A fibrinogen precipitation apparatus and purification process

Abstract

The application provides a fibrinogen precipitation device and a purification process. The fibrinogen precipitation device comprises a shell, a driving device and a feed pipe. A liquid storage cavity is arranged in the shell to accommodate a first liquid containing fibrinogen. The driving device is connected to the shell to ensure that the first liquid is in a flowing state. The feed pipe is arranged through the shell to deliver a second liquid. The feed pipe comprises an extension arranged obliquely. The extension is partially inserted into the first liquid. An angle alpha is formed between the direction of the extension and the flowing direction of the first liquid flowing through the feed pipe, wherein 0 < alpha < 90 degrees. The fibrinogen precipitation device can significantly reduce the proportion of precipitate A generated after the addition of ethanol, reduce protein denaturation to improve product stability, significantly reduce the residual amount of inactivator in the product, and be beneficial to the control of the impurity content of human fibrinogen process.

Description

Technical Field

[0001] This invention relates to the field of bio-equipment technology, and more specifically, to an apparatus and purification process for fibrinogen precipitation. Background Technology

[0002] Fibrinogen, also known as coagulation factor I, is the most abundant coagulation factor in blood plasma and a central protein in the coagulation system. In the final stage of coagulation, soluble fibrinogen is converted into thrombin and Ca2+. + Under the influence of fibrinogen, fibrin monomers are formed. These monomers covalently bind to fibrin under the action of fibrin stabilizing factors to form stable fibrin, thus coagulating the blood. Human fibrinogen is suitable for coagulation disorders caused by fibrinogen deficiency due to congenital fibrinogen deficiency, severe liver damage, cirrhosis, disseminated intravascular coagulation, postpartum hemorrhage, and major surgery, trauma, or internal bleeding. It is an essential emergency drug for treating massive hemorrhage and stopping bleeding in clinical practice.

[0003] Currently, the main inactivation process for lipid-enveloped viruses uses the S / D inactivation method, which effectively inactivates lipid-enveloped viruses and is widely used in the production of blood products such as fibrinogen. However, added inactivating agents such as polysorbate 80 and tributyl phosphate need to be removed during purification. The low-temperature ethanol method is a common method for the separation and purification of human fibrinogen. Ethanol is added to the fibrinogen solution to produce a precipitate, which is then separated from the solution by centrifugation, thereby reducing the content of S / D inactivating agents such as polysorbate 80 and tributyl phosphate in the product. However, the removal efficiency of S / D inactivating agents is correlated with the dissolution factor or the number of precipitation cycles. Increasing the dissolution factor or the number of precipitation cycles will increase the solution volume and reduce the recovery rate of the target protein.

[0004] Furthermore, when ethanol precipitates fibrinogen, due to its inherent properties, the precipitate readily adheres to protruding equipment and pipe surfaces. Over time, this accumulation increases, forming larger clumps or lumps of precipitate. The portions of the pipes in contact with the solution are prone to precipitate buildup; as ethanol is slowly added, the precipitate gradually enlarges, forming clumps or lumps. When the precipitate adhering to the outer wall of the pipe is exposed to air for an extended period, it easily denatures and becomes inactive.

[0005] Developing precipitation equipment that balances yield and purification efficiency is clearly essential for improving product competitiveness and achieving cost reduction and efficiency improvement. Therefore, this invention is proposed. Summary of the Invention

[0006] The problem addressed by this invention is how to reduce the dissolution factor and / or precipitation number when treating fibrinogen solutions using the low-temperature ethanol precipitation method, in order to balance yield and purification efficiency.

[0007] To address the aforementioned problems, this invention provides a fibrinogen precipitation device, comprising a housing, a drive unit, and a feed pipe. The housing contains a liquid storage chamber for holding a first liquid containing fibrinogen. The drive unit is connected to the housing to ensure the first liquid is in a flowing state. The feed pipe penetrates the housing and is used to transport a second liquid. The feed pipe includes an inclined extension portion that partially extends into the first liquid. The direction of the extension portion forms an angle α with the flow direction of the first liquid flowing through the feed pipe, where 0 < α < 90°. This design reduces the formation of "precipitate A," resulting in good purification. It also features a simple structure, facilitating production and processing. The flow direction of the first liquid flowing through the feed pipe refers to the overall direction of movement within the horizontal plane of the first liquid.

