CN107467013A - Preparation method of quality control product universal plasma matrix capable of being stored for long time - Google Patents

Abstract

The invention discloses a preparation method of a quality control product universal plasma matrix capable of being stored for a long time, wherein the universal plasma matrix comprises but is not limited to plasma which can be used for preparing a blood coagulation quality control product, an anticoagulant drug quality control product and an antithrombin quality control product. The invention also relates to a freeze-drying protective agent and a freeze-drying process of the universal plasma. The freeze-drying protective agent is prepared by mixing polyalcohol, amino acid and inorganic salt according to a certain proportion.

Description

Preparation method of quality control product universal plasma matrix capable of being stored for long time

Technical Field

The invention belongs to the technical field of biological detection. More particularly, the invention relates to a preparation method of a general quality control product plasma matrix.

Background

Cardiovascular and cerebrovascular diseases are major diseases which harm the health of the public in modern society and live at the first position of various causes of death. The cardiovascular and cerebrovascular diseases have the characteristics of high morbidity, high disability rate, high mortality rate, high recurrence rate and many complications. According to statistics, more than 2.7 hundred million patients with cardiovascular and cerebrovascular diseases in China die of nearly 300 million patients with cardiovascular and cerebrovascular diseases every year, and accounts for more than 30% of the total causes of death every year in China. Therefore, it is very important to prevent the cardiovascular and cerebrovascular diseases.

The core pathological symptom of cardiovascular and cerebrovascular diseases is the formation of thrombus, and the blood coagulation-hemostasis function is an important self-protection mechanism of the organism. The blood coagulation function test mainly comprises plasma Prothrombin Time (PT), Fibrinogen (FIB), Activated Partial Thromboplastin Time (APTT) and plasma Thrombin Time (TT), and the blood coagulation function test can be used for detecting whether an internal and external blood coagulation path is abnormal or not, whether hyperfibrinolysis and thrombosis exist or not, and is a necessary monitoring item for the screening of a blood coagulation mechanism before operation, the early diagnosis of a thrombotic disease and the monitoring of thrombolytic therapy. In order to prevent thrombosis, patients need to use anticoagulant drugs, which mainly comprise common unfractionated heparin, low molecular weight heparin, fondaparinux, rivaroxaban, edoxaban, apixaban and the like, and some patients have weak reaction to antithrombotic drugs and still have strong blood coagulation tendency, and others have obvious reaction to drugs, can generate bleeding and have serious life risks. Therefore, monitoring of antithrombotic agents is very important. Antithrombin assays are commonly used to measure antithrombin activity in a patient to assist the clinician in determining whether the patient has normal anticoagulant function and in determining the appropriate amount of anticoagulant medication to use.

When the PT, APTT, FIB, anticoagulation drug concentration monitoring and antithrombin activity test are clinically carried out, corresponding quality control products are needed to ensure the reliability of detection. The matrix of the quality control products is blood plasma, the chemical components of the blood plasma contain 90-92% of water, and the solute mainly contains blood plasma protein, which is the most main solid component in the blood plasma and has the content of 60-80 g/L. In order to prevent the specific spatial conformation of the plasma from being changed under the action of certain physical and chemical factors, thereby causing the change of the physicochemical property and the loss of the biological activity of the plasma, the vacuum freeze-drying method is optimally adopted to freeze and store the plasma. The freeze-dried blood plasma can be stored for a long time at normal temperature, occupies small space, has light weight, is convenient to transport, and is easy to rehydrate again to recover the activity.

The freeze drying of plasma refers to a drying method in which a plasma solution is frozen at a low temperature, then is subjected to sublimation drying under a vacuum condition to remove ice crystals, and is subjected to desorption drying after sublimation is finished to remove part of bound water. Various stresses exist throughout the freeze-drying process, including low temperature stresses, freezing stresses (including dendritic ice crystal formation, increased ionic strength, pH change, phase separation, etc.), and drying stresses (removal of a monolayer of water molecules from the surface of the protein), which are often factors that directly or indirectly lead to protein denaturation. In order to maintain the normal function of plasma, it is often necessary to add a certain amount of protective agent during the freeze-drying process.

