An activated prothrombin complex aPCC, its preparation method and application

Abstract

This invention provides an activated prothrombin complex (aPCC), its preparation method, and its application, belonging to the field of hemostatic drug preparation technology. This invention involves crude extraction of cryoprecipitated plasma using DEAE-Sephadex A-50 gel, SD virus inactivation, and further purification using DEAE-Sephadex A-50 gel. Activation is then performed after crude extraction or further purification to prepare activated prothrombin complex (aPCC). The activated prothrombin complex (aPCC) prepared by this invention exhibits high FEIBA activity, all above 25 IU / mL, with a maximum reaching 101.64, demonstrating good hemostatic effect; it also has low FIIa activity, all below 2 IU / mL, significantly reducing the risk of thrombosis in patients after use.

Description

Technical Field

[0001] This invention relates to the field of hemostatic drug preparation technology, and in particular to an activated prothrombin complex aPCC, its preparation method and application. Background Technology

[0002] Human prothrombin complex concentrates (PCC) are prepared from mixed plasma of healthy individuals. They primarily contain four vitamin K-dependent coagulation factors: factor II (FII), factor VII (FVII), factor IX (FIX), and factor X (FX), as well as small amounts of anticoagulation factors. They possess certain coagulation activity and are mainly used to treat factor IX deficiency (hemophilia B), and deficiencies of factors II, VII, and X. Activated prothrombin complex concentrates (aPCC) are prepared by activating the coagulation factors, especially FVII, within PCC. aPCC mainly contains large amounts of FVIIa, FII, FVII, FIX, and FX, as well as small amounts of activated FIIa, FIXa, and FXa. aPCC preparations are mainly used for the treatment of bleeding in patients with hemophilia A who are hemophilia A patients. They exert their hemostatic effect by supplementing the coagulation factors required in the exogenous or common coagulation pathway to form thrombin, thereby avoiding FVIII. They also have good efficacy in anticoagulant reversal, surgical and traumatic bleeding.

[0003] aPCCs have a complex composition, containing various activated and inactivated coagulation factors. Therefore, the process control of the activation step during preparation is extremely important, and controlling the activation conditions is crucial for the preparation of aPCCs. An excessive amount of activated coagulation factors (especially FIIa and FXa) in aPCCs poses a potential risk of thrombosis; conversely, an insufficient amount of activated coagulation factors in aPCCs affects their hemostatic function. FVIII bypass activity (FEIBA activity) is a standardized reference indicator for evaluating aPCCs, established through collaboration between international regulatory agencies and multi-center laboratories. It reflects the drug's ability to bypass the FVIII coagulation pathway and initiate coagulation, and is an important indicator for evaluating aPCC function. High FVIII bypass activity (FEIBA activity) indicates good hemostatic function.

[0004] Therefore, the development of aPCC concentrate with good hemostatic function and low thrombosis risk is crucial for the research and development of hemostatic drugs. Summary of the Invention

[0005] The purpose of this invention is to provide an activated prothrombin complex aPCC, its preparation method and application. The obtained aPCC product has both high FEIBA activity and good hemostatic effect, as well as low FIIa activity, and the risk of thrombosis is also significantly reduced.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing activated prothrombin complex aPCC, comprising the following steps:

[0008] (1) After removing cryoprecipitate plasma and adjusting protein concentration and pH, the plasma was adsorbed, washed and eluted by DEAE-SephadexA-50 gel to obtain crude PCC extract.

[0009] (2) PCC crude extract is processed and activated to obtain PCC activator, and the activation can be performed before or after the processing.

[0010] (3) PCC activator was ultrafiltration dialysis and concentration to obtain activated prothrombin complex aPCC.

[0011] Further, in step (1), the adjustment of protein concentration is as follows: the protein concentration of the de-chirpedoprecipitated plasma is adjusted to 3%-5% using physiological saline and sodium acetate buffer, and the pH is 6.8-7.2; the equilibration solution used for DEAE-Sephadex A-50 gel adsorption contains 0.012 mol / L sodium citrate and 0.1 mol / L NaCl, with a pH of 6.8-7.2; the washing solution used for washing contains 0.02 mol / L sodium citrate and 0.15 mol / L NaCl, with a pH of 6.8-7.2; the elution solution used for elution contains 0.0075-0.015 mol / L sodium citrate and 2.4 mol / L NaCl, with a pH of 6.6-7.0.

