A method for simultaneously separating canine fibrinogen and canine prothrombin complex from canine plasma

Abstract

The present invention discloses a method for simultaneously separating canine fibrinogen and canine prothrombin complex from canine plasma. First, rough separation and fine separation are carried out by gel column chromatography and anion column chromatography. Then, further purification is carried out by DEAE Sephadex A-50 gel adsorption. Through the combination of multiple steps of processes and their parameters, high-purity and high-yield canine fibrinogen, prothrombin and other components can be extracted from canine plasma simultaneously. The purity of both canine fibrinogen and canine prothrombin complex can reach over 95%, and the extraction rates of canine fibrinogen and canine prothrombin complex reach over 90% and over 85% respectively, greatly improving the comprehensive utilization rate of canine plasma. At the same time, by adjusting the formula and ratio of the stabilizer, the freeze-dried products of canine fibrinogen and canine prothrombin complex have good solubility, clear solutions, and short reconstitution times, which are 10-15 minutes and 5-10 minutes respectively. In summary, the method of the present invention has stable processes, high raw material utilization rate, and good product quality.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary drugs, and particularly relates to a method for simultaneously separating canine fibrinogen and canine prothrombin complex from canine plasma. Background Art

[0002] Fibrinogen (Fg) is a protein with blood coagulation function, and its main function is to promote blood coagulation. It is synthesized by the liver and is the most abundant of all blood coagulation factors in the human body. The content in human plasma is 2 - 4 g / L, and the half-life is 4 - 6 days. Lack of Fg will lead to various blood coagulation diseases, common ones including: congenital fibrinogen reduction or deficiency, and acquired fibrinogen deficiency caused by liver injury, cirrhosis, disseminated intravascular coagulation, external injury, postpartum hemorrhage, major surgery, internal hemorrhage, etc. For Fg reduction or deficiency caused by congenital factors, supplementing Fg is currently the only reliable treatment method, while for Fg reduction or deficiency caused by acquired factors, different doses of Fg need to be infused according to different causes.

[0003] Blood coagulation factors are various protein components participating in the blood coagulation process, which are activated during bleeding, adhere to platelets and plug the wounds on blood vessels. Currently, coagulation factor VII, VIII, IX and prothrombin complex (mainly containing coagulation factors II, IX, VII, X) have been developed into drugs in human medicine for the treatment of hemophilia A or B.

[0004] Currently, there are no available procoagulant products suitable for pet dogs and cats in pet clinical medicine. Canine plasma is a limited and precious resource, and the utilization level of canine plasma in China is still very low. Generally, only one product is extracted, such as the preparation method and product of canine fibrinogen disclosed in CN113214384 A. Not only a large amount of precious resources are discarded, but also the production cost of existing products is increased, the efficiency is low, and the waste is large, which is very regrettable. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for simultaneously separating canine fibrinogen and canine prothrombin complex from canine plasma, with stable process, high product purity, high yield, short reconstitution time, greatly improving the utilization rate of canine blood resources and reducing production costs.

[0006] The method for simultaneously separating canine fibrinogen and canine prothrombin complex from canine plasma of the present invention includes the following steps:

[0007] S1. Cryoprecipitation: Thaw fresh frozen plasma, centrifuge at 16000 r / min under the condition of 0℃ - 5℃, collect the supernatant and warm it up to 4℃ - 8℃, filter it with a 0.22 μm filter element, take the supernatant and set it aside;

[0008] S2, Inactivation: Take the supernatant from step S1 and inactivate it with S / D. Add Tween 80 to 1.0% and TNBP to 0.3% to the supernatant from step S1, stir well, then raise the temperature to 24 - 26°C, keep warm for 6 - 8 hours, and then filter through a 0.45μm filter membrane to collect the filtrate;

[0009] S3, Add the pre - equilibrated DEAE - Sephadex - A50 gel to the filtrate in step S2 for adsorption. The addition amount per liter of supernatant is 0.5g - 2.0g, mix well, and adsorb for 50 - 80 minutes; After the adsorption is completed, collect the liquid and the gel respectively;

[0010] S4, Wash the gel collected in step S3 with 2 - 3 times the gel volume of equilibration buffer 1 for 2 - 4 times, collect the flow - through liquid, and combine it with the liquid collected in step S3 for standby, which is used for preparing canine fibrinogen; Elute the gel with 1 - 3 times the gel volume of elution buffer 1 for 3 - 5 times, collect the eluate, which is used for preparing canine prothrombin complex; Equilibration buffer 1 is a 10 - 20mM citric acid - sodium citrate buffer containing 50 - 100mM sodium chloride, with a pH of 6.5 - 7.5; Elution buffer 1 is a 10 - 20mM citric acid - sodium citrate buffer containing 400 - 600mM sodium chloride, with a pH of 6.5 - 7.5;

[0011] S5, Concentrate the combined liquid collected in step S4 to obtain concentrate 1, with a protein content of 2 - 3mg / ml; Concentrate the collected eluate to obtain concentrate 2, with a protein content of 2 - 3mg / ml;

[0012] S6, First, fully equilibrate the anion - exchange column with equilibration buffer 2, then load concentrate 1 onto the column. During the loading process, control the flow rate at 30 - 40ml / h and collect the flow - through liquid; After the loading is completed, wash the column with 2 - 3 times the equilibration buffer 2 for 2 - 4 times, and combine the washing liquid with the flow - through liquid for standby, which is used for preparing fibrinogen; Filter the combined liquid through a 0.45μm filter membrane to collect the filtrate, and operate the filtrate according to steps S7A - S10A; Equilibration buffer 2 contains 0.01M - 0.02M TRIS, 10 - 20mM citric acid - sodium citrate, 0.1M - 0.3M arginine hydrochloride, and the rest is water, with a pH of 6.5 - 7.5;