Suitable lyoprotectants are important in order to obtain the desired lyophilized product. The freeze-drying protective agent can be generally divided into polyhydroxy compounds, sugar, amino acid, alcohol, protein, polymer, inorganic salt and the like, and not only needs to have the effect of protecting the biological activity of a product, but also needs to have freeze-drying excipient function, but any single protective agent hardly has all the characteristics, and a proper freeze-drying protective agent needs to be screened according to the product characteristics.

The freeze-drying protective agent has good protective effect in the freezing process, the drying process and the long-term storage process of freeze-dried products

During freezing of the product, the more accepted "preferential action" mechanism is believed that the protein solution preferentially interacts with water before the maximum freezing concentration is reached, while the protectant is preferentially rejected at the protein region. This is due to the addition of the protectant which increases the surface tension of the water molecules, causing the protein molecules to preferentially interact with the water molecules. In this case, the outer surface of the protein molecule has relatively more water molecules and relatively fewer protective molecules than in its bulk phase, thus also preserving the native conformation of the protein.

The "glassy state hypothesis" is a relatively well-recognized protection mechanism during drying. During the drying of the solution containing the protective agent, the mixture of the protective agent and the active ingredient forms a glass state when the concentration is sufficiently high and the crystallization of the protective agent does not occur. Glass states are classified into two types, i.e., strong glass and weak glass. Lowering the temperature below the glass transition temperature increases the viscosity of the weak glass more rapidly than that of the strong glass. Thus, excipients form a weak glass much better than a strong glass. Sucrose and trehalose are very protective because they form a weak glass.

The time scale occurring during drying, which causes protein deterioration, is hours, while for storage the time scale is months or years. In a proper freeze-drying process, the product temperature is required to be close to its glass transition temperature and the ambient temperature should be much lower than its glass transition temperature under proper storage conditions to obtain a long relaxation time.

In addition to suitable lyoprotectants, the lyophilization process is another factor in determining whether a lyophilized product is successful. In order to obtain the optimal freeze-drying process, several key factors such as the pre-freezing temperature and time, the main drying temperature and time, and the analytical drying temperature and time of the product need to be screened. The pre-freezing can adopt slow freezing or quick freezing, and the slow freezing has the advantages that the freeze-dried block of the obtained product is loose and has good re-solubility, and the defect that the structure of the product is easy to damage, thereby influencing the property of the product. The quick freezing has the advantages of better maintaining the property of the product to be freeze-dried and has the defects of relatively compact structure and poor re-solubility of the freeze-dried block after freeze-drying. When the primary drying temperature is established, the eutectic point of the product to be lyophilized is an important reference factor. The eutectic point can be obtained by a resistance method. The temperature of the second drying is generally the highest temperature which can be endured by the product to be freeze-dried, so that the performance of the freeze-dried product can be well protected, and the bound water in the product can be removed to the greatest extent. The time of each factor involved in the freeze-drying process must be groped and verified according to the appearance reconstitution performance of the freeze-dried block in the freeze-drying process.

Disclosure of Invention

In view of the above problems, it is an object of the present invention to provide a method for preparing a quality control plasma matrix which can be stored stably for a long period of time. In order to achieve the purpose, the technical scheme provided by the invention is as follows:

a method for preparing a quality control plasma matrix, the method comprising the steps of:

(a) preparing blood plasma;

(b) adding a lyoprotectant to the plasma obtained in step (a); and

(c) freeze-drying the plasma mixture obtained in step (b), thereby obtaining the quality control plasma matrix. The lyophilized quality control plasma matrix can be reconstituted with a suitable liquid (e.g., water) at the time of use.

In a preferred embodiment of the method of the present invention, the quality control substance plasma matrix is used for the preparation of a coagulation quality control substance, an antithrombin quality control substance and an anticoagulant drug quality control substance, preferably wherein the quality control substance is used for detecting coagulation function, including plasma Prothrombin Time (PT), Fibrinogen (FIB), Activated Partial Thromboplastin Time (APTT) and plasma Thrombin Time (TT).

In another preferred embodiment of the method of the present invention, said anticoagulant drug is selected from the group consisting of: common unfractionated heparin (e.g., molecular weight of 15000), low molecular weight heparin (e.g., molecular weight of 4000 to 6500), fondaparinux, rivaroxaban, edoxaban, and apixaban; or the antithrombin is antithrombin III.

