Process for preparing cryoprecipitate from plasma
The cold precipitation was isolated from plasma by cellulose adsorption, which solved the problems of high cost and low yield in the prior art, and achieved efficient and safe large-scale production of coagulation factor VIII and fibrinogen, which was suitable for the preparation of blood products.
Patent Information
- Application Number
- CN202410026788.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-08
- Publication Date
- 2025-07-08
AI Technical Summary
The method for preparing cold precipitation from plasma in the prior art has the problems of high process costs, difficult to expand production, and low yields of target substances, especially the separation efficiency of coagulation factor VIII and fibrinogen is not high.
Cellulose adsorption method is used to mix cellulose with plasma, cellulose precipitation is separated by filtration, and rinse and dissolve it using a specific formula solution to obtain a protein solution rich in coagulation factor VIII, vasculophilia factor and fibrinogen.
It reduces the cost of equipment investment, simplifies the operation process, improves the yield of coagulation factor VIII to 70%, and the yield of fibrinogen reaches 40%. It can meet the requirements of vasculophilia factors at the same time, and is suitable for large-scale production.
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Figure CN120271694A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of blood product preparation, and particularly relates to a process for preparing cryoprecipitate from plasma, based on cellulose adsorption, for preparing coagulation factor VIII or fibrinogen from plasma. Background Art
[0002] Plasma cryoprecipitate refers to the cold-insoluble precipitate formed when plasma is under low temperature conditions. In 1959, American doctor J.G. Pool first observed that a small amount of cold-insoluble precipitate appeared when frozen plasma was melted at 4°C. Pool also further developed a method for separating and preparing cryoprecipitate preparations from single-donor plasma in a closed sterile system in 1965 together with Shannon, etc. The cryoprecipitate mainly contains coagulation factor VIII and fibrinogen. This method is simple and easy to implement, and was quickly popularized in many hospitals and central blood stations. After numerous research and improvements over the years, it has been adopted by some countries for a relatively long time. The cryoprecipitate preparation has thus become a coagulation factor VIII preparation for the treatment of hemophilia A. The main disadvantages of cryoprecipitate used in clinical treatment are the uncertain unit titer of its preparation, the relatively large amount of each infusion, low purity of the preparation, containing particulate matter and blood group antibodies, with allergic reactions occurring from time to time, and moreover, the unprelyophilized cryoprecipitate preparations prepared by general blood banks and central blood stations also need to be refrigerated at low temperature (-20°C), etc.
[0003] In order to further improve the quality of the preparation, factor VIII preparations processed and purified using cryoprecipitate as the raw material were developed after the 1970s. Such preparations not only have a small volume and high titer, but also have stable and reliable curative effects. Moreover, due to the continuous improvement of the purity of factor VIII preparations in the past decade or so, and the implementation of strict virus inactivation treatment during the production process, there are fewer adverse reactions and higher safety during clinical use. However, due to the relatively large viscosity of fibrinogen in plasma cryoprecipitate, it is not easy to separate by filtration method. Currently, blood product manufacturers at home and abroad still use the centrifugation method to separate cryoprecipitate. The principle of the centrifugation separation technology is that the differences in sedimentation coefficient, buoyancy, and density of each component in the mixed plasma can be expanded to a separable state under the action of centrifugal force. The centrifuge used for plasma cryoprecipitate separation is a tubular freezing continuous flow centrifuge. The main control parameters during centrifugation include the rotation speed of the centrifuge, the inlet liquid temperature, the outlet liquid temperature, the outlet liquid speed, and the refrigerant temperature, etc. The specific operation is to transfer plasma with a temperature controlled between 0 - 5°C from the plasma inlet below the centrifuge to the centrifuge cylinder. The centrifuge cylinder rotates at a high speed of more than 10000 r / min, and the temperature of the centrifuged plasma is controlled by the refrigerant around the centrifuge cylinder. The centrifuged plasma flows out from the plasma outlet above, and the temperature of the flowing-out plasma is controlled between 0 - 5°C, while the cryoprecipitate is left in the centrifuge cylinder. After centrifugation is completed, the cryoprecipitate in the centrifuge cylinder is collected. The cryoprecipitate centrifugation separation method has expensive equipment, complex operation, slow centrifugation speed, and long process time, which seriously restricts the processing of a large number of raw plasma and the separation of cryoprecipitate.