[0012] Furthermore, in step (2), the treatment is ultrafiltration dialysis, S / D virus inactivation, and re-purification; the activation method is: adding an activator for activation, and EDTA sodium terminating the activation after the activation is completed.

[0013] Furthermore, the activator is CaCl2, the final concentration of the activator is 5-10 mM, the activation temperature is 12-25°C, and the activation time is 8-32 h.

[0014] Furthermore, the final concentration of the sodium EDTA is 5–10 mM, and the activation termination time is 20–40 min.

[0015] Furthermore, in step (2), the ultrafiltration dialysis is performed until the conductivity is below 14 ms / cm; the method for inactivating the S / D virus is as follows: add S / D inactivating agent to make the final concentrations 0.1% Tween80 and 0.3% tributyl phosphate, and inactivate the S / D virus at a constant temperature of 24°C for 6-8 hours.

[0016] Further, in step (2), the re-purification is performed using DEAE-Sephadex A-50 gel for adsorption, washing, and elution. The purification conditions are as follows: the equilibration solution used for DEAE-Sephadex A-50 gel adsorption contains 0.02 mol / L sodium citrate and 0.1 mol / L NaCl, with a pH of 6.8-7.2; the washing solution used for washing contains 0.02 mol / L sodium citrate and 0.16 mol / L NaCl, with a pH of 6.8-7.2; and the eluent used for elution contains 0.0075-0.015 mol / L sodium citrate and 2.4 mol / L NaCl, with a pH of 6.6-7.0.

[0017] Furthermore, in step (3), the ultrafiltration is performed until the conductivity is below 11 ms / cm, and the concentration is performed until the protein concentration is 2.0%-4.0%.

[0018] The present invention also provides an activated prothrombin complex aPCC prepared by the above-described method.

[0019] The present invention also provides an application of the above-mentioned activated prothrombin complex aPCC in the preparation of hemostatic drugs.

[0020] The method for preparing activated prothrombin complex aPCC provided by this invention has the following advantages compared with the prior art:

[0021] This invention involves crude extraction of cryoprecipitated plasma using DEAE-Sephadex A-50 gel, SD virus inactivation, and further purification using DEAE-Sephadex A-50 gel. Activation is then performed after crude extraction or further purification to prepare activated prothrombin complex aPCC. The activated prothrombin complex aPCC prepared by this invention exhibits high FEIBA activity, all above 25 IU / mL, with a maximum reaching 101.64 IU / mL, demonstrating good hemostatic effect; it also has low FIIa activity, all below 2 IU / mL, significantly reducing the risk of thrombosis in patients after use. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a flowchart illustrating the process of first activating and then processing the activated prothrombin complex aPCC according to the present invention;

[0024] Figure 2 The flowchart for the preparation of activated prothrombin complex aPCC by pretreatment and then activation is shown in this invention. Detailed Implementation

[0025] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0026] This invention is based on Figure 1 , 2 The provided flowchart describes the preparation of activated prothrombin complex aPCC, which exhibits high FEIBA activity, good hemostatic effect, and low FIIa activity, significantly reducing the risk of thrombosis in patients after use. The specific steps include:

[0027] (1) After removing cryoprecipitate plasma and adjusting protein concentration and pH, the plasma was adsorbed, washed and eluted by DEAE-SephadexA-50 gel to obtain crude PCC extract.

[0028] (2) PCC crude extract is processed and activated to obtain PCC activator, and the activation can be performed before or after the processing;

[0029] (3) PCC activator was ultrafiltration dialysis and concentration to obtain activated prothrombin complex aPCC.

[0030] Example 1

[0031] The method for preparing activated prothrombin complex aPCC in Example 1 includes the following steps:

[0032] (1) The protein concentration of the de-crystal plasma was adjusted to 3% with physiological saline (0.9% NaCl), and the pH was adjusted to 6.8 with sodium acetate buffer (0.02M, pH 6.8). The plasma was equilibrated with DEAE-Sephadex A-50 gel with equilibration buffer of 0.012 mol / L sodium citrate and 0.1 mol / L NaCl at pH 7.0. The de-crystal plasma was added to the equilibrated gel and stirred for 50 min for adsorption. After adsorption, it was washed twice with washing buffer of 0.02 mol / L sodium citrate and 0.15 mol / L NaCl at pH 7.0 for 20 min each time. Then it was eluted twice with elution buffer of 0.01 mol / L sodium citrate and 2.4 mol / L NaCl at pH 7.0 for 15 min each time to obtain the crude PCC extract.