[0008] Preferably, the driving device is a stirring assembly, which includes a stirring paddle vertically disposed within the housing. The stirring paddle is driven by a motor, enabling the liquid to flow clockwise or counterclockwise along the inner wall of the housing. This arrangement makes full use of existing precipitation equipment, requires minimal equipment modification, and achieves excellent purification results.

[0009] Preferably, the feed pipe further includes a connecting portion connected to the extension portion. The extension portion is straight and inclined downwards, and the inclination direction of the extension portion forms an angle α with the flow direction of the first liquid flowing through the extension portion, where 0 < α < 60°. This configuration has a simple structure and is easy to modify and process.

[0010] Preferably, the feed pipe further includes a connecting portion connected to the extension, and the extension has a liquid outlet at one end away from the connecting portion. The extension is arc-shaped, and the tangent direction of the circle containing the extension at the liquid outlet forms an angle α with the flow direction of the first liquid flowing through the extension, where 0 < α < 60°. This configuration can reduce the generation of "precipitate A" and achieve better purification effect.

[0011] Preferably, the connecting portion is located close to the inner wall surface of the housing, and the connecting portion and the extension portion are integrally formed. This arrangement can effectively prevent the feed pipe from interfering with the drive device, and the second liquid such as ethanol in the feed pipe can diffuse rapidly after entering the first liquid, avoiding excessively high local temperatures that could lead to fibrinogen inactivation.

[0012] Preferably, the included angle α is 10°≤α≤60°. When the included angle α is 60°, the ratio of "precipitate A" to "precipitate B" is 1.5%; preferably, when the included angle α is 45°, the ratio of "precipitate A" to "precipitate B" is 0.4%, resulting in good purification effect.

[0013] Preferably, the feed pipe is made of silicone. Since silicone has a higher density than the solution, even if the feed pipe is vertically installed, the outlet will tilt to one side under the impact of the first liquid and will always be below the surface of the first liquid. If the first liquid is flowing in a turbulent state, the feed pipe may be in a swinging state in the first liquid, which is not conducive to the formation of precipitate A.

[0014] Preferably, the feed pipe includes a connecting part and an extension part, with a rotating part disposed between the extension part and the connecting part, enabling the extension part to tilt downwards after the first liquid flows. By providing the rotating part, the extension part can tilt at a certain angle under the impact of the first liquid, ensuring that it is roughly consistent with the flow direction of the first liquid, thereby achieving the purpose of automatic pipe setting, saving labor, and reducing manual setting errors. This design is simple and low-cost. Preferably, the rotating part enables the extension part to form an angle α with the flow direction of the first liquid flowing through the feed pipe. That is, when the first liquid flows clockwise, the extension part tilts to the left of the vertical line; when the first liquid flows counterclockwise, the extension part tilts to the right of the vertical line. After each ethanol precipitation, the feed pipe needs to be disassembled, cleaned, and reinstalled for the next use. Special attention needs to be paid to the tilting direction of the extension part during installation.

[0015] Preferably, the rotating part is a connecting hose. This configuration is simple in structure, low in cost, easy to assemble and disassemble, and has high cleaning efficiency. Preferably, a support is provided at the bottom of the housing, and a jacket is provided around the periphery of the housing for adjusting or maintaining the temperature of the first liquid.

[0016] Compared with the prior art, the fibrinogen precipitation equipment of the present invention has the following beneficial effects: (1) The fibrinogen precipitation equipment of the present invention can significantly reduce the proportion of precipitate A after adding ethanol, reduce protein denaturation and improve product stability; (2) The fibrinogen precipitation equipment of the present invention can significantly reduce the residual amount of inactivating agent in the product, which is conducive to the control of the impurity content of human fibrinogen process, and realizes the ethanol precipitation purification requirements under the condition of low solubility and / or few precipitation times, taking into account both yield and purification efficiency; (3) The structure is simple and easy to produce and process.