[0004] The cryoprecipitate collected by centrifugation contains 60% of the total amount of coagulation factor VIII in plasma and 60% of the total amount of fibrinogen in plasma. The cryoprecipitate is further separated by pre-thawing, pulverization, washing, dissolution, ethanol precipitation or acid precipitation, ion exchange chromatography, etc. The specific activity of the final product of coagulation factor VIII can reach 20 - 30 IU / mg protein. Due to the presence of fibrinogen and coagulation factor VIII in the cryoprecipitate, it has high viscosity and is colloidal. When washing and dissolving again, it needs to be pulverized first and cannot be completely decomposed. It is very easy to precipitate and activate during the production process, resulting in a low yield of the final product of coagulation factor VIII, and the yield can only reach 10% - 30%.
[0005] It can be seen that the method of obtaining cryoprecipitate by traditional centrifugation and then obtaining coagulation factor VIII by separation through a chromatographic column, etc. has problems such as high process cost, difficulty in expanding production, and low yield of the target substance. There is an urgent need to develop a new method for efficient extraction, separation, and purification of coagulation factor VIII in plasma. Summary of the Invention
[0006] The present invention aims to provide a process for preparing cryoprecipitate from plasma to solve the technical problems of high process cost, difficulty in expanding production, and low yield of the target substance in the existing preparation methods.
[0007] To achieve the above object, the present invention adopts the following technical solutions:
[0008] A process for preparing cryoprecipitate from plasma, wherein the cellulose is balanced and then mixed with the raw plasma, filtered after adsorption to obtain cellulose precipitate; after rinsing the cellulose precipitate, it is soaked in a precipitate dissolution solution; then the filtrate is taken by filtration to obtain a protein solution rich in coagulation factor VIII, von Willebrand factor, and human fibrinogen.
[0009] Further, the method for balancing the cellulose is: soaking the cellulose with a balance solution; the balance solution contains 10 mM - 20 mM sodium citrate and 0.1 M - 0.14 M sodium chloride, with a pH value of 7.50 - 7.90 and a conductivity of 11 mS / cm - 14 mS / cm.
[0010] Further, the cellulose after being balanced is mixed with the raw plasma in an environment of -2°C - 2°C for 10 - 30 min.
[0011] Further, the dosage of cellulose is 2 - 4 g of cellulose / L of plasma.
[0012] Further, the method for rinsing the cellulose precipitate is: rinsing the cellulose precipitate successively with a first top rinse solution and a second top rinse solution.
[0013] Further, the first washing solution is a 10 mM sodium citrate solution with a pH value of 7.50 - 7.80; the second washing solution is formulated as a solution containing 10 mM sodium citrate and 2 M glycine with a pH value of 7.50 - 7.80.
[0014] Further, the precipitation dissolution solution contains 0.015 - 0.025 M tris(hydroxymethyl)aminomethane, 10 - 20 mM sodium citrate, 0.05 - 0.15 M alanine, 0.09 M - 0.12 M sodium chloride, with a pH value of 6.50 - 7.00 and a conductivity of 10.0 mS / cm - 15.0 mS / cm.
[0015] Further, the dosage ratio of the cellulose precipitate after elution to the precipitation dissolution solution is 1:8 - 12.
[0016] Further, the cellulose precipitate after elution is immersed in the precipitation dissolution solution at 31 - 35 °C for 10 - 50 min.
[0017] This technical solution also provides a protein solution rich in coagulation factor VIII, von Willebrand factor, and human fibrinogen prepared by a method for preparing coagulation factor VIII or fibrinogen from plasma based on cellulose adsorption.
[0018] The principle of this solution is as follows:
[0019] In this technical solution, cellulose is added to low-temperature plasma, and the cellulose precipitate adsorbed with plasma cryoprecipitate is separated by filtration or pressure filtration, that is, a fiber precipitate adsorbed with cryo-insoluble proteins such as factor VIII, vWF factor, fibrinogen, and fibronectin is obtained. After the cellulose precipitate is dissolved, conventional purification of factor VIII, etc. is carried out to obtain high-purity coagulation factor VIII and fibrinogen products.