In another preferred embodiment of the method of the invention, said plasma is obtained by centrifugation after mixing the collected blood, optionally with a citric acid solution, in step (a). The blood may be collected from normal healthy persons, or may be obtained commercially.

In another preferred embodiment of the method of the present invention, the lyoprotectant is a mixture of amino acids, polyols and inorganic salts; preferably wherein the amino acids are selected from the group consisting of: glycine, arginine, histidine and combinations thereof, preferably glycine, more preferably glycine added to plasma at a final concentration of 5-40g/L, most preferably at a final concentration of 20 g/L; and/or preferably, wherein the polyols are selected from the group consisting of: arabitol, mannitol, sorbitol and combinations thereof, preferably mannitol, more preferably mannitol is added to plasma at a final concentration of 20-100g/L, most preferably at a final concentration of 50 g/L; and/or preferably, wherein the inorganic salt is selected from the group consisting of: sodium chloride, potassium chloride and combinations thereof, preferably potassium chloride, more preferably potassium chloride added to plasma at a final concentration of 1-20g/L, most preferably at a final concentration of 10 g/L.

In another preferred embodiment of the process of the present invention, the freeze-drying in step (c) comprises the steps of:

A) freezing the plasma mixture obtained in step (b) to-50 to-35 ℃ for at least 1 hour (e.g., 1 to 12 hours, 1, 2, 3, 4, 5, 6, 7, 8 hours) to obtain a pre-frozen plasma mixture;

B) heating the pre-frozen blood plasma mixture obtained in the step A) at a temperature of-45 to-25 ℃ for 2 to 4 hours under a vacuum condition;

C) heating the blood plasma mixture heated in the step B) for 8-15 hours at the temperature of-30 to-25 ℃ under the vacuum condition;

D) heating the plasma mixture heated in the step C) at a temperature of-10 ℃ for 10-60 min under a vacuum condition;

E) heating the plasma mixture heated in the step D) at the temperature of 5-10 ℃ for 3-5 hours under a vacuum condition;

F) heating the plasma mixture heated in the step E) at the temperature of 10-37 ℃ for 10-60 min under a vacuum condition; and

G) and F) preserving the heat of the plasma mixture obtained in the step F) for 1-2 hours at the temperature of 20-37 ℃ under the vacuum condition to obtain a freeze-dried plasma mixture.

In a further preferred embodiment of the process according to the invention, the vacuum (vacuum conditions) in step B) and step C) is 200-; and/or the vacuum in steps D), E), F) and G) (vacuum conditions) is 0 to 10 mbar.

In another preferred embodiment of the method of the present invention, the freezing in step A) is in a quick freezing form, and the cooling rate is 100-130 ℃/h.

In another preferred embodiment of the process of the invention, the rate of temperature rise in step B) is 5 to 10 ℃/h; the heating time in step C) is 10 hours; the temperature rise rate in the step D) is 50-100 ℃/h; the heating time in step E) was 4 hours; the temperature rise rate in the step F) is 50-100 ℃/h; and/or the incubation time in step G) is 1.5 hours.

The invention also provides a quality control plasma matrix obtainable according to the method of the invention.

In a particular embodiment, the present invention provides the following aspects:

plasma acquisition: using a CTAD blood collection tube, venous blood was collected by venipuncture as 9: 1 and citric acid anticoagulant (0.11mol/L) are mixed evenly, and then are centrifuged for 15min at 18 ℃ within 1h under the centrifugal force of at least 2500g, and plasma is obtained through separation. The plasma here is normal human plasma.

The invention aims to prepare a general plasma matrix which can be stably stored for a long time by a vacuum freeze drying mode. In the preparation process, a proper freeze-drying protective agent and a freeze-drying process are key factors for the success of the preparation of the blood plasma.

Adding a freeze-drying protective agent: adding lyophilized protectant glycine, mannitol, and potassium chloride into blood plasma, wherein the glycine concentration is preferably 5-40g/L, and the optimal concentration is 20 g/L. The preferable concentration of mannitol is 20-100g/L, and the optimal concentration is 50 g/L. The preferred concentration of potassium chloride is 1-20g/L, with the most preferred concentration being 10 g/L.