[0013] Another anion exchange column was first fully equilibrated with equilibration solution 2, and then the concentrated solution 2 was loaded onto the column. During the loading process, the flow rate was controlled at 30 - 40 ml / h. After the loading was completed, the column was eluted with 1 - 3 times the volume of eluent 2 for 3 - 5 times, and the eluate was collected and reserved for the preparation of prothrombin complex; the eluate was filtered through a 0.45 μm filter element, and the filtrate was collected. The filtrate was operated according to steps S7B - S10B; eluent 2 contained 0.01M - 0.02M TRIS, 10 - 20 mM citric acid - sodium citrate, 400 mM - 600 mM sodium chloride, and the rest was water, with a pH of 6.50 - 7.50;

[0014] S7A. The combined solution collected in step S6 was adsorbed using DEAE - Sephadex - A50 as the gel medium;

[0015] S8A. Before the gel was loaded onto the column, it was equilibrated with equilibration solution 1, then loaded and adsorbed. The loading speed was 30 - 40 ml / h. After the loading was completed, the gel column was washed with 2 - 3 times the volume of equilibration solution 1 for 2 - 4 times, and the flow - through liquid and the washing liquid were collected and mixed evenly;

[0016] S9A. The mixed solution of S8A was ultrafiltered and concentrated with a 30KD ultrafiltration membrane until the protein content reached 53 mg / ml, and glycine, sucrose, Tween 80, and sodium chloride were added as stabilizers, so that the final concentration of glycine was 1% - 2%, the final concentration of sucrose was 4% - 6%, the final concentration of Tween 80 was 0.01% - 0.02%, and the final concentration of sodium chloride was 0.6% - 0.9%;

[0017] S10A. The liquid in step S9A was sterilized by filtration, aliquoted, freeze - dried, and capped, and dry - heat virus inactivation was used to obtain canine fibrinogen;

[0018] S7B. The eluate collected in step S6 was adsorbed using DEAE - Sephadex - A50 as the gel medium;

[0019] S8B. Before the gel was loaded onto the column, it was equilibrated with equilibration solution 1, then loaded and adsorbed. The loading speed was 30 - 40 ml / h. After the loading was completed, the gel column was eluted with 1 - 3 times the volume of eluent 1 for 3 - 5 times, and the eluate was collected;

[0020] S9B. The eluate of S8B was ultrafiltered and concentrated with a 10KD ultrafiltration membrane to make the titers of coagulation factor IX, II, and X not less than 200 IU / ml, and the titer of coagulation factor VII not less than 50 IU / ml. Then, glycine, sucrose, and Tween 80 were added as stabilizers, so that the final concentration of glycine was 1% - 2%, the final concentration of sucrose was 4% - 6%, and the final concentration of Tween 80 was 0.01% - 0.02%;

[0021] S10B, sterilizing and filtering the liquid in step S9B, packaging, freeze-drying, capping, and inactivating the virus by dry heat to obtain a prothrombin complex.

[0022] Furthermore, in step S3, the amount of gel added is 1.0 g / L serum, and the adsorption time is 70 min.

[0023] Furthermore, the gel in step S4 is washed three times with equilibration solution 1 having an amount twice the gel volume.

[0024] Furthermore, in step S4, the gel is eluted 4 times with elution solution 1 in an amount 3 times the volume of the gel.

[0025] Furthermore, in step S6, the anion exchange column is selected from Capto DAEA or Capto Q.

[0026] Furthermore, the flow rate during column loading in step S6 is 30 ml / h.

[0027] Furthermore, in the stabilizer of step S9A, the final concentration of glycine is 1%, the final concentration of sucrose is 4%, the final concentration of Tween 80 is 0.01%, and the final concentration of sodium chloride is 0.9%; or the final concentration of glycine is 1%, the final concentration of sucrose is 5%, the final concentration of Tween 80 is 0.02%, and the final concentration of sodium chloride is 0.6%; or the final concentration of glycine is 2%, the final concentration of sucrose is 6%, the final concentration of Tween 80 is 0.01%, and the final concentration of sodium chloride is 0.9%.

[0028] Furthermore, in the stabilizer of step S9B, the final concentration of glycine is 1%, the final concentration of sucrose is 4%, and the final concentration of Tween 80 is 0.01%; or the final concentration of glycine is 1%, the final concentration of sucrose is 5%, and the final concentration of Tween 80 is 0.02%; or the final concentration of glycine is 2%, the final concentration of sucrose is 4%, and the final concentration of Tween 80 is 0.02%; or the final concentration of glycine is 2%, the final concentration of sucrose is 6%, and the final concentration of Tween 80 is 0.01%.

[0029] Compared with the prior art, the technical effects of the present invention are:

[0030] The present invention provides a method for simultaneously separating canine fibrinogen and canine prothrombin complex from canine plasma. First, rough separation and fine separation are carried out by gel column chromatography and anion column chromatography, and then further purified by DEAE Sephadex A-50 gel adsorption respectively. Through the combination of multi-step processes and their parameters, high-purity and high-yield canine fibrinogen, prothrombin and other components are extracted from canine plasma simultaneously. The purity of canine fibrinogen and canine prothrombin complex can both reach over 95%, and the extraction rates of canine fibrinogen and prothrombin complex reach over 90% and over 85% respectively, greatly improving the comprehensive utilization rate of canine plasma. At the same time, by adjusting the formula and ratio of the stabilizer, the freeze-dried products of canine fibrinogen and canine prothrombin complex have good solubility, clear solution and short reconstitution time, which are 10-15 minutes and 5-10 minutes respectively. In summary, the method of the present invention has stable process, high raw material utilization rate and good product quality, filling the domestic blank of large-scale production of procoagulant products for pet dogs and cats. Brief Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings.