[0020] This technical solution provides a simple and feasible method for separating plasma cryoprecipitate from raw plasma. Compared with the conventional low-temperature centrifugation method, the cost is greatly reduced. This separation method does not require a low-temperature centrifuge, reducing equipment investment, and the centrifuge is a high-speed rotating device with a relatively high operation risk.
[0021] Cold-insoluble proteins such as factor VIII, vWF factor, fibrinogen, and fibronectin adsorbed on cellulose have good solubility in the precipitation and dissolution solution of this protocol. However, the solubility of plasma cryoprecipitate obtained by the low-temperature centrifugation method is very poor, and it requires steps such as pre-thawing, crushing, washing, and dissolution to achieve dissolution. Since cellulose is added to the cryoprecipitate separated by this method, loose pores will form during the formation of the cryoprecipitate, so it is easy to disperse during dissolution. The fiber precipitate containing factor VIII does not require pre-thawing and crushing, has simple operation, a more controllable production environment, a low possibility of introducing microorganisms, and guaranteed product quality. Cellulose is widely distributed in nature, insoluble in water and common organic solvents, and is the main component of plant cell walls. Cellulose is the most abundant natural organic matter in the world. Cellulose is a fibrous, multi-capillary linear polymer formed by connecting many β-D-glucosyl groups through 1,4-glycosidic bonds. It has porosity, a large surface area, and an amphiphilic structure. Therefore, it has certain adsorption properties. The cost of obtaining cellulose is relatively low, making it particularly suitable for industrial production. The cellulose referred to in this invention is not limited to a certain type of cellulose, nor to a certain form of cellulose.
[0022] At the same time, the recovery rate of factor VIII in the cryoprecipitate separated by this method can reach 70%, and the fibrinogen can reach more than 40%. It can be used for the separation and production of both factor VIII and fibrinogen at the same time. Currently, the cryoprecipitate separated by the centrifugation method is generally used for the production of factor VIII, and fibrinogen is treated as waste in the production, resulting in a large loss. The European Pharmacopoeia stipulates that if the factor VIII product is used to treat von Willebrand disease, the content of von Willebrand factor (vWF) is also specified. The recovery rate of von Willebrand factor (vWF) in the cryoprecipitate produced by this method can also reach more than 70%.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] (1) The separation method does not require the use of a centrifuge, reducing equipment investment and saving costs.
[0025] (2) This protocol can use the pressure filtration method or the ordinary filtration method, with simple operation, being safer and easier for large-scale and batch production.
[0026] (3) The cryoprecipitate produced by this method can be directly dissolved without pre-thawing and crushing, with simple operation, a more controllable production environment, a low possibility of introducing microorganisms, and more guaranteed product quality.
[0027] (4) The cryoprecipitate separated by this method has a higher recovery rate of factor VIII, reaching 70%.
[0028] (5) In addition to the high recovery rate of factor VIII, the cryoprecipitate separated by this method can also meet the recovery requirements for von Willebrand factor (vWF), and its active recovery can reach 70%.
[0029] (6) It can also be used for the production of fibrinogen at the same time, with a yield of 40%. Description of the Drawings
[0030] Figure 1 Photograph of typical cellulose precipitation (fiber adsorption precipitation) in Example 1.
[0031] Figure 2 Photograph of typical cellulose precipitation in Example 1 dispersed in the precipitation dissolution solution.
[0032] Figure 3 Photograph of the solution state after dissolution and filtration of typical cellulose precipitation in Example 1.
[0033] Figure 4 Photograph of typical plasma centrifugal cryoprecipitate in Comparative Example 1. Detailed Description of the Invention
[0034] The present invention will be further described in detail below in conjunction with the embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.
[0035] Example 1
[0036] A process for making cryoprecipitate from plasma, and the general process flow is as follows:
[0037] (1) Raw plasma treatment: After the raw plasma stored at -30°C is taken out of the warehouse, the surface of the plasma bag is disinfected with 70%-75% ethanol solution, and then the plasma bag is broken, and the temperature is controlled to melt at 0-4°C. The melted plasma (hereinafter referred to as mixed plasma) is taken for detection of plasma coagulation factor VIII, fibrinogen, fibronectin and von Willebrand factor (vWF).
