CN102441172A

CN102441172A - High-purity prothrombin complex product freeze-drying stabilizer

Info

Publication number: CN102441172A
Application number: CN2011104004506A
Authority: CN
Inventors: 曹海军; 黄亮; 李长清; 何琦; 赵红卫; 邱家山; 林方昭; 李志雄
Original assignee: CHENGDU YINGDE BIO-ENGINEERING CO LTD; Sichuan University; Institute of Hematology and Blood Diseases Hospital of CAMS and PUMC; China Bluestar Chengrand Research Institute of Chemical Industry Co Ltd
Current assignee: CHENGDU YINGDE BIO-ENGINEERING CO LTD; Sichuan University; Institute of Hematology and Blood Diseases Hospital of CAMS and PUMC; China Bluestar Chengrand Research Institute of Chemical Industry Co Ltd
Priority date: 2011-12-06
Filing date: 2011-12-06
Publication date: 2012-05-09
Anticipated expiration: 2031-12-06
Also published as: CN102441172B

Abstract

The invention relates to the field of medicinal biotechnology, and in particular to a high-purity prothrombin complex product stabilizer capable of effectively preventing the inactivation of a blood coagulation factor IX with a main effect and blood coagulation factors II, VII and X in a product in a freeze-drying process, wherein the specific activity of each of four blood coagulation factors II, VII, IX and X is greater than or equal to 3.5IU/mg protein. The stabilizer provided by the invention contains arginine or/and trehalose or/and histidine or/and glycine, and also contains common sodium citrate and NaCl. The tests show that so long as the high-purity prothrombin complex contains 0.1-10% of arginine or/and 0.1-10% of trehalose or/and 0.1-10% of histidine or/and 0.1-10% of glycine, the activity of the blood coagulation factor IX and the activity of the blood coagulation factors II, VII and X can be effectively protected in the freeze-drying process. Thus, the stabilizer provided by the invention can be applied to the freeze-drying process of the high-purity prothrombin complex.

Description

The former composite article lyophilization of high-purity thrombase stabilizing agent

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) at freezing dry process, can effectively prevent the stabilizing agent of main effect plasma thromboplastin component and prothrombin in the goods, VII, X inactivation.In more detail; In the former complex of high-purity thrombase, add the 0.1%-10% arginine or/and the 0.1%-10% trehalose or/and the 0.1%-10% histidine, can effectively be protected the activity of goods plasma thromboplastin component and prothrombin, VII, X or/and the 0.1%-10% glycine in freezing dry process.

Background technology

Human Factor (Prothrombin Complex Concentrate; PCC) be from the healthy subjects pooled plasma, to separate a kind of blood products that can promote blood coagulation that is prepared into, mainly contain the synthetic prothrombin of vitamin K dependent, VII, IX, X.Clinically, PCC is mainly used in the hemophilia A of hemophilia B, the generation of proconvertin antibody and the hemorrhage treatment that is lowly caused by hepatic disease, the caused thrombin level of vitamin K deficiency.China is hepatitis B patient big country, adds other types hepatic disease, and the demand of PCC goods is had bigger patient colony, and along with the raising of clinical degree of recognition, in recent years, the PCC goods have bigger user demand.

From the 1950's PCC be used for clinical since, cause thrombotic report after venoclysis is constantly arranged, therefore, reducing the potential exploitation that causes the high-end PCC goods of bolt property generation is the research emphasis of these goods.High-end PCC goods should have less short bolt protein factor, and four kinds of prothrombins, VII, IX, X should have purity and equilibrium degree preferably.The PCC goods of domestic market molding at present, between 0.3-1.0IU/mg, purity is relatively low mostly for the specific activity of prothrombin, VII, IX, X.In addition, prothrombin, VII, IX, X equilibrium degree also remain to be improved, from certain meaning, and the hidden danger that domestic present PCC goods exist certain thrombosis side reaction to take place.