[0032] Figure 1 It is a flowchart of the method provided by the embodiment of the present invention. Detailed Embodiments

[0033] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with the embodiments and the drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[0034] The experimental methods in the following embodiments are all conventional methods unless otherwise specified.

[0035] The test materials used in the following embodiments are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0036] Example 1 Determination of Gel Addition Amount and Adsorption Time

[0037] Thaw the fresh frozen plasma, centrifuge it at 16,000 r / min under the condition of 0°C - 5°C, collect the supernatant and warm it up to 4°C - 8°C, filter it with a 0.22 μm filter element, and take the supernatant. Add Tween 80 to 1.0% and TNBP (tributyl phosphate) to 0.3% to the supernatant, stir well and then warm it up to 24 - 26°C, incubate for 6 - 8 hours for inactivation, and then filter it with a 0.45 μm filter element to collect the filtrate. Add the pre-equilibrated DEAE-Sephadex-A50 gel to the filtrate for adsorption. The addition amount of the gel is set as 0.25 g / L, 0.5 g / L, 1.0 g / L, 2.0 g / L, and the adsorption time for each gel amount is also set as 40 min, 50 min, 60 min, 70 min, 80 min. After the adsorption is completed, measure the contents of fibrinogen (Fg) and coagulation factors VII, II, IX, and IV in the liquid before and after adsorption, and determine the addition amount of the gel and the adsorption time through the adsorption rate.

[0038] Adsorption rate of Fg = (Fg content before adsorption - Fg content after adsorption) / Fg content before adsorption × 100%

[0039] Adsorption rate of each coagulation factor = (content of each coagulation factor before adsorption - content of each coagulation factor after adsorption) / content of each coagulation factor before adsorption × 100%

[0040] Screening criteria: The lower the adsorption rate of Fg, the better, and the higher the adsorption rate of each coagulation factor, the better.

[0041] The test results show (as shown in Table 1) that as the gel dosage increases and the adsorption time prolongs, the gel will adsorb part of Fg. The influence of the gel dosage from 0.25 g / L to 2.0 g / L and the adsorption time from 40 min to 80 min on Fg is not significant. When the gel addition amount is 0.25 g / L, as the adsorption time prolongs, the adsorption rates of each coagulation factor gradually increase, but the maximum adsorption rate does not reach more than 90%. When the gel addition amount is 0.5 g / L - 2.0 g / L, as the adsorption time prolongs, the adsorption rates of each coagulation factor gradually increase, and the adsorption rates are all above 85% when the adsorption time is from 50 min to 80 min. Therefore, it is determined that the amount of gel added is 0.5 g / L - 2.0 g / L of serum, and the adsorption time is 50 - 80 min.

[0042] Table 1 Determination of gel addition amount

[0043]

[0044] Example 2 Determination of the amount and number of washes of the gel washing solution

[0045] Thaw the fresh frozen plasma, centrifuge it at 16,000 r / min under the condition of 0°C - 5°C, collect the supernatant and warm it up to 4°C - 8°C, filter it with a 0.22 μm filter element, and take the supernatant. Add Tween 80 to 1.0% and TNBP (tributyl phosphate) to 0.3% to the supernatant, stir well and then warm it up to 24 - 26°C, incubate for 6 - 8 hours for inactivation, and then filter it with a 0.45 μm filter element to collect the filtrate. Add the pre-equilibrated DEAE-Sephadex-A50 gel to the filtrate for adsorption, set the addition amount of the gel to 1.0 g / L, and set the adsorption time of the gel amount to 70 min. After the adsorption is completed, filter it and take the filtrate for standby. Load the gel onto the column, and wash the column with washing solutions of 1 column volume, 2 column volumes, and 3 column volumes respectively, and each column volume is washed 1, 2, 3, 4, and 5 times respectively. Collect the washing solutions for each washing separately, and measure the fibrinogen content in the liquid collected each time for each column volume.

[0046] The test results show that the fibrinogen content is still very high at 0.11 mg when washing for the 5th time with 1 column volume, the fibrinogen contents are relatively low at 0.04 and 0.008 mg respectively when washing for the 4th and 5th times with 2 column volumes, and the fibrinogen contents are relatively low and all lower than 0.01 mg when washing for the 3rd, 4th, and 5th times with 3 column volumes. Therefore, it is determined to wash with 2 - 3 column volumes for 2 - 4 times. As shown in Table 2.

[0047] Table 2 Determination of the amount of washing solution and the number of washing times

[0048]

[0049] Example 3 Eluent - Determination of the amount and number of elution times of the eluent

[0050] Thaw the fresh frozen plasma, centrifuge it at 16,000 r / min under the condition of 0°C - 5°C, collect the supernatant and warm it up to 4°C - 8°C, filter it with a 0.22 μm filter element, and take the supernatant. Add Tween 80 to 1.0% and TNBP (tributyl phosphate) to 0.3% to the supernatant, stir well and then warm it up to 24 - 26°C, incubate for 6 - 8 hours for inactivation, and then filter it with a 0.45 μm filter element to collect the filtrate. Add the pre-equilibrated DEAE-Sephadex-A50 gel to the filtrate for adsorption, set the addition amount of the gel to 1.0 g / L, and set the adsorption time of the gel amount to 70 min. After the adsorption is completed, filter it and take the filtrate for standby. Load the gel onto the column, and wash the column with 2 column volumes of equilibration solution 1, wash it 3 times in total, and each washing solution is combined with the filtrate for standby.