[0017] This invention also provides a fibrinogen purification process, which uses the aforementioned fibrinogen precipitation equipment for ethanol precipitation, comprising the following steps: S1, taking the precipitate of component I, adding 2-6 times (V / M) of dissolving solution to dissolve for 1-3 hours, filtering to the fibrinogen precipitation equipment; adding polysorbate 80 and / or tributyl phosphate, adjusting the pH to 7.15, and then inactivating at room temperature for 3-6 hours; S2, cooling to 0-2°C, adding pre-cooled ethanol solution through feed pipe 2, so that the final ethanol content in the solution is 8% (V / V); S3, adjusting the pH to 6.95, stirring at 0.2°C for 1 hour, letting stand for 2 hours, and then centrifuging to collect the precipitate. The purification process has the same beneficial effects as the precipitation equipment, and will not be elaborated here. Preferably, steps S2 and S3 are repeated 1-3 times. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the fibrinogen precipitation device described in an embodiment of the present invention;

[0019] Figure 2 This is a schematic diagram of a feeding pipe according to an embodiment of the present invention;

[0020] Figure 3 This is a schematic diagram of another structure of the feed pipe according to an embodiment of the present invention;

[0021] Figure 4 This is a schematic diagram of another structure of the feed pipe described in an embodiment of the present invention.

[0022] Explanation of reference numerals in the attached figures:

[0023] 1-Shell; 11-Liquid storage chamber; 2-Feed pipe; 21-Connecting part; 22-Extension part; 23-Rotating part; 24-Liquid outlet; 3-Drive device; 4-Jacket; 5-Support. Detailed Implementation

[0024] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the described embodiments are only some, not all, of the embodiments of the present invention. The specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0025] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0026] Fibrinogen is often purified using low-temperature ethanol precipitation to remove residual inactivating agents such as polysorbate 80 and tributyl phosphate. Since ethanol releases heat during its hydration reaction with water, the ethanol pipeline is usually submerged below the liquid surface when adding ethanol to facilitate diffusion and avoid excessively high local temperatures. For ease of explanation, the solution containing fibrinogen is defined as the first solution, and the organic solvent used for precipitation, such as ethanol, is defined as the second solution; the precipitate adhering to feed pipeline 2 is named "precipitate A," and the particulate precipitate dispersed in the solution is named "precipitate B."

[0027] The applicant's research found that the residual amount of inactivating agent in "precipitate A" was significantly higher than that in "precipitate B," and the larger the clumps of "precipitate A," the higher the residual amount of inactivating agent. A possible reason is that, compared to "precipitate B," the clump-like or blocky state of "precipitate A" causes the precipitate to adhere to and encapsulate the solution, hindering the separation of the precipitate from the solution and impeding the removal of polysorbate 80 and tributyl phosphate, resulting in high residual amounts of inactivating agents such as polysorbate 80 and tributyl phosphate. Compared to the existing vertically arranged feed pipe 2, when the flow direction of the second liquid flowing out of the outlet of feed pipe 2 forms an acute angle with the flow direction of the first liquid flowing through the outlet, the precipitate is less likely to adhere to the ethanol pipe 2 under the flushing action of the first liquid, resulting in less "precipitate A." This allows for the achievement of ideal purification effects with lower solubility and / or fewer precipitation cycles, while also preventing the precipitate from being exposed to air for extended periods.

[0028] like Figure 1-4 As shown, a fibrinogen precipitation device includes a housing 1, a drive unit 3, and a feed pipe 2. The housing 1 has a storage chamber 11 for containing a first liquid containing fibrinogen. The drive unit 3 is connected to the housing 1 to ensure the first liquid is in a flowing state. The feed pipe 2 penetrates the housing 1 and is used to transport a second liquid. The outlet end of the feed pipe 2 extends into the first liquid, and the flow direction of the second liquid at the outlet end forms an angle α with the flow direction of the first liquid, where 0 < α < 90°. This design significantly reduces the formation of "precipitate A," resulting in good purification; it also has a simple structure and is easy to manufacture.

[0029] As an example of the present invention, the driving device 3 is a pump body used to drive the first liquid to flow continuously; preferably, the driving device 3 is a stirring assembly, the stirring assembly including a stirring paddle, the stirring paddle being vertically disposed inside the housing 1, the stirring paddle being driven by a motor, enabling the liquid to flow clockwise or counterclockwise along the inner wall surface of the housing 1. This arrangement requires minimal modification to existing precipitation equipment and achieves good purification effect. Preferably, the stirring paddle is a double-layer three-bladed stirring paddle. As an example of the present invention, the driving device 3 includes a stirring paddle, a gearbox, and a motor, the gearbox being fixed to the top of the housing 1, the motor being fixed to the gearbox, the output shaft of the motor being poweredly connected to the input end of the gearbox, the stirring paddle being disposed inside the housing 1, the root of the stirring paddle extending out of the housing 1 and being poweredly connected to the output end of the gearbox.