The freeze-drying scheme of the prepared plasma matrix comprises three stages of pre-freezing, main drying and resolution drying. In the pre-freezing stage, a quick freezing method is adopted, the pre-freezing temperature is preferably-50 ℃ to-35 ℃, and the optimal temperature is-40 ℃. The pre-freezing temperature reduction rate is 100-130 ℃/h, and the temperature is kept for 1 hour to ensure that all liquid is frozen into solid. The temperature of the eutectic point of the prepared plasma is-28 ℃ by using a resistance method, the temperature of primary drying is preferably-45 to-25 ℃, the optimal primary drying temperature is-30 ℃, the temperature rising rate is 5-10 ℃/h, and the holding time is 8-15 hours. And (4) analyzing and drying the components, namely, increasing the temperature to 5-10 ℃ at a heating rate of 50-100 ℃/h, and keeping the temperature for 3-5 hours. And secondly, raising the temperature to 20-37 ℃ at a temperature raising rate of 50-100 ℃/h, and keeping the temperature for 2-3 hours.

Drawings

FIG. 1 is a follow-up monitoring of activity change of low molecular weight heparin quality control product for 15 months.

Detailed Description

The present invention will be described more fully with reference to the following examples and comparative examples. Unless otherwise indicated, all reagents and equipment were commercially available and were operated according to the instructions.

The present invention will be further described with reference to specific examples, but the present invention should not be construed as being limited thereto.

Example 1

Preparation of a Universal plasma matrix

Obtaining of plasma: using a CTAD blood collection tube, venous blood was collected by venipuncture as 9: 1 and citric acid anticoagulant (0.11mol/L) are mixed evenly, and then are centrifuged for 15min at 18 ℃ within 1h under the centrifugal force of at least 2500g, and plasma is obtained through separation. The blood sampling object is a healthy normal person.

Adding a lyoprotectant to the plasma: 20g/L of glycine, 50g/L of mannitol and 10g/L of potassium chloride. Subpackaging into 1 ml/bottle.

The freeze-drying process provided by the invention adopts a SmartLyo 2 freeze dryer of Youey company. The specific process comprises the following steps:

A) freezing the plasma to-45 deg.C to obtain prefreezed plasma, and maintaining for 1 hr; B) heating the pre-frozen plasma obtained in step A) at-30 ℃ for 3 hours under vacuum; C) heating the plasma heated in the step B) at the temperature of-30 ℃ for 10 hours under vacuum; D) heating the plasma heated in the step C) at the temperature of 5 ℃ for 60min under vacuum condition; E) heating the plasma heated in the step D) at the temperature of 5 ℃ for 4 hours under vacuum condition; F) heating the plasma heated in the step E) at the temperature of 25 ℃ for 60min under vacuum condition; and G) keeping the heated blood plasma in the step F) at the temperature of 25 ℃ for 1.5 hours under vacuum conditions to obtain a freeze-dried product. The vacuum in step B) and step C) was 200 mbar; and/or the vacuum in steps D), E), F) and G) is 10 mbar. The temperature rise rate in step B) is 8 ℃/h; the temperature rise rate in step D) is 80 ℃/h; the temperature increase rate in step F) was 80 ℃/h.

In the step A), semi-pressing, feeding into a cabinet at normal temperature, and reducing the temperature of the plasma to-45 ℃ within 30min by adopting a quick-freezing mode (the temperature reduction rate is 100-130 ℃/h). Under the condition of quick freezing, the plasma can be kept in the original state, the internal components can be kept in the original biological activity better, the blood plasma can be kept for 1 hour, and the whole blood plasma is guaranteed to be frozen. The eutectic point of the prepared plasma is about-28 ℃, the main drying temperature is-30 ℃, and the temperature rise process from pre-freezing to main drying is adopted, because a large amount of water exists in the plasma, if the temperature rises too fast, the bottle spraying phenomenon is easy to cause, if the temperature rises too slow, the energy waste is caused, and according to the experimental result, the temperature rise rate is most suitable under the condition of 5-10 ℃/h. The main drying phase was maintained for 10 hours to ensure that all free water in the plasma was removed. The desorption drying adopts a mode of 2 times of sublimation. In the process of 2 times of sublimation, the temperature of 2 times of sublimation drying is gradually increased, and the stability of the final freeze-dried product can be improved. The first sublimation is carried out, the heating rate is 80 ℃/h, the temperature is kept for 4 hours, the second sublimation is carried out, the heating rate is 80 ℃/h, and the temperature is kept for 1.5 hours.