[0051] After washing the gel column with the washing solution, elute it with the elution solution. Elute the column with 1 column volume, 2 column volumes, and 3 column volumes of the elution solution respectively. Each column volume is eluted 1, 2, 3, 4, and 5 times respectively. Collect the elution solution of each elution separately, and measure the contents of coagulation factor VII, II, IX, and IV in the liquid collected each time for each column volume.

[0052] Use the recovery rates of coagulation factor VII, II, IX, and IV as evaluation indicators.

[0053] Recovery rate of the first elution = Content of coagulation factor in the first elution solution / Content of the corresponding coagulation factor in plasma × 100%

[0054] Recovery rate of the second elution = (Content of coagulation factor in the first + second elution solutions) / Content of the corresponding coagulation factor in plasma × 100%

[0055] Recovery rate of the third elution = (Content of coagulation factor in the first + second + third elution solutions) / Content of the corresponding coagulation factor in plasma × 100%

[0056] Recovery rate of the fourth elution = (Content of coagulation factor in the first + second + third + fourth elution solutions) / Content of the corresponding coagulation factor in plasma × 100%

[0057] Recovery rate of the fifth elution = (Content of coagulation factor in the first + second + third + fourth + fifth elution solutions) / Content of the corresponding coagulation factor in plasma × 100%

[0058] Evaluation index: Compared with before adsorption, when the recovery rates of each coagulation factor reach over 90%, the elution times for each column volume can be determined.

[0059] The test results show that when eluting with 1 column volume, when eluting 5 times, the recovery rate of coagulation factor VII can reach over 90%, and the recovery rates of the other three coagulation factors are over 85%; when eluting with 2 column volumes, when washing 4 times, the recovery rate of coagulation factor VII can reach over 90%, and the recovery rates of the other three coagulation factors are over 85%; when eluting with 3 column volumes, when eluting 3 times, the recovery rate of coagulation factor VII can reach over 90%, and the recovery rates of the other three coagulation factors are over 85%. Based on the above analysis, determine the elution dosage as 1 - 3 column volumes and the elution times as 3 - 5 times. As shown in Table 3.

[0060] Table 3 Determination of elution solution dosage and elution times

[0061]

[0062]

[0063] Example 4 Anion Exchange Column - Determination of Flow Rate of Flowthrough

[0064] Thaw fresh frozen plasma, centrifuge at 16000r / min at 0℃~5℃, collect the supernatant and heat it to 4℃~8℃, filter it with a 0.22μm filter, and take the supernatant. Add Tween80 to 1.0% and TNBP (tributyl phosphate) to the supernatant to 0.3%, stir well, heat it to 24~26℃, keep it warm for 6-8 hours to inactivate, then filter it with a 0.45μm filter, and collect the filtrate. Add the equilibrated DEAE-Sephadex-A50 gel to the filtrate for adsorption, the amount of gel added is set to 1.0g / L, and the adsorption time of the gel is set to 70min. After the adsorption is completed, filter, take the filtrate, and set it aside. Put the gel on the column, wash the column with 2 times the column volume of equilibrium solution 1, wash it 3 times in total, and combine the washing solution with the filtrate each time for standby.

[0065] After washing the gel column with the washing solution, the column was eluted with the eluent. The amount of the eluent used was 3 times the column volume, and the number of washes was 4 times.

[0066] The collected washing liquid and eluate were concentrated and filtered respectively to make the protein content of them 2-3 mg / ml.

[0067] The concentrated solution of the eluate is chromatographed on an anion exchange column. The column is fully equilibrated with equilibration solution 2 in advance, and then loaded onto the column. The flow rates during loading are set to 20 ml / h, 30 ml / h, 40 ml / h, and 50 ml / h, respectively. The flow-through solution is collected, and the adsorption rate of coagulation factor VII in the flow-through solution is used as an evaluation index. If the flow rate is too low, the test time will be extended, and if the flow rate is too fast, the coagulation factor adsorption will be incomplete.

[0068] The test results show that when the flow rate is set to 20 ml / h, the test time is long due to the slow speed, and when the speed is set to 50 ml / h, the flow rate is too fast, resulting in a decrease in the adsorption rate of coagulation factor VII. Therefore, the optimal flow rate is determined to be 30-40 ml / h. As shown in Table 4.

[0069] Table 4 Determination of flow rate of flow-through liquid

[0070] Flow rate Adsorption rate of coagulation factor VII 20 ml / h 90.2％ 30 ml / h 91.5％ 40 ml / h 90.7％ 50 ml / h 80.4％

[0071] Example 5: Selection basis of freeze-drying protective agent and determination basis of dosage

[0072] Thaw fresh frozen plasma, centrifuge at 16,000 r / min under the condition of 0℃ - 5℃, collect the supernatant and warm it up to 4℃ - 8℃, filter it with a 0.22μm filter element, and take the supernatant. Add Tween80 to 1.0% and TNBP (tributyl phosphate) to 0.3% to the supernatant, stir well and then warm it up to 24 - 26℃, keep warm for 6 - 8 hours for inactivation, and then filter it with a 0.45μm filter element to collect the filtrate. Add the well - balanced DEAE - Sephadex - A50 gel to the filtrate for adsorption, set the addition amount of the gel to 1.0 g / L, and set the adsorption time of the gel amount to 70 min. After the adsorption is completed, filter it and take the filtrate for standby. Take the gel onto the column, wash the column with 2 column volumes of equilibration buffer 1 for 3 times in total, and each washing solution is combined with the filtrate for standby.

