product activity detection

Use the Sysmex CA-1500 automatic coagulation instrument and the supporting coagulation factor IX detection reagent to detect the activity of the blood coagulation factor IX quality control product in the embodiment.

Table 1 coagulation factor activity determination table of coagulation factor IX quality control product

Test items II V VII x VIII IX XI XII Factor activity (%) 99.8 92.6 98.3 96.5 93.4 1.2 100.5 93.2

It can be seen from the above table that the product obtained by the preparation method has good specificity, and can specifically remove blood coagulation factor IX without substantially affecting the content of other blood coagulation factors.

(2) Determination of product uniformity

10 of the coagulation factor IX quality control products prepared in the examples were randomly selected for accurate reconstitution and reconstitution.

With Sysmex CA-1500 automatic coagulation instrument and supporting coagulation factor IX detection reagent, the activity of the blood coagulation factor IX quality control product of the embodiment is detected, and the results are as follows in order: percent activity (%) 29.9, 30.2, 30.0, 29.8, 29.0, 29.5, 30.3, 30.1, 30.2, 29.7, with an average of 29.9.

Get 3 bottles of the blood coagulation factor IX quality control product prepared in the example, carry out accurate reconstitution and reconstitution, detect FIX activity continuously 10 times after mixing, the results are as follows: percent activity (%) 29.4, 30.5, 29.9, 29.8, 30.0, 30.5 , 30.6, 30.1, 30.3, 29.9; average 30.1.

After calculation, the repeatability CV value between CV bottles of the coagulation factor IX quality control product bottle is 0.39%, which shows that this level of blood coagulation factor IX quality control product reaches the general requirements of clinical trials for the uniformity of quality control products (bottles in batches). Between CV≤5.0%).

(3) Stability testing of freeze-dried products

The blood coagulation factor IX quality control substance and SIEMENS P quality control substance prepared in the example were stored at 37°C, and samples were taken at 1 day, 3 days, 5 days, and 7 days respectively, and accurate reconstitution was carried out at the same time.

Use Sysmex CA-1500 automatic coagulation instrument and supporting coagulation factor IX detection reagent to detect the activity of the blood coagulation factor IX quality control product of the embodiment, the results are as follows:

Table 2 Stability of freeze-dried products at 37°C

Example The coagulation factor IX quality control product was placed at 37°C for 7 days, and the activity fluctuation range before and after placement was <5%, which was equivalent to the commercially available imported SIEMENS abnormal quality control plasma. It can be considered that the coagulation factor VIII quality control product prepared by the present invention was placed at 37°C for 7 days. The sky is stable.

(4) Product reconstitution stability testing

The blood coagulation factor IX quality control product and SIEMENS P quality control product prepared in the example were accurately reconstituted and reconstituted, divided into two parts and stored at 2°C-8°C and room temperature (20°C-25°C), and kept at regular intervals Samples were taken for coagulation factor IX activity testing.

Use Sysmex CA-1500 automatic coagulation instrument and supporting coagulation factor IX detection reagent to detect the activity of the blood coagulation factor IX quality control product of the embodiment, the results are as follows:

Table 3 reconstitution stability

Example The blood coagulation factor IX quality control product was reconstituted and placed at 2°C to 8°C and room temperature. There was no significant change in FIX:C between 24 hours and 0 hours, and the activity fluctuation range was less than 5%, which was abnormal with the commercially available imported SIEMENS. Quality control plasma was comparable. It can be considered that the product can remain stable within 24 hours after reconstitution at 2°C to 8°C and at room temperature. It can be seen that the blood coagulation factor IX prepared by the present invention has better stability when opening a bottle.

The above is only an example of a concentration level, which is a preferred embodiment of the present invention. The present invention protects the entire technical process and products. It should be pointed out that for those of ordinary skill in the art, without departing from the present invention Under the premise of the principle, some improvements and modifications can also be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (4)

1. blood coagulation factor IX quality control product preparation method, it is characterized in that: comprise the following steps: (1) Add and mix blood collected from human veins and anticoagulant at a volume ratio of 9:1, centrifuge at 2°C to 8°C, separate and absorb the upper layer of plasma, and mix the upper layer of plasma after separation of several people, that is Multiple servings of pooled plasma; (2) The immunoaffinity chromatography column is pre-balanced with buffer solution at 10°C to 18°C; (3) After adding MgCl ₂ to the raw plasma, the concentration of MgCl ₂ in the raw plasma is 4.5-5.5mmol/L, put it on the immunoaffinity chromatography column, and collect the plasma flowing out from the bottom of the immunoaffinity chromatography column, which means lack of coagulation factor IX plasma; (4) Mix the blood coagulation factor IX-deficient plasma with the mixed blood plasma of several people according to the required ratio, that is, prepare blood coagulation factor IX quality control products with different concentration levels; (5) The prepared blood coagulation factor IX quality control substance was added into a lyoprotectant, subpackaged, and freeze-dried. 2. The method for preparing blood coagulation factor IX quality control product according to claim 1, characterized in that: the anticoagulant is trisodium citrate solution with a concentration of 0.109mol/L-0.129mol/L. 3. The method for preparing blood coagulation factor IX quality control product according to claim 1, characterized in that: said buffer solution comprises 0.02mol/L Tris, 0.15mol/L NaCl, 8mmol/L MgCl ₂ , pH7.25. 4. The preparation method of blood coagulation factor IX quality control product according to claim 1, characterized in that: said lyoprotectant comprises 0.5-1.5% Hepes, 1-5% of blood coagulation factor IX quality control product in mass proportion Glycine, 0.5-5% mannitol.