[0038] Among them, the raw plasma is the supernatant after centrifuging blood to remove cells, containing proteins, inorganic salts, water, etc., and no blood cells. More specifically, the raw plasma is the human plasma referred to in the "Chinese Pharmacopoeia": The human plasma used for the production of blood products is the healthy human plasma collected by apheresis for the production of plasma protein products.
[0039] (2) The plasma temperature is lowered to -2°C - 2°C, and cellulose that has been balanced with the balance solution (in this technical solution, a common paper fiber filter plate produced by Shenyang Great Wall Filtration is specifically used) is added under stirring. The cellulose is added and stirred for adsorption for 10-30 minutes (15 minutes is specifically used in subsequent experimental studies), and the temperature is maintained at -2°C - 2°C during the stirring process.
[0040] The balanced solution formula is: 10 mM - 20 mM sodium citrate + 0.1 M - 0.14 M sodium chloride, with a pH value of 7.50 - 7.90 and a conductivity of 11 mS / cm - 14 mS / cm. The ratio of cellulose to plasma is 2 - 4 g of cellulose per liter of plasma.
[0041] (3) Filter the plasma containing cellulose using a cellulose filter plate or membrane, and perform subsequent processing on the separated components that cannot pass through the membrane (named cellulose precipitate). The membrane material is not limited (it can be polyethersulfone, cellulose acetate, etc., and a cellulose acetate membrane is specifically used in subsequent experiments), and the pore size of the membrane can be 1 - 10 μm. Take a photo of a typical cellulose precipitate (fiber adsorption precipitate), see Figure 1 . After filtration, use the first top wash solution and the second top wash solution to perform top washing (rinsing) on the cellulose precipitate part that cannot pass through the membrane. And take samples for detection of plasma coagulation factor VIII, fibrinogen, fibronectin, and von Willebrand factor (vWF).
[0042] The dosage of the first top wash solution is 150 - 300 mL per 1 L of raw plasma, and the flow rate is 200 - 400 mL / min; the composition is 10 mM sodium citrate, and the pH is adjusted to 7.50 - 7.80 using citric acid. The solution is pre-cooled in a 2 - 8 °C refrigerator and then used.
[0043] The dosage of the second top wash solution is 200 - 450 mL per 1 L of raw plasma, and the flow rate is 200 - 400 mL / min; the composition is 10 mM sodium citrate + 2 M glycine, and the pH value is adjusted to 7.20 (at 25 °C) using sodium hydroxide. The solution is pre-cooled in a 2 - 8 °C refrigerator and then used.
[0044] (4) Take out the top-washed cellulose precipitate and use a precipitation dissolution solution to dissolve the soluble components in the cellulose precipitate. The dissolution temperature is 31 - 35 °C (water bath), and continuous stirring is carried out during the dissolution process for 10 - 50 min (specifically stir for 45 min in subsequent experimental studies). The dosage of the precipitation dissolution solution is 8 - 12 times the weight of the precipitate, and the formula is: 0.015 - 0.025 M tris(hydroxymethyl)aminomethane + 10 - 20 mM sodium citrate + 0.05 - 0.15 M alanine + 0.09 M - 0.12 M sodium chloride, with a pH value of 6.50 - 7.00 and a conductivity of 10.0 mS / cm - 15.0 mS / cm. See the picture of the fiber adsorption plasma and then dissolution in Figure 2 . After the above process, use silk to filter to remove cellulose, and the filtrate is a protein solution rich in coagulation factor VIII and human fibrinogen (see Figure 3 , that is, the filtrate after filtration), and it is used for further separation of coagulation factor VIII and fibrinogen.