[0073] Concentrate the collected liquid washing solution and equilibration buffer, define it as concentrate 1, and its protein content is 2 - 3 mg / ml; concentrate the collected eluate, define it as concentrate 2, and its protein content is 2 - 3 mg / ml.

[0074] (1) Screening of fibrinogen lyoprotectant. First, fully equilibrate the anion - exchange column with equilibration buffer 2, and then load concentrate 1 onto the column. During the loading process, control the flow rate at 30 - 40 ml / h, collect the flow - through liquid. After the loading is completed, wash the column with 2 - 3 times of equilibration buffer 2 for 2 - 4 times, and combine the washing solution with the flow - through liquid for standby. Prepare fibrinogen. Filter the combined liquid with a 0.45μm filter element and collect the filtrate.

[0075] The filtrate is adsorbed with the gel medium DEAE - Sephadex - A50. The gel is equilibrated with equilibration buffer 1 before loading onto the column, sample and adsorb, and the sample loading speed is 30 - 40 ml / h. After the sample loading is completed, wash the gel column with 2 gel volumes of equilibration buffer 1 for 3 times, and collect the flow - through liquid and the washing solution and mix them evenly. Ultrafilter and concentrate the flow - through liquid and the washing solution with a 30KD ultrafiltration membrane until the protein content reaches 53 mg / ml, and then design the prescription for screening fibrinogen lyoprotectant according to the following table.

[0076] Screen glycine, sucrose, Tween80, and sodium chloride as the lyoprotectants of the product, design 9 test schemes with a three - factor and three - level orthogonal test, and evaluate the type and dosage of the selected lyoprotectants based on the appearance and solubility after lyophilization.

[0077] The test results show that: taking the appearance and solubility of the solution after lyophilization as the inspection indexes, the combinations of A2B1C2D3, A2B2C3D2, and A3B3C2D3 have good solubility for fibrinogen, that is, the content of glycine is 1% - 2%, the content of sucrose is 4% - 6%, the content of Tween80 is 0.01% - 0.02%, and the content of sodium chloride is 0.6% - 0.9%. As shown in Table 5 and Table 6.

[0078] Table 5 Factor levels of orthogonal test for fibrinogen

[0079]

[0080] Table 6 Fibrinogen orthogonal test results

[0081]

[0082]

[0083] (2) Screening of prothrombin complex lyophilization protective agent: Take another anion exchange column and fully equilibrate it with equilibration solution 2, then load the concentrate 2 onto the column. During the loading process, the flow rate is controlled at 30-40 ml / h. After the loading is completed, the column is eluted 4 times with 3 times eluent 2, and the eluent is collected for use to prepare the prothrombin complex. The eluent is filtered with a 0.45 μm filter element and the filtrate is collected.

[0084] The filtrate is adsorbed with DEAE-Sephadex-A50 as gel medium. The gel is balanced with balance solution 1 before loading, and the sample is adsorbed at a loading speed of 30-40ml / h. After loading, the gel column is eluted 3 times with eluent 1 of 2 times the gel volume, and the eluent is collected. The eluent is ultrafiltered and concentrated with a 10KD ultrafiltration membrane until the titer of factors IX, II, and X is not less than 200IU / ml, and the titer of factor VII is not less than 50IU / ml, and then the prescription of the prothrombin complex lyophilization protective agent is designed and screened according to the table below.

[0085] Glycine, sucrose and Tween 80 were selected as lyoprotectants for the product. Nine experimental schemes were designed with three factors and three levels in an orthogonal experiment. The type and dosage of the selected lyoprotectants were evaluated based on their appearance and solubility after lyophilization.

[0086] From the appearance and solubility of the freeze-dried solution, the combination of A2B1C2, A2B2C3, A3B1C3, and A3B3C2 has good solubility for the prothrombin complex, that is, the content of glycine is 1% to 2%, the content of sucrose is 4% to 6%, and the content of Tween 80 is 0.01% to 0.02%, as shown in Tables 7 and 8.

[0087] Table 7 Factor levels of orthogonal test for prothrombin complex

[0088]

[0089] Table 8 Prothrombin complex orthogonal test results

[0090]

[0091]

[0092] Example 6 Determination of the anion exchange column filler

[0093] Capo DAEA, Q-Sepharose FF, Q-Sepharose 4FF, Q-Sepharose HP and Capo Q were used as anion exchange column fillers, and 5 portions of fresh frozen plasma were thawed. Each portion of plasma was purified as follows.

[0094] (1) Centrifuge at 16000 r / min at 0℃~5℃, collect the supernatant and heat it to 4℃~8℃, filter it with a 0.22μm filter element, and take the supernatant for later use.

[0095] (2) Inactivation: The supernatant of step (1) is inactivated with S / D. Tween 80 is added to 1.0% and TNBP (tributyl phosphate) is added to 0.3% of the supernatant of step (1). After stirring, the temperature is raised to 24-26° C. and kept warm for 6-8 hours. Then, the supernatant is filtered through a 0.45 μm filter element and the filtrate is collected.

[0096] (3) Add the equilibrated DEAE-Sephadex-A50 gel to the supernatant in step (2) for adsorption, with the amount added being 1.0 g / L per L of supernatant, mix well, and adsorb for 60 minutes. After the adsorption is completed, collect the liquid and gel separately.