[0033] (2) Add calcium chloride to the crude PCC extract to a final concentration of 5 mM, activate at 16°C for 24 h, add sodium EDTA to a final concentration of 5 mM, stir for 30 min to stop the activation, and then dialyze using an ultrafiltration concentrator with 0.01 mol / L sodium citrate as the dialysate and pH 7.0 until the conductivity is below 13 mS / cm to obtain the PCC activated product.

[0034] (3) S / D inactivating agent was added to the PCC activator to achieve final concentrations of 0.1% Tween 80 and 0.3% tributyl phosphate, respectively. The PCC activator was inactivated by S / D virus inactivation at 24℃ for 6 hours. After inactivation, the PCC activator was purified again by adsorption, washing, and elution using DEAE-Sephadex A-50 gel, equilibration buffer, washing buffer, and elution buffer. The activator was then equilibrated with 0.02 mol / L sodium citrate and 0.1 mol / L NaCl at pH 7.0. The live organism was added to a well-equilibrated gel and stirred for 50 min for adsorption. After adsorption, it was washed twice with washing buffer of 0.02 mol / L sodium citrate and 0.16 mol / L NaCl at pH 7.0 for 20 min each time. Then it was eluted twice with elution buffer of 0.01 mol / L sodium citrate and 2.4 mol / L NaCl at pH 7.0 for 15 min each time. After purification, it was ultrafiltered and dialyzed until the conductivity was 11 mS / cm. The protein concentration was concentrated to 2.0% to obtain activated prothrombin complex aPCC.

[0035] The FEIBA and FIIa activities of the activated prothrombin complex aPCC prepared in Example 1 are shown in Table 1.

[0036] Table 1. FEIBA and FIIa activities of activated prothrombin complex aPCC in Example 1

[0037] sample Incubation time (h) FEIBA activity (IU / ml) FIIa activity (IU / ml) Before incubation 0 <0.32 <0.3 After incubation 24 22.15 0.78

[0038] Example 2

[0039] The method for preparing activated prothrombin complex aPCC in Example 2 includes the following steps:

[0040] (1) The protein concentration of the de-crystal plasma was adjusted to 4% with physiological saline (0.9% NaCl), and the pH was adjusted to 6.8 with sodium acetate buffer (0.02M, pH 6.8). The plasma was equilibrated with DEAE-Sephadex A-50 gel with 0.012 mol / L sodium citrate and 0.1 mol / L NaCl at pH 7.0. The de-crystal plasma was added to the equilibrated gel and stirred for 50 min for adsorption. After adsorption, it was washed twice with washing buffer of 0.02 mol / L sodium citrate and 0.15 mol / L NaCl at pH 7.0 for 20 min each time. Then it was eluted twice with elution buffer of 0.01 mol / L sodium citrate and 2.4 mol / L NaCl at pH 7.0 for 15 min each time to obtain the crude PCC extract.

[0041] (2) Add calcium chloride to the crude PCC extract to a final concentration of 5.5 mM, activate at 12°C for 32 h, add sodium EDTA to a final concentration of 5.5 mM, stir for 20 min, terminate the activation, and ultrafilter dialyze until the conductivity is 12 ms / cm to obtain the PCC activator.

[0042] (3) S / D inactivating agent was added to the PCC activator to achieve final concentrations of 0.1% Tween 80 and 0.3% tributyl phosphate, respectively. The PCC activator was inactivated by S / D virus inactivation at 24℃ for 6 hours. After inactivation, the PCC activator was purified again by adsorption, washing, and elution using DEAE-Sephadex A-50 gel, equilibration buffer, washing buffer, and elution buffer. The activator was then equilibrated with 0.02 mol / L sodium citrate and 0.1 mol / L NaCl at pH 7.0. The live organism was added to a well-equilibrated gel and stirred for 50 min for adsorption. After adsorption, it was washed twice with washing buffer of 0.02 mol / L sodium citrate and 0.16 mol / L NaCl at pH 7.0 for 20 min each time. Then it was eluted twice with elution buffer of 0.01 mol / L sodium citrate and 2.4 mol / L NaCl at pH 7.0 for 15 min each time. After purification, it was ultrafiltered and dialyzed until the conductivity was 10 mS / cm. The protein concentration was concentrated to 2.0% to obtain activated prothrombin complex aPCC.