Thrombin is prone to the inactivation degeneration, and this based article needs lyophilization be treated as powder injection formulation in the preparation process for the ease of preserving, transporting.In refrigerating process, ice crystal is separated out from solution, and many factors such as spatial induction destruction that the goods salt ionic concentration increases sharply, salting-out process causes the variation of goods internal pH-values, refrigerating process again and cryogenic conditions all have negative effect to protein stability; In dry run, the factors such as direct exposure of vitrification of goods solution and glass transition, water sublimed hydrogen bond also cause proteinic degeneration easily, and for easy inactivation Denatured protein based article, freezing dry process must add certain stabilizing agent.

High-purity PCC protein content is less relatively; Requirement to stabilizing agent is more higher; So far do not see the report of relevant PCC lyophilization stabilizing agent, more do not see arginine or/and trehalose or/and histidine or/and glycine is used for high-purity PCC lyophilization Study of Stabilizers report.

Summary of the invention

The technical problem that solves

In order effectively to protect high-purity PCC coagulation factor protein in freezing dry process active; Reduce the loss of activity of thrombin; The present invention provides the stabilizing agent of several kinds and several groups high-purity PCC; Handle the activity that can protect prothrombin, VII, IX, X effectively through lyophilization, wherein the plasma thromboplastin component activity yield all＞70%.

Technical scheme

The present invention is a material with high-purity PCC, relatively obtains 3 kinds and 6 groups of high-purity PCC freezing drying protective agents through lyophilization, the comparison of shaping outward appearance, determination of activity, redissolution clarity, and concrete steps are following:

A) high-purity PCC preparation

Removing the cryoprecipitate supernatant with the healthy subjects pooled plasma is raw material, utilizes expansion bed technology preparation high-purity PCC.The eluting peak solution of collecting with contained 0.01M sodium citrate, 0.5%NaCl, the ultrafiltration in Millipore Labscale ultrafiltration and concentration system of pH7.0 ultrafiltration buffer, desalination, concentrated and determination of activity, is obtained high-purity PCC.

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

B) interpolation of lyophilization stabilizing agent

With the plasma thromboplastin component is standard, suitably adjusts concentrated solution coagulation factor activity unit to 9-15IU/ml, add arginine in proportion or/and trehalose or/and histidine or/and the glycine stabilizing agent, adjustment pH to 7.0-7.2, aseptic filtration, packing.

C) lyophilization

Goods after the packing-40 ℃ pre-freeze 6hr-9hr, the vacuum lyophilization of Labconco FreeZone freeze dryer is carried out lyophilization by Fig. 1 freeze-drying curve.

D) the shaping outward appearance relatively

Goods shaping outward appearance after the lyophilizing is compared.

E) determination of activity and analysis

Water for injection dissolving freeze-dried products, the first phase method is measured coagulation factor activity, relative analysis.

F) redissolve clarity relatively

Freeze-dried products clarity after redissolving is compared analysis.

Beneficial effect

The invention has the beneficial effects as follows; 3 kinds and 6 groups of high-purity PCC lyophilization stabilizing agents are provided; Prothrombin, VII, IX, X there is the better protect effect; Wherein the activity yield of plasma thromboplastin component is all more than 70%, and four kinds of coagulation factor activity response rate of optimum protective agent combination reach more than 84%.

The specific embodiment

In conjunction with embodiment stabilizing agent of the present invention is elaborated in the protective effect of freezing dry process to high-purity PCC, embodiment of the present invention is not limited thereto.

Embodiment 1: arginine, trehalose, histidine mainly act on the protection effect of plasma thromboplastin component to high-purity PCC

The blank goods that do not add any stabilizing agent are after lyophilization, and the plasma thromboplastin component response rate is 59.5%, and add the goods of variable concentrations (0.5%, 1.5%) trehalose used as stabilizers, and the plasma thromboplastin component activity recovery is more than 70%; Add the goods of variable concentrations (0.5%, 1.5%) arginine used as stabilizers, the plasma thromboplastin component activity recovery is more than 76%; Add the goods of variable concentrations (0.5%, 1.5%) histidine used as stabilizers; The plasma thromboplastin component activity recovery (is being seen table 1) more than 77%, explains that protective agent of the present invention can mainly act on the plasma thromboplastin component activity to high-purity PCC and protect under single factors.