[0097] (4) The gel collected in step (3) is washed four times with balancing solution 1 having a volume 3 times that of the gel, the washing solution is collected, and combined with the liquid collected in step 3 for later use in preparing canine fibrinogen; the gel is eluted four times with elution solution 1 having a volume 2 times that of the gel, and the eluate is collected for preparing prothrombin complex.

[0098] (5) The liquid washing solution and the equilibrium solution collected in step (4) are concentrated to have a protein content of 2-3 mg / ml.

[0099] (6) Take the anion exchange columns containing Capo DAEA, Q-Sepharose FF, Q-Sepharose 4FF, Q-Sepharose HP and Capo Q, and fully balance them with equilibration solution 2 respectively, then load the above 5 concentrated solutions onto the columns respectively. During the loading process, the flow rate is controlled at 30-40 ml / h, and the flow-through liquid is collected. After the loading is completed, wash the column 3 times with 2 times equilibration solution 2, and combine the washing liquid with the flow-through liquid for use. Prepare fibrinogen. Filter the combined liquid with a 0.45 μm filter element and collect the filtrate.

[0100] (7) The filtrate collected in step (6) is adsorbed using DEAE-Sephadex-A50 as the gel medium. The gel is equilibrated with equilibration buffer 1 before loading onto the column. The sample is loaded for adsorption at a rate of 30 - 40 ml / h. After the sample loading is completed, the gel column is washed 3 times with 2 gel volumes of equilibration buffer 1, and the flow-through and washing solutions are collected. The flow-through and washing solutions are ultrafiltered and concentrated using a 30 KD ultrafiltration membrane until the protein content reaches 53 mg / ml. Glycine, sucrose, Tween 80, and sodium chloride are added as stabilizers, with the final concentration of glycine being 1%, sucrose being 5%, Tween 80 being 0.02%, and sodium chloride being 0.6%. The liquid is sterilized by filtration, aliquoted, freeze-dried, and capped, and dry heat virus inactivation (100 °C for 30 min) is carried out.

[0101] The most suitable packing material is determined by the appearance and purity of the finished product after purification through different chromatographic columns. From the analysis of appearance and purity, the best packing materials for the anion chromatographic column when purifying fibrinogen are Capto DAEA and Capto Q (a small amount of particulate matter is allowed during the reconstitution of fibrinogen). The results are shown in Table 9.

[0102] Table 9 Results of packing material screening for fibrinogen

[0103] Packing material Appearance Purity Capto DAEA Clear liquid, with a small amount of particulate matter precipitating 96.6％ Q-Sepharose FF Clear liquid, with a small amount of particulate matter precipitating 93.4％ Q-Sepharose 4FF Turbid liquid, with more particles precipitating 86.0％ Q-Sepharose HP Clear liquid, with more filamentous substances precipitating 72.3％ Capto Q Clear liquid, with a small amount of particulate matter precipitating 97.7％

[0104] Example 7 Three batches of products are prepared according to the established preparation process.

[0105] S1. Cold precipitation: Take three portions of fresh frozen plasma, 50 kg each. Centrifuge at 16000 r / min under the condition of 0 °C - 5 °C, collect the supernatant and warm it up to 4 °C - 8 °C, filter it through a 0.22 μm filter element, and take the supernatant for standby.

[0106] S2. Inactivation: Take the supernatant from step S1 and inactivate it with S / D. Add Tween 80 to 1.0% and TNBP to 0.3% in the supernatant from step S1, stir well and then warm it up to 24 - 26 °C, keep it warm for 6 - 8 hours, and then filter it through a 0.45 μm filter element to collect the filtrate.

[0107] S3. Add the pre-equilibrated DEAE-Sephadex-A50 gel to the filtrate in step S2 for adsorption, with the addition amount being 1.0 g / L per L of supernatant. Mix well and adsorb for 70 minutes. After the adsorption is completed, collect the liquid and the gel respectively.

[0108] S4. The gel collected in step S3 is washed 3 times with equilibration buffer 1 in an amount of 2 times the gel volume, and the flow-through is collected and combined with the liquid collected in step S3 for standby use in the preparation of canine fibrinogen. The gel is eluted 4 times with elution buffer 1 in an amount of 3 times the gel volume, and the eluate is collected for the preparation of canine prothrombin complex. Equilibration buffer 1 is a 10 - 20 mM citric acid - sodium citrate buffer containing 50 - 100 mM sodium chloride, with a pH of 6.5 - 7.5. Elution buffer 1 is a 10 - 20 mM citric acid - sodium citrate buffer containing 400 - 600 mM sodium chloride, with a pH of 6.5 - 7.5.

[0109] S5. The combined liquid collected in step S4 is concentrated to obtain concentrated solution 1 with a protein content of 2 - 3 mg / ml. The collected eluate is concentrated to obtain concentrated solution 2 with a protein content of 2 - 3 mg / ml.

[0110] S6. The anion exchange column is first fully equilibrated with equilibration buffer 2, and then concentrated solution 1 is loaded onto the column. During the loading process, the flow rate is controlled at 30 - 40 ml / h, and the flow-through is collected. After the loading is completed, the column is washed 3 times with 2 times the equilibration buffer 2, and the washing solution is combined with the flow-through for standby use in the preparation of canine fibrinogen. The combined solution is filtered through a 0.45 μm filter element, and the filtrate is collected and designated as filtrate 1. Filtrate 1 is operated according to steps S7A - S10A. Equilibration buffer 2 contains 0.01 M - 0.02 M TRIS, 10 - 20 mM citric acid - sodium citrate, 0.1 M - 0.3 M arginine hydrochloride, and the rest is water, with a pH of 6.5 - 7.5.