[0043] The FEIBA and FIIa activities of the activated prothrombin complex aPCC prepared in Example 2 are shown in Table 2.

[0044] Table 2. FEIBA and FIIa activities of activated prothrombin complex aPCC in Example 2

[0045] sample Incubation time (h) FEIBA activity (IU / ml) FIIa activity (IU / ml) Before incubation 0 <0.32 <0.3 After incubation 32 23.91 0.89

[0046] Example 3

[0047] The method for preparing activated prothrombin complex aPCC in Example 3 includes the following steps:

[0048] (1) The protein concentration of the de-precipitated plasma was adjusted to 3% and the pH to 6.8 with physiological saline (0.9% NaCl) and sodium acetate buffer (0.02M, pH 6.8). The plasma was then equilibrated with DEAE-Sephadex A-50 gel in equilibration buffer of 0.012 mol / L sodium citrate and 0.1 mol / L NaCl at pH 7.0. The de-precipitated plasma was added to the equilibrated gel and stirred for 50 min for adsorption. After adsorption, the plasma was washed twice with washing buffer of 0.02 mol / L sodium citrate and 0.15 mol / L NaCl at pH 7.0 for 20 min each time. The plasma was then eluted twice with elution buffer of 0.01 mol / L sodium citrate and 2.4 mol / L NaCl at pH 7.0 for 15 min each time to obtain the crude PCC extract.

[0049] (2) The crude PCC extract was dialyzed until the conductivity was 12 mS / cm, and then S / D inactivation agents were added to make the final concentrations 0.1% Tween 80 and 0.3% tributyl phosphate, respectively. The S / D virus was inactivated at 24℃ for 6 h. After inactivation, the PCC activator after S / D inactivation was adsorbed, washed, and eluted again using DEAE-Sephadex A-50 gel and equilibration buffer, washing buffer, and elution buffer: equilibration was performed with 0.02 mol / L sodium citrate and 0.1 mol / L NaCl, pH 7.0. The equilibrated gel was added to the crude PCC extract and stirred for 50 min for adsorption. After adsorption, the activator was purified with 0.02 mol / L sodium citrate and 0.16 mol / L tributyl phosphate. Wash twice with NaCl and a washing buffer at pH 7.0 for 20 min each time. Then elute twice with a 0.01 mol / L sodium citrate and 2.4 mol / L NaCl and a washing buffer at pH 7.0 for 15 min each time. Collect the eluent to obtain the PCC repurification solution.

[0050] (3) Add calcium chloride to the PCC repurification solution to a final concentration of 5mM, activate at 25℃ for 8h, add sodium EDTA to a final concentration of 5mM, stir for 30min, terminate the activation, ultrafilter dialyze to a conductivity of 10ms / cm, concentrate to a protein concentration of 4.0%, and obtain activated prothrombin complex aPCC.

[0051] The FEIBA and FIIa activities of the activated prothrombin complex aPCC prepared in Example 3 are shown in Table 3.

[0052] Table 3. FEIBA and FIIa activities of activated prothrombin complex aPCC in Example 3

[0053] sample Incubation time (h) FEIBA activity (IU / ml) FIIa activity (IU / ml) Before incubation 0 <0.32 <0.3 After incubation 8 101.64 1.09

[0054] Example 4

[0055] The method for preparing activated prothrombin complex aPCC in Example 4 includes the following steps:

[0056] (1) The protein concentration of the de-precipitated plasma was adjusted to 3% and the pH to 6.8 with physiological saline (0.9% NaCl) and sodium acetate buffer (0.02M, pH 6.8). The plasma was then equilibrated with DEAE-Sephadex A-50 gel in equilibration buffer of 0.012 mol / L sodium citrate and 0.1 mol / L NaCl at pH 7.0. The de-precipitated plasma was added to the equilibrated gel and stirred for 50 min for adsorption. After adsorption, the plasma was washed twice with washing buffer of 0.02 mol / L sodium citrate and 0.15 mol / L NaCl at pH 7.0 for 20 min each time. The plasma was then eluted twice with elution buffer of 0.01 mol / L sodium citrate and 2.4 mol / L NaCl at pH 7.0 for 15 min each time to obtain the crude PCC extract.