Comparative example

To further illustrate the present invention, the same lyophilization process as in example 1 was employed except that plasma without adding a lyoprotectant was used, and the obtained lyophilizate was compared with the lyophilizate obtained in example 1 of the present invention. The results of the comparison are shown in Table 1 below.

By comparing the two freeze-dried products, the freeze-drying protective agent adopted by the invention has good protective effect on freeze-dried blood plasma.

Example 2

And evaluating PT, APTT and FIB performances of quality control plasma.

After reconstitution of the lyophilized product prepared according to example 1, PT, APTT and FIB (PT, APTT, FIB measurement kit, purchased from shanghai sun biotechnology limited) were tested 20 times each, and the results of the test and the homogeneity of the lyophilized product were observed. See table 2, below. The results show that the freeze-dried products PT, APTT and FIB are all in qualified ranges, have good uniformity and can meet the requirements of clinical quality control.

Example 3

And evaluating the antithrombin III performance of the quality control plasma.

After reconstitution of the lyophilized product prepared according to example 1, antithrombin iii (AT iii assay kit, available from shanghai sun biotechnology limited) was tested 20 times, and the results of the test were observed for acceptability and homogeneity of the lyophilized product, and are shown in the following table 3. The results show that the freeze-dried antithrombin III product has within the range of the detection results of normal people, has good uniformity and can meet the requirements of clinical quality control.

Example 4

For anticoagulant drugs, plasma is used as a substrate to prepare quality control products, and antithrombin III is the most important influencing factor in plasma. The plasma matrix capable of ensuring the stability of the antithrombin III is the basic requirement for preparing the quality control product matrix of the anticoagulant drug.

Using a low molecular weight heparin quality control as an example, plasma matrices supplemented with protective agents were prepared as in example 1, to which various amounts of enoxaparin (from a commercial source) were added) The potency was measured by the anti-Xa assay kit disclosed in Chinese patent CN 104048931A, and the results are shown in Table 4 below. The prepared quality control product is lyophilized by the lyophilization process provided in example 1. The results were examined after lyophilization and are shown in table 4. According to the detection results, the activity of the low molecular weight heparin quality control product is well maintained before and after freeze-drying, and the uniformity of the quality control product is good. Can meet the clinical requirements on quality control.

Example 5

Long-term stability tracking was performed on the quality controls of examples 2, 3 and 4 above. The test is carried out several times in 3 months, and the test results are shown in the attached tables 5 and 6. As can be seen from the detection results in Table 5, the detection results of the PT, APTT and FIB items of the prepared quality control product still meet the requirements of normal people after being placed at 4 ℃ for 15 months. As can be seen from the detection results shown in the attached Table 6 and FIG. 1, the antithrombin III of the prepared quality control product still meets the requirements of normal people after being placed at 4 ℃ for 15 months. The prepared low molecular weight heparin quality control product is placed for one year, the activity change is within 8 percent, and the requirement of clinical quality control is met.

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Table 1 comparison of the lyophilisates of the invention with the lyophilisates of the comparative examples

	The freeze-dried product of the invention	Comparative example
			Appearance of lyophilized product	The outer surface is smooth	Rough outer surface
Reconstitution time	60s	180s
			Clarity of the product	Is very clear	Slightly turbid

TABLE 2 PT, APTT, FIB quality control test results

Item	Number of detections	Results	Normal range	CV％
					PT	20	14.4±0.13(s)	25-37s	0.82
APTT	20	31.5±0.28(s)	11-14s	0.92
					FIB	20	3.2±0.08	2-4g/L	2.3

TABLE 3 antithrombin III quality control test results

Item	Number of detections	Results	Normal range	CV％
					Antithrombin III	20	106.5％±3.5％	108.5％+5.3％	1.65

TABLE 4 detection results of low-molecular heparin quality control products

TABLE 5 Long-term stability tracking and monitoring results of PT, APTT and FIB quality control products