[0045] A protein solution rich in coagulation factor VIII and human fibrinogen was prepared in the following specific manner:
[0046] The raw plasma is melted in the above conventional manner, and the melted raw plasma is sampled to detect the content of coagulation factor VIII, human fibrinogen and von Willebrand factor (vWF). The plasma is taken into a container, and the container containing the plasma product is placed in an ice bath to stir the plasma sample in a water bath and control the temperature. The temperature control range of the adsorption and filtration process is within -2°C-2°C, and the amount of cellulose added is 2g cellulose / L plasma (or 3g cellulose / L plasma, or 4g cellulose / L plasma). The cellulose (paper fiber filter plate) is fully infiltrated with the balancing solution so that the cellulose (paper fiber) is evenly dispersed in the balancing solution, and then the balancing solution is drained, and the cellulose that has been drained of the balancing solution is added to the plasma, and adsorbed with low temperature stirring for 15 minutes. The plasma containing cellulose adsorbed was filtered with a 10 μm filter membrane to separate the adsorbed plasma and fiber precipitate (i.e., two parts were obtained: cellulose precipitate and filtered liquid part), and the precipitate was top-washed with the first top-wash solution (260 mL per 1L of raw plasma, flow rate of 300 mL / min) and the second top-wash solution (430 mL per 1L of raw plasma, flow rate of 300 mL / min). The filtered plasma was sampled and the content of coagulation factor VIII, human fibrinogen and von Willebrand factor (vWF) was tested. The cellulose precipitate after top-washing was taken out, and the precipitate was dissolved in a precipitate dissolving solution 10 times the weight of the precipitate at 34°C in a stirring water bath for 45 minutes. The cellulose was removed by filtration with silk, and the filtrate was a protein solution rich in coagulation factor VIII and human fibrinogen, which was used for further separation of coagulation factor VIII and fibrinogen.
[0047] In the above process, the specific conditions of the solutions used are as follows:
[0048] The specific formula of the balancing solution is: 15 mM sodium citrate, 0.12 M sodium chloride, the pH value is adjusted to 7.70 with citric acid, the conductivity is about 13 mS / cm, and the temperature is 2°C (refrigerated).
[0049] The specific formula of the first top wash solution is: 10 mM sodium citrate, pH adjusted to 7.70 with citric acid, temperature 2°C (refrigerated).
[0050] The specific formula of the second top wash solution is: 10 mM sodium citrate, 2 M glycine, using sodium hydroxide to adjust the pH value to 7.20 (at 25°C), the temperature is 2°C (refrigerated).
[0051] The specific formula of the precipitation dissolving solution is: 0.020M tris(hydroxymethyl)aminomethane, 15mM sodium citrate, 0.1M alanine, 0.1M sodium chloride, pH value 6.50, conductivity 14.0mS / cm.
[0052] The experimental results are shown in Table 1. The detection of factor VIII was carried out using a STAGO automatic blood coagulation analyzer, fibrinogen and fibronectin were detected using an ELISA kit, and the percentage activity of von Willebrand factor (vWF) was detected using a STAGO blood coagulation analyzer.
[0053] The proportions of factor VIII, fibrinogen and vWF in the filtered plasma were calculated as follows:
[0054] They were respectively the ratios of the amounts of factor VIII, fibrinogen and vWF in the filtered plasma to the total amounts of these proteins in the mixed plasma (obtained and detected in step (1)).
[0055] The proportions of factor VIII, fibrinogen and vWF dissolved in the precipitate were calculated by the following method:
[0056] They were the ratios of the amounts of factor VIII, fibrinogen and vWF in the precipitate dissolution solution to the amounts of factor VIII, fibrinogen and vWF in the mixed plasma. The values calculated in this part can also be called the yields of factor VIII, fibrinogen and vWF.
[0057] Among them, the filtered plasma included the part that passed through the filter membrane after filtering the plasma containing cellulose, and the part that passed through the filter membrane after two top washes. The parts that passed through the filter membrane several times were collected and combined, and the amounts of factor VIII, fibrinogen, fibronectin and vWF in the combined liquid were detected and calculated. After the cellulose precipitate was taken out after top washing, it was placed in a precipitate dissolution solution and dissolved for a period of time, and then silk-screen filtered to take the filtrate part. This filtrate part was used for the detection of components, and the amounts of factor VIII, fibrinogen and vWF factor in it were detected, which were used to calculate the proportions of factor VIII, fibrinogen and vWF dissolved in the precipitate.