[0111] Another anion exchange column is first fully equilibrated with equilibration buffer 2, and then concentrated solution 2 is loaded onto the column. During the loading process, the flow rate is controlled at 30 - 40 ml / h. After the loading is completed, the column is eluted 4 times with 3 times the elution buffer 2, and the eluate is collected for standby use in the preparation of canine prothrombin complex. The eluate is filtered through a 0.45 μm filter element, and the filtrate is collected and designated as filtrate 2. Filtrate 2 is operated according to steps S7B - S10B. Elution buffer 2 contains 0.01 M - 0.02 M TRIS, 10 - 20 mM citric acid - sodium citrate, 400 mM - 600 mM sodium chloride, and the rest is water, with a pH of 6.50 - 7.50.

[0112] S7A. The filtrate 1 collected in step S6 is adsorbed using DEAE - Sephadex - A50 as the gel medium.

[0113] S8A. Before loading onto the column, the gel is equilibrated with equilibration buffer 1. The sample is loaded for adsorption at a loading rate of 30 - 40 ml / h. After the loading is completed, the gel column is washed 3 times with 2 times the gel volume of equilibration buffer 1, and the flow-through and the washing solution are collected and mixed evenly.

[0114] S9A. Ultrafilter and concentrate the mixture of S8A with a 30KD ultrafiltration membrane until the protein content reaches 53 mg / ml, and add glycine, sucrose, Tween 80, and sodium chloride as stabilizers, so that the final concentration of glycine is 1% - 2%, the final concentration of sucrose is 4% - 6%, the final concentration of Tween 80 is 0.01% - 0.02%, and the final concentration of sodium chloride is 0.6% - 0.9%.

[0115] S10A. Sterilize and filter the liquid in step S9A, subpackage, freeze-dry, and crimp the cap, and use dry heat virus inactivation (100°C for 30 min) to obtain canine fibrinogen.

[0116] S7B. Adsorb the filtrate 2 collected in step S6 with DEAE-Sephadex-A50 as the gel medium.

[0117] S8B. Balance the gel column with equilibration buffer 1 before loading the sample, load the sample for adsorption, and the sample loading speed is 30 - 40 ml / h. After the sample loading is completed, elute the gel column 4 times with 3 gel volumes of elution buffer 1, and collect the eluate.

[0118] S9B. Ultrafilter and concentrate the eluate of S8B with a 10KD ultrafiltration membrane to make the potencies of coagulation factor IX, II, and X not less than 200 IU / ml, and the potency of coagulation factor VII not less than 50 IU / ml. Then add glycine, sucrose, and Tween 80 as stabilizers, so that the final concentration of glycine is 1% - 2%, the final concentration of sucrose is 4% - 6%, and the final concentration of Tween 80 is 0.01% - 0.02%.

[0119] S10B. Sterilize and filter the liquid in step S9B, subpackage, freeze-dry, and crimp the cap, and use dry heat virus inactivation (100°C for 30 min) to obtain prothrombin complex.

[0120] The detection indexes of fibrinogen and prothrombin complex prepared from three batches of plasma all include recovery rate, appearance of freeze-dried powder, solubility, reconstitution time, and purity. The results are shown in Table 10.

[0121] Table 10 List of recovery rate and key technical indexes of trial preparations

[0122]

[0123]