[0030] As an example of the present invention, the feed pipe 2 includes a connecting portion 21 and an extension portion 22 connected together. The extension portion 22 is straight and inclined downwards. The inclination direction of the extension portion 22 forms an angle α with the flow direction of the first liquid flowing through the extension portion 22, where 0 < α < 60°. This configuration has a simple structure and is easy to modify and process. Preferably, the feed pipe 2 includes a connecting portion 21 and an extension portion 22 connected together. The extension portion 22 has an outlet 24 at the end away from the connecting portion 21. The extension portion 22 is arc-shaped, and the tangent direction of the circle containing the outlet 24 forms an angle α with the flow direction of the first liquid flowing through the extension portion 22, where 0 < α < 60°. This configuration can reduce the impact of the first liquid on the outer wall of the feed pipe 2, resulting in better purification effect.

[0031] Preferably, the connecting portion 21 is disposed close to the inner wall surface of the housing 1, and the connecting portion 21 and the extension portion 22 are integrally formed. This arrangement can prevent the feed pipe 2 from interfering with the drive device 3, and the second liquid such as ethanol in the feed pipe 2 can diffuse rapidly after entering the first liquid, avoiding excessive local temperature and thus preventing fibrinogen inactivation.

[0032] Preferably, the included angle α is 45°≤α≤60°. When the included angle α is 60°, the ratio of "precipitate A" to "precipitate B" is 1.5%; preferably, when the included angle α is 45°, the ratio of "precipitate A" to "precipitate B" is 0.4%, resulting in good purification effect.

[0033] Preferably, the feed pipe 2 is made of silicone. Since silicone has a higher density than the solution, even if the feed pipe is vertically installed, the outlet 24 will tilt to one side under the impact of the first liquid and will always be below the surface of the first liquid. If the first liquid is flowing in a turbulent state, the feed pipe 2 may be in a swinging state in the first liquid, which is not conducive to the formation of precipitate A.

[0034] Preferably, the feed pipe 2 includes a connecting part 21 and an extension part 22. A rotating part 23 is provided between the extension part 22 and the connecting part 21, which allows the extension part 22 to tilt downwards under the impact of the first liquid flow. Human fibrinogen is a pharmaceutical product, and the pipe needs to be disassembled and cleaned after each separation and purification, and then reinstalled for the next use. Special attention must be paid to ensuring that the outlet direction of the extension part 22 is roughly consistent with the flow direction of the first liquid; opposite directions will produce the opposite effect. By setting the rotating part 23, the extension part 22 can be tilted at a certain angle under the impact of the first liquid, ensuring that it is roughly consistent with the flow direction of the first liquid. Preferably, the rotating part 23 is a connecting hose. This design is simple in structure, low in cost, easy to disassemble and assemble, and has high cleaning efficiency.

[0035] Preferably, a support 5 is provided at the bottom of the housing 1, and a jacket 4 is provided around the periphery of the housing 1 for adjusting or maintaining the temperature of the first liquid. Preferably, the housing 1 is equipped with a pressure gauge, a thermometer, and a level gauge, etc., the specific structure and connection relationship of which are existing technologies and will not be described in detail here.

[0036] In the following examples, the main component of component I is fibrinogen, which is a common name in this technical field. In the following examples, the solution used was: 15 g / L sodium citrate + 9 g / L sodium chloride + 5 g / L tromethorphan, pH 6.80 ± 0.20, with a stirring speed of 50 rpm, and the volume of the fibrinogen precipitation device was 1200 L.

[0037] Example 1

[0038] A fibrinogen ethanol precipitation process, comprising:

[0039] S1. Take 25.6 kg of component I, add 3.5 times (V / M) of dissolving solution, stir and dissolve at 25.5℃ for 1.5 hours, clarify and filter to fibrinogen precipitation equipment, push the solution to 300L, add 3000g of polysorbate 80 and 900g of tributyl phosphate, adjust pH to 6.89, stir at 25℃ for 6 hours to inactivate.

[0040