[0058] Table 1:
[0059]
[0060] It can be seen from the above experimental results that by using the preparation method of the present technical solution, factor VIII, fibrinogen and vWF factor can be enriched in the precipitate dissolution solution, especially factor VIII and vWF factor. And the above process is simple, the equipment used is simple, safer and easier for large-scale and batch production. The factor VIII, fibrinogen and vWF factor in the precipitate dissolution solution can be separated by conventional methods of the existing technology, and then the corresponding products can be obtained. The European Pharmacopoeia stipulates that if the factor VIII product is used to treat von Willebrand disease, the content of von Willebrand factor (vWF) is also specified. Through this process, factor VIII and vWF factor can be enriched at the same time, and products with higher contents of the two factors can be obtained, which further meets the international requirements.
[0061] Comparative Example 1
[0062] In this comparative example, centrifugation was used to collect cryoprecipitate, and the specific operation was as follows: After the frozen raw plasma was taken out of the warehouse, the surface of the plasma bag was disinfected with 70%-75% ethanol solution, and then the plasma bag was broken, and the temperature was controlled at 0-4°C for melting. The melted plasma was taken for detection of plasma coagulation factor VIII, fibrinogen, fibronectin and von Willebrand factor (vWF). Then the plasma was centrifuged, and the specific centrifugation parameters were: 12,000 rpm / min, 0-2°C. The precipitate part taken after centrifugation was the cryoprecipitate collected by the centrifugation method. Different from the cryoprecipitate collected by the cellulose-assisted adsorption method in Example 1, the cryoprecipitate obtained in this comparative example had the characteristics of non-loose precipitate and difficult dissolution. See the photo of typical plasma centrifuged cryoprecipitate in Figure 4 .
[0063] In order to compare the performance of the cryoprecipitate obtained in this comparative example with that of the foregoing examples, the cryoprecipitate obtained in this comparative example was added to the precipitate dissolution solution used in Experiment Nos. 1-3 of Example 1 at a weight ratio of 1:10, and after stirring and water bathing at 34°C for 45 min, the dissolution of the cryoprecipitate was observed. It was shown that the precipitate was not fully dissolved and could not be dissolved, and there were many cryoprecipitates remaining in the precipitate dissolution solution in the form of large particles. The above results illustrate that it is difficult to dissolve the cryoprecipitate obtained by the traditional method by simply mixing it with the dissolution solution, and operations such as pre-melting, crushing, washing, and dissolution are required to ensure the dissolution of the cryoprecipitate. In Example 1, after top washing, the cellulose precipitate could be dissolved in the stirring water bath at 34°C for 45 min, and the protein substances could be fully dissolved in the precipitate dissolution solution. According to the experimental results in Table 1, most of the factor VIII, fibrinogen and vWF factors were in a dissolved state (dissolved in the filtered plasma or the precipitate dissolution solution), and the loss was very small. This also shows from the side that very few factor VIII, fibrinogen and vWF factors were adsorbed on the cellulose and could not be dissolved.
[0064] In addition, this technical solution was the first to use cellulose as an adsorption material to enrich coagulation factor VIII, von Willebrand factor (vWF) and fibrinogen. Before the research and development of this process, those skilled in the art did not know that cellulose had an affinity for these cold-insoluble proteins, and the research results exceeded the inventor's expectations. Using cellulose, the yields of coagulation factor VIII, von Willebrand factor (vWF) and fibrinogen can reach an ideal level (more than 60%, more than 70% and more than 40% respectively).
[0065] In addition, the two top-washing processes are very crucial for removing impurity proteins under the process conditions of this process. The top-washing process can effectively remove albumin and IgG (which are impurity proteins relative to the target protein of this solution). Through top-washing, the albumin and IgG in the cellulose precipitate can be eluted. The proportion of this part of albumin and IgG both reaches about 4% of the total amount of albumin and IgG in the raw material plasma. Removing this part of impurity proteins such as albumin / IgG through top-washing enters the subsequent experiments. Cellulose is used to adsorb cold-insoluble protein (the target protein of this solution), and under certain elution conditions, cellulose can stably adsorb cold-insoluble protein while eluting the impurity proteins. This is also first discovered in this solution, and the above phenomenon is successfully used to enrich cold-insoluble protein.
[0066] Comparative Example 2: Comparison of precipitation dissolution solutions
[0067] In this comparative example, the precipitation dissolution solution was tested. The experimental process was carried out with reference to No. 2 of Example 1, only the type of precipitation dissolution solution was replaced. The specific experimental results are shown in Table 2.