[0057] (2) After dialysis of the crude PCC extract until the conductivity is below 13 mS / cm, S / D inactivation agents are added to achieve final concentrations of 0.1% Tween 80 and 0.3% tributyl phosphate. The mixture is kept at 24°C for 8 hours to inactivate the S / D virus. After inactivation, the PCC activator after S / D inactivation is purified again by adsorption, washing, and elution using DEAE-Sephadex A-50 gel and equilibration buffer, washing buffer, and elution buffer. The mixture is then equilibrated with 0.02 mol / L sodium citrate and 0.1 mol / L NaCl at pH 7.0. The crude PCC extract is added to the equilibrated gel and stirred for 50 minutes for adsorption. After adsorption, the mixture is purified with 0.02 mol / L sodium citrate and 0.16 mol / L tributyl phosphate. Wash twice with NaCl and a washing buffer at pH 7.0 for 20 min each time. Then elute twice with a 0.01 mol / L sodium citrate and 2.4 mol / L NaCl and a washing buffer at pH 7.0 for 15 min each time. Collect the eluent to obtain the PCC repurification solution.

[0058] (3) Add calcium chloride to the PCC repurification solution to a final concentration of 10 mM, activate at 25°C for 8 h, add sodium EDTA to a final concentration of 10 mM, stir for 30 min, terminate the activation, ultrafilter dialyze until the conductivity is 11 ms / cm, concentrate to a protein concentration of 4.0%, and obtain activated prothrombin complex aPCC.

[0059] The FEIBA and FIIa activities of the activated prothrombin complex aPCC prepared in Example 4 are shown in Table 4.

[0060] Table 4. FEIBA and FIIa activities of activated prothrombin complex aPCC in Example 4

[0061] sample Incubation time (h) FEIBA activity (IU / ml) FIIa activity (IU / ml) Before incubation 0 <0.32 <0.3 After incubation 8 76.23 1.25

[0062] Example 5

[0063] Example 5 verifies the effects of activation after crude extraction and activation after re-purification on FEIBA and FIIa activities. The optimal conditions for activation after crude extraction are compared with the optimal conditions after re-purification, while all other conditions remain the same. This example involves activation of the crude extract, including the following steps:

[0064] (1) The protein concentration of the de-precipitated plasma was adjusted to 5% and the pH to 6.8 with physiological saline (0.9% NaCl) and sodium acetate buffer (0.02M, pH 6.8). The plasma was then equilibrated with DEAE-Sephadex A-50 gel in equilibration buffer of 0.012 mol / L sodium citrate and 0.1 mol / L NaCl at pH 7.0. The de-precipitated plasma was added to the equilibrated gel and stirred for 50 min for adsorption. After adsorption, the plasma was washed twice with washing buffer of 0.02 mol / L sodium citrate and 0.15 mol / L NaCl at pH 7.0 for 20 min each time. The plasma was then eluted twice with elution buffer of 0.01 mol / L sodium citrate and 2.4 mol / L NaCl at pH 7.0 for 15 min each time to obtain the crude PCC extract.

[0065] (2) Add calcium chloride to the crude PCC extract to a final concentration of 5mM, activate at 16℃ for 16h, add sodium EDTA to a final concentration of 5mM, stir for 30min, terminate the activation, and ultrafilter dialyze until the conductivity is 13ms / cm to obtain PCC activator.

[0066] (3) S / D inactivating agent was added to the PCC activator to achieve final concentrations of 0.1% Tween 80 and 0.3% tributyl phosphate, respectively. The PCC activator was inactivated by S / D virus inactivation at 24℃ for 6 hours. After inactivation, the PCC activator was purified again by adsorption, washing, and elution using DEAE-Sephadex A-50 gel, equilibration buffer, washing buffer, and elution buffer. The activator was then equilibrated with 0.02 mol / L sodium citrate and 0.1 mol / L NaCl at pH 7.0. The live organism was added to a well-equilibrated gel and stirred for 50 min for adsorption. After adsorption, it was washed twice with washing buffer of 0.02 mol / L sodium citrate and 0.16 mol / L NaCl at pH 7.0 for 20 min each time. Then it was eluted twice with elution buffer of 0.01 mol / L sodium citrate and 2.4 mol / L NaCl at pH 7.0 for 15 min each time. After purification, it was ultrafiltered and dialyzed until the conductivity was 11 mS / cm. The protein concentration was concentrated to 4.0% to obtain activated prothrombin complex aPCC.