Storage time (moon)	PT(s)	APTT(s)	FIB(g/L)
				0	12.1	24.2	2.52
3	12.2	24.1	2.53
				6	12.1	24.3	2.55
9	12.3	24.2	2.55
				12	12.5	24.1	2.56
15	12.4	24.3	2.56

TABLE 6 Long-term stability tracking and monitoring results of antithrombin III quality control product and low-molecular-weight heparin quality control product

Claims (10)

1. A method for preparing a quality control plasma matrix, the method comprising the steps of:

(a) preparing blood plasma;

(b) adding a lyoprotectant to the plasma obtained in step (a); and

(c) freeze-drying the plasma mixture obtained in step (b), thereby obtaining the quality control plasma matrix.

2. The method of claim 1, wherein the quality control plasma matrix is used in the preparation of a coagulation quality control product, an antithrombin quality control product, and an anticoagulant drug quality control product, preferably wherein the quality control product is used to detect coagulation function, including plasma Prothrombin Time (PT), Fibrinogen (FIB), Activated Partial Thromboplastin Time (APTT), and plasma Thrombin Time (TT).

3. The method of claim 2, wherein said anticoagulant drug is selected from the group consisting of: common unfractionated heparin, low molecular weight heparin, fondaparinux, rivaroxaban, edoxaban, and apixaban; or the antithrombin is antithrombin III.

4. A process according to any one of claims 1 to 3, wherein the plasma is obtained by centrifugation after mixing the collected blood, optionally with a citric acid solution, in step (a).

5. The method of any one of claims 1 to 4, wherein the lyoprotectant is a mixture of amino acids, polyols, and inorganic salts; preferably wherein the amino acids are selected from the group consisting of: glycine, arginine, histidine and combinations thereof, preferably glycine, more preferably glycine added to plasma at a final concentration of 5-40g/L, most preferably at a final concentration of 20 g/L; and/or preferably, wherein the polyols are selected from the group consisting of: arabitol, mannitol, sorbitol and combinations thereof, preferably mannitol, more preferably mannitol is added to plasma at a final concentration of 20-100g/L, most preferably at a final concentration of 50 g/L; and/or preferably, wherein the inorganic salt is selected from the group consisting of: sodium chloride, potassium chloride and combinations thereof, preferably potassium chloride, more preferably potassium chloride added to plasma at a final concentration of 1-20g/L, most preferably at a final concentration of 10 g/L.

6. The method according to any one of claims 1 to 5, wherein the freeze-drying in step (c) comprises the steps of:

A) freezing the plasma mixture obtained in step (b) to-50 to-35 ℃ for at least 1 hour to obtain a pre-frozen plasma mixture;

B) heating the pre-frozen blood plasma mixture obtained in the step A) at a temperature of-45 to-25 ℃ for 2 to 4 hours under a vacuum condition;

C) heating the blood plasma mixture heated in the step B) for 8-15 hours at the temperature of-30 to-25 ℃ under the vacuum condition;

D) heating the plasma mixture heated in the step C) at a temperature of-10 ℃ for 10-60 min under a vacuum condition;

E) heating the plasma mixture heated in the step D) at the temperature of 5-10 ℃ for 3-5 hours under a vacuum condition;

F) heating the plasma mixture heated in the step E) at the temperature of 10-37 ℃ for 10-60 min under a vacuum condition; and

G) and F) preserving the heat of the plasma mixture obtained in the step F) for 1-2 hours at the temperature of 20-37 ℃ under the vacuum condition to obtain a freeze-dried plasma mixture.

7. The process as claimed in claim 6, wherein the vacuum in steps B) and C) is 200 and 300 mbar; and/or the vacuum in steps D), E), F) and G) is 0 to 10 mbar.

8. The method as claimed in claim 6 or 7, wherein the freezing in step A) is in the form of quick freezing, and the cooling rate is 100-130 ℃/h.

9. The process according to any one of claims 6 to 8, wherein the ramp rate in step B) is 5-10 ℃/h; the heating time in step C) is 10 hours; the temperature rise rate in the step D) is 50-100 ℃/h; the heating time in step E) was 4 hours; the temperature rise rate in the step F) is 50-100 ℃/h; and/or the incubation time in step G) is 1.5 hours.

10. A quality control plasma matrix obtainable according to the method of any one of claims 1 to 9.