[0068] The precipitation dissolution solution of No. 2 in Table 1 is as follows:
[0069] The A precipitation dissolution solution is: 0.020 M tris(hydroxymethyl)aminomethane, pH 6.80.
[0070] The B precipitation dissolution solution is: 0.020 M tris(hydroxymethyl)aminomethane + 15 mM sodium citrate + 0.1 M alanine + 0.12 M sodium chloride, pH 6.80, and the conductivity is 14 mS / cm.
[0071] The C precipitation dissolution solution is: 0.020 M tris(hydroxymethyl)aminomethane + 15 mM sodium citrate + 0.1 M alanine + 0.12 M sodium chloride, adjusted to pH 6.80 with hydrochloric acid, and the conductivity is 15 mS / cm.
[0072] The D precipitation dissolution solution is: 0.020 M tris(hydroxymethyl)aminomethane + 15 mM sodium citrate + 0.1 M alanine + 0.12 M sodium chloride, pH 6.50, and the conductivity is 15 mS / cm.
[0073] Table 2:
[0074]
[0075] The above precipitation dissolution solutions can all dissolve the target protein to varying degrees. Among them, the use of tris(hydroxymethyl)aminomethane is crucial for the dissolution of the target protein from the cellulose precipitate. Adding alanine, sodium citrate, and sodium chloride, etc. to it can further maintain the activity of the target protein.
[0076] The above are only embodiments of the present invention. Specific technical solutions and / or common knowledge such as characteristics well known in the art are not described in detail herein. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope claimed in this application shall be subject to the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.
Claims
1. A process for preparing cryoprecipitate from plasma, characterized in that: After the cellulose is balanced, it is mixed with raw plasma, filtered after adsorption to obtain a cellulose precipitate; after the cellulose precipitate is rinsed, it is soaked in a precipitate dissolution solution; then the filtrate is filtered to obtain a protein solution rich in factor VIII, von Willebrand factor, and human fibrinogen.
2. The process for preparing cryoprecipitate from plasma according to claim 1, characterized in that: The method for balancing the cellulose is: soaking the cellulose with a balance solution; the balance solution contains 10 mM - 20 mM sodium citrate and 0.1 M - 0.14 M sodium chloride, with a pH value of 7.50 - 7.90 and a conductivity of 11 mS / cm - 14 mS / cm.
3. A process for preparing cryoprecipitate from plasma according to claim 2, characterized in that: After the cellulose is balanced, it is mixed with raw plasma in an environment of -2°C - 2°C for 10 - 30 min.
4. A process for preparing cryoprecipitate from plasma according to claim 3, characterized in that: The dosage of cellulose is 2 - 4 g of cellulose / L of plasma.
5. A process for preparing cryoprecipitate from plasma according to claim 4, characterized in that: The method for rinsing the cellulose precipitate is: rinsing the cellulose precipitate successively with a first top rinse solution and a second top rinse solution.
6. The process for preparing cryoprecipitate from plasma according to claim 5, wherein: The first top rinse solution is a 10 mM sodium citrate solution with a pH value of 7.50 - 7.80; the formula of the second top rinse solution is a solution containing 10 mM sodium citrate and 2 M glycine with a pH value of 7.50 - 7.
80.
7. A process for preparing cryoprecipitate from plasma according to claim 6, characterized in that: The precipitate dissolution solution contains 0.015 - 0.025 M tris(hydroxymethyl)aminomethane, 10 - 20 mM sodium citrate, 0.05 - 0.15 M alanine, 0.09 M - 0.12 M sodium chloride, with a pH value of 6.50 - 7.00 and a conductivity of 10.0 mS / cm - 15.0 mS / cm.
8. A process for preparing cryoprecipitate from plasma according to claim 7, characterized in that: The dosage ratio of the rinsed cellulose precipitate to the precipitate dissolution solution is 1:8 - 12.
9. A process for preparing cryoprecipitate from plasma according to claim 8, characterized in that: The rinsed cellulose precipitate is soaked in the precipitate dissolution solution at 31 - 35°C for 10 - 50 min.
10. A protein solution rich in factor VIII, von Willebrand factor, and human fibrinogen prepared by the process of making cryoprecipitate from plasma according to any one of claims 1 - 9.