[0067] The FEIBA and FIIa activities of the activated prothrombin complex aPCC prepared in Example 5 are shown in Table 5.

[0068] Table 5. FEIBA and FIIa activities of activated prothrombin complex aPCC in Example 5

[0069] sample Incubation time (h) FEIBA activity (IU / ml) FIIa activity (IU / ml) Before incubation 0 <0.32 <0.3 After incubation 16 43.53 0.93

[0070] Example 6

[0071] Example 6 verifies the effects of activation after crude extraction and activation after re-purification on FEIBA and FIIa activities. The optimal conditions for activation after crude extraction are compared with the optimal conditions after re-purification, while all other conditions remain the same. This example uses activation after re-purification, and the method is as follows:

[0072] A concentrated aPCC solution was prepared as a control group using a purification-activation method, as follows:

[0073] (1) The protein concentration of the de-precipitated plasma was adjusted to 5% and the pH to 6.8 with physiological saline (0.9% NaCl) and sodium acetate buffer (0.02M, pH 6.8). The plasma was then equilibrated with DEAE-Sephadex A-50 gel in equilibration buffer of 0.012 mol / L sodium citrate and 0.1 mol / L NaCl at pH 7.0. The de-precipitated plasma was added to the equilibrated gel and stirred for 50 min for adsorption. After adsorption, the plasma was washed twice with washing buffer of 0.02 mol / L sodium citrate and 0.15 mol / L NaCl at pH 7.0 for 20 min each time. The plasma was then eluted twice with elution buffer of 0.01 mol / L sodium citrate and 2.4 mol / L NaCl at pH 7.0 for 15 min each time to obtain the crude PCC extract.

[0074] (2) After dialysis of the crude PCC extract until the conductivity was 13 mS / cm, S / D inactivation agents were added to achieve final concentrations of 0.1% Tween 80 and 0.3% tributyl phosphate. The mixture was kept at 24°C for 6 hours to inactivate the S / D virus. After inactivation, the PCC activator was purified again by adsorption, washing, and elution using DEAE-Sephadex A-50 gel and equilibration buffer, washing buffer, and elution buffer. The mixture was equilibrated with 0.02 mol / L sodium citrate and 0.1 mol / L NaCl at pH 7.0. The crude PCC extract was added to the equilibrated gel and stirred for 50 minutes for adsorption. After adsorption, the mixture was purified with 0.02 mol / L sodium citrate and 0.16 mol / L... Wash twice with NaCl and a washing buffer at pH 7.0 for 20 min each time. Then elute twice with a 0.01 mol / L sodium citrate and 2.4 mol / L NaCl and a washing buffer at pH 7.0 for 15 min each time. Collect the eluent to obtain the PCC repurification solution.

[0075] (3) 5 mM CaCl2 was added to the purified solution, and the solution was activated at 16°C for 16 h. Activation was terminated with 5 mM sodium EDTA for 30 min. The terminated solution was subjected to ultrafiltration dialysis until the conductivity reached 11 mS / cm, and then concentrated to a protein concentration of 4.0% to obtain the aPCC concentrate of the control group. The FEIBA and FIIa activities of the activated prothrombin complex aPCC prepared in Example 6 are shown in Table 6. The components and FVIII bypass activity in the prepared aPCC concentrate of the control group were evaluated and compared with those in Example 5. The test results are shown in Table 7.

[0076] Table 6. FEIBA and FIIa activities of activated prothrombin complex aPCC in Example 6

[0077] sample Incubation time (h) FEIBA activity (IU / ml) FIIa activity (IU / ml) Before incubation 0 <0.32 <0.3 After incubation 8 78.21 1.82

[0078] Table 7. Components of PCC concentrate and FVIII bypass activity.

[0079]

[0080] As shown in Table 6, the FEIBA activity was higher in the aPCC concentrate prepared by the activation method after re-purification.

[0081] Example 7

[0082] Example 7 was designed to verify the effects of different activators on the activities of FEIBA and FIIa. The difference between Example 7 and Example 1 was the type of activator and the activation temperature. The results are shown in Table 7.