[0124] Only some exemplary embodiments of the present invention are described by way of illustration above. Undoubtedly, for those of ordinary skill in the art, without departing from the spirit and scope of the present invention, the described embodiments can be modified in various different ways. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for simultaneously separating canine fibrinogen and canine prothrombin complex from canine plasma, characterized in that, It includes the following steps: S1. Cryoprecipitation: Thaw fresh frozen plasma, centrifuge at 16000 r / min under the condition of 0℃ - 5℃, collect the supernatant and warm it up to 4℃ - 8℃, filter it with a 0.22 µm filter element, take the supernatant and reserve it; S2. Inactivation: Take the supernatant of step S1 and inactivate it with S / D. Add Tween 80 to 1.0% and TNBP to 0.3% in the supernatant of step S1, stir well and then warm it up to 24 - 26℃, keep warm for 6 - 8 hours, and then filter it with a 0.45 µm filter element to collect the filtrate; S3. Add the well - balanced DEAE - Sephadex - A50 gel to the filtrate in step S2 for adsorption. The addition amount per liter of supernatant is 0.5 g - 2.0 g, mix well, and adsorb for 50 - 80 minutes; After the adsorption ends, collect the liquid and the gel respectively; S4. Wash the gel collected in step S3 with 2 - 3 times the gel volume of equilibration buffer 1 for 2 - 4 times, collect the flow - through liquid, combine it with the liquid collected in step S3, reserve it for the preparation of canine fibrinogen; Elute the gel with 1 - 3 times the gel volume of elution buffer 1 for 3 - 5 times, collect the eluate for the preparation of prothrombin complex; S5. Concentrate the combined liquid collected in step S4 and define it as concentrated liquid 1, with a protein content of 2 - 3 mg / ml; Concentrate the collected eluate and define it as concentrated liquid 2, with a protein content of 2 - 3 mg / ml; S6. First, fully equilibrate the anion - exchange column with equilibration buffer 2, then load concentrated liquid 1 onto the column. During the loading process, control the flow rate at 30 - 40 ml / h and collect the flow - through liquid; After the loading ends, wash the column with 2 - 3 times the equilibration buffer 2 for 2 - 4 times, and combine the washing liquid with the flow - through liquid and reserve it for the preparation of fibrinogen; Filter the combined liquid with a 0.45 µm filter element, collect the filtrate, and operate the filtrate according to steps S7A - S10A; Take another anion - exchange column and fully equilibrate it with equilibration buffer 2, then load concentrated liquid 2 onto the column. During the loading process, control the flow rate at 30 - 40 ml / h. After the loading ends, elute the column with 1 - 3 times the elution buffer 2 for 3 - 5 times, collect the eluate and reserve it for the preparation of prothrombin complex; Filter the eluate with a 0.45 µm filter element, collect the filtrate, and operate the filtrate according to steps S7B - S10B; S7A. Adsorb the combined liquid collected in step S6 with DEAE - Sephadex - A50 as the gel medium; S8A. Equilibrate the gel with equilibration buffer 1 before loading onto the column, load the sample for adsorption, and the loading speed is 30 - 40 ml / h. After the loading ends, wash the gel column with 2 - 3 times the gel volume of equilibration buffer 1 for 2 - 4 times, and collect the flow - through liquid and mix it evenly with the washing liquid; S9A. Ultrafilter and concentrate the mixed liquid in S8A with a 30KD ultrafiltration membrane until the protein content reaches 53 mg / ml, and add glycine, sucrose, Tween 80 and sodium chloride as stabilizers to make the final concentration of glycine 1% - 2%, the final concentration of sucrose 4% - 6%, the final concentration of Tween 80 0.01% - 0.02%, and the final concentration of sodium chloride 0.6% - 0.9%; S10A. Sterilize, filter, aliquot, freeze-dry, and crimp the vial of the liquid from step S9A, and perform dry heat virus inactivation to obtain canine fibrinogen; S7B. Adsorb the eluate collected in step S6 using DEAE-Sephadex-A50 as the gel medium; S8B. Equilibrate the gel column with equilibration buffer 1 before loading the sample. Load the sample for adsorption at a rate of 30 - 40 ml / h. After the sample loading is completed, elute the gel column 3 - 5 times with 1 - 3 gel volumes of elution buffer 1, and collect the eluate; S9B. Ultrafilter and concentrate the eluate from S8B using a 10KD ultrafiltration membrane to ensure that the titers of coagulation factors IX, II, and X are not less than 200 IU / ml, and the titer of coagulation factor VII is not less than 50 IU / ml. Then add glycine, sucrose, and Tween 80 as stabilizers, with the final concentration of glycine being 1% - 2%, the final concentration of sucrose being 4% - 6%, and the final concentration of Tween 80 being 0.01% - 0.02%; S10B. Sterilize, filter, aliquot, freeze-dry, and crimp the vial of the liquid from step S9B, and perform dry heat virus inactivation to obtain prothrombin complex; The equilibration buffer 1 is a 10 - 20 mM citric acid - sodium citrate buffer containing 50 - 100 mM sodium chloride, with a pH of 6.5 - 7.5; The elution buffer 1 is a 10 - 20 mM citric acid - sodium citrate buffer containing 400 - 600 mM sodium chloride, with a pH of 6.5 - 7.5; The equilibration buffer 2 is composed of 0.01M - 0.02M TRIS, 10 - 20 mM citric acid - sodium citrate, 0.1M - 0.3M arginine hydrochloride, with the rest being water, and a pH of 6.5 - 7.5; The anion exchange column is selected from Capto DAEA or Capto Q; The elution buffer 2 is composed of 0.01M - 0.02M TRIS, 10 - 20 mM citric acid - sodium citrate, 400 mM - 600 mM sodium chloride, with the rest being water, and a pH of 6.50 - 7.

50.

2. The method for simultaneously separating canine fibrinogen and canine prothrombin complex from canine plasma according to claim 1, characterized in that, Wash the gel in step S4 three times with 2 gel volumes of equilibration buffer 1.

3. The method for simultaneously separating canine fibrinogen and canine prothrombin complex from canine plasma according to claim 1, wherein in step S4, the gel is eluted 4 times with an eluent 1 having a volume 3 times that of the gel.

4. The method for simultaneously separating canine fibrinogen and canine prothrombin complex from canine plasma according to claim 1, wherein the flow rate during column loading in step S6 is 30 ml / h.

5. The method for simultaneously separating canine fibrinogen and canine prothrombin complex from canine plasma according to claim 1, wherein in the stabilizer in step S9A, the final concentration of glycine is 1%, the final concentration of sucrose is 4%, the final concentration of Tween 80 is 0.01%, the final concentration of sodium chloride is 0.9% or the final concentration of glycine is 1%, the final concentration of sucrose is 5%, the final concentration of Tween 80 is 0.02%, the final concentration of sodium chloride is 0.6% or the final concentration of glycine is 2%, the final concentration of sucrose is 6%, the final concentration of Tween 80 is 0.01%, the final concentration of sodium chloride is 0.9%.

6. According to the method for simultaneously separating canine fibrinogen and canine prothrombin complex from canine plasma as described in claim 1, in the stabilizer of step S9B, the final concentration of glycine is 1%, the final concentration of sucrose is 4%, and the final concentration of Tween 80 is 0.01%; or the final concentration of glycine is 1%, the final concentration of sucrose is 5%, and the final concentration of Tween 80 is 0.02%; or the final concentration of glycine is 2%, the final concentration of sucrose is 4%, and the final concentration of Tween 80 is 0.02%; or the final concentration of glycine is 2%, the final concentration of sucrose is 6%, and the final concentration of Tween 80 is 0.01%.

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