Table 1 arginine, trehalose, histidine are to the influence of freezing dry process high-purity PCC plasma thromboplastin component activity recovery

Embodiment 2: glycine and glycine+trehalose or arginine or histidine are to the protection effect of four kinds of thrombins of high-purity PCC

The goods that only add 2% glycine stabilizing agent, after the lyophilization, four kinds of high activity yields of thrombin are 75.4%; On the basis of 2% glycine, add 0.5% trehalose stabilizing agent, four kinds of coagulation factor activity yields are minimum to be 81.2%; On the basis of 2% glycine, add 0.5% arginine stabilizing agent, four kinds of coagulation factor activity yields are minimum to be 84.6%; On the basis of 2% glycine, add 0.5% histidine stabilizing agent, four kinds of coagulation factor activity yields are minimum to be 83.5% (seeing table 2); In addition; Compare with 2% glycine; The goods lyophilizing shaping outward appearance, the clarity all better (seeing table 3) of redissolving that have trehalose, arginine, histidine to exist; Can find out, on the basis that glycine exists, trehalose, arginine, any equal can further effectively protection of histidine to four kinds of coagulation factor activities of high-purity PCC in the freezing dry process.

Table 2 glycine and glycine+trehalose or arginine or histidine are to the influence of four kinds of coagulation factor activity response rate of freezing dry process high-purity PCC

Table 3 glycine and glycine+trehalose or arginine or histidine are to the influence of high-purity PCC lyophilization postforming outward appearance, redissolution clarity

Type	Lyophilizing postforming outward appearance	Redissolve clarity after the lyophilizing
			The contrast of 2% glycine	Whole atrophy	Fine hair is arranged
2.0% glycine+0.5% trehalose	No atrophy is shaped	Clarification
			2.0% glycine+0.5% arginine	No atrophy is shaped	Clarification
2.0% glycine+0.5% histidine	No atrophy is shaped	Clarification

Embodiment 3: glycine and glycine+trehalose or/and arginine or/and histidine to the protection effect of four kinds of thrombins of high-purity PCC

Add 1.0% glycine+0.5% arginine+0.5% trehalose stabilizing agent, after the lyophilization, four kinds of coagulation factor activity yields are minimum to be 79.6%; Add 1.0% glycine+0.5% histidine+0.5% trehalose stabilizing agent, after the lyophilization, four kinds of coagulation factor activity yields are minimum to be 77.9%; Only add 1.0% glycine protective agent and be contrast, four kinds of coagulation factor activity yields are up to 69.2% (seeing table 4); In addition; There are arginine and trehalose or histidine and trehalose stabilizing agent to exist; Goods lyophilizing shaping outward appearance, the clarity of redissolving all are superior to glycine matched group (seeing table 5), can find out, on the basis that glycine exists; There are arginine or histidine and trehalose to exist, all can improve four kinds of thrombin stability of high-purity PCC in the freezing dry process.Table 4 glycine and glycine+trehalose or/and arginine or/and histidine to the influence of four kinds of coagulation factor activity response rate of freezing dry process high-purity PCC

Table 5 glycine and glycine+trehalose or/and arginine or/and histidine to high-purity PCC lyophilization postforming outward appearance, the influence of clarity of redissolving

Claims

Arginine or/and trehalose or/and histidine or/and glycine as the former complex lyophilization of high-purity thrombase stabilizing agent; Arginine content is 0.1%-10%; Content of trehalose is 0.1%-10%, and histidine content is 0.1%-10%, and glycine content is 0.1%-10%.
According to the said arginine of claim 1 or/and trehalose or/and histidine or/and glycine, is characterized in that four kinds of prothrombins of prothrombin complex, VII, IX, X are than the equal >=3.5IU/mg that lives as the former complex freezing dry process of high-purity thrombase stabilizing agent.
According to the said arginine of claim 1 or/and trehalose or/and histidine or/and glycine as the former complex freezing dry process of high-purity thrombase stabilizing agent, is characterized in that also containing in the stabilizing agent sodium citrate and NaCl.
According to the said arginine of claim 1 or/and trehalose or/and histidine or/and glycine as the former complex freezing dry process of high-purity thrombase stabilizing agent, is equally applicable to other kinds prothrombin complex goods.