[0083] Table 7. Effects of different activators on FEIBA and FIIa activities.

[0084]

[0085]

[0086] As shown in Table 7, diatomaceous earth and kaolinite could not activate PCC under the experimental conditions, and therefore could not obtain aPCC with FVIII bypass activity. Only CaCl2 could activate PCC and obtain aPCC with relatively high FVIII bypass activity.

[0087] Example 8

[0088] Example 8 was designed to verify the effects of different activator concentrations, activation times, and activation temperatures on the activities of FEIBA and FIIa. The difference between Example 8 and Example 1 was the different activator concentrations, activation times, and activation temperatures. The results are shown in Table 8.

[0089] Table 8. Effects of activator concentration, activation time, and activation temperature on FEIBA and FIIa activities.

[0090]

[0091]

[0092] As shown in Table 8, the activator concentration, activation time, and activation temperature have extremely significant effects on the activities of FEIBA and FIIa.

[0093] Experimental Example 1

[0094] Experimental Example 1 compares the components and FVIII bypass activity of domestic PCC and the aPCC concentrate prepared in Example 5. The comparison results are shown in Table 9.

[0095] Table 9. Components and FVIII bypass activity in PCC concentrate.

[0096]

[0097] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing activated prothrombin complex aPCC, characterized in that, The steps include: (1) After removing the cryoprecipitate plasma and adjusting the protein concentration and pH, the plasma was adsorbed, washed and eluted using a DEAE-Sephadex A-50 gel to obtain a crude PCC extract. (2) PCC crude extract was processed and activated sequentially to obtain PCC activator; (3) PCC activator was ultrafiltration dialysis and concentration to obtain activated prothrombin complex aPCC; In step (1), the protein concentration adjustment is as follows: the protein concentration of the de-chirpedoprecipitated plasma is adjusted to 3%-5% using physiological saline and sodium acetate buffer, with a pH of 6.8-7.2; the equilibration buffer used for DEAE-Sephadex A-50 gel adsorption contains 0.012 mol / L sodium citrate and 0.1 mol / L NaCl, with a pH of 6.8-7.2; the washing buffer used for washing contains 0.02 mol / L sodium citrate and 0.15 mol / L NaCl, with a pH of 6.8-7.2; the elution buffer used for elution contains 0.01 mol / L sodium citrate and 2.4 mol / L NaCl, with a pH of 6.6-7.

0. In step (2), the treatment is ultrafiltration dialysis, S / D virus inactivation, and re-purification; the activation method is: adding an activator for activation, and EDTA sodium to terminate the activation after the activation is completed; In step (2), the ultrafiltration dialysis is performed until the conductivity is below 14 ms / cm; the method for inactivating the S / D virus is as follows: add S / D inactivating agent to make the final concentrations 0.1% Tween 80 and 0.3% tributyl phosphate, and inactivate the S / D virus at 24℃ for 6-8 hours. In step (2), the re-purification involves adsorption, washing, and elution using a DEAE-Sephadex A-50 gel. The purification conditions are as follows: the equilibration solution used for DEAE-Sephadex A-50 gel adsorption contains 0.02 mol / L sodium citrate and 0.1 mol / L NaCl, with a pH of 6.8-7.2; the washing solution used for washing contains 0.02 mol / L sodium citrate and 0.16 mol / L NaCl, with a pH of 6.8-7.2; and the eluent used for elution contains 0.01 mol / L sodium citrate and 2.4 mol / L NaCl, with a pH of 6.6-7.

0. In step (2), the activator is CaCl2, the final concentration of the activator is 5~10mM, the activation temperature is 12~25℃, and the activation time is 8~32h.

2. The method for preparing the activated prothrombin complex aPCC according to claim 1, characterized in that, In step (2), the final concentration of sodium EDTA is 5~10mM, and the time required to terminate the activation is 20~40min.

3. The method for preparing the activated prothrombin complex aPCC according to claim 1, characterized in that, In step (3), the ultrafiltration is performed until the conductivity is below 11 ms / cm, and the concentration is performed until the protein concentration is 2.0%-4.0%.

4. An activated prothrombin complex aPCC prepared by the method of any one of claims 1 to 3.

5. The use of the activated prothrombin complex aPCC as described in claim 4 in the preparation of hemostatic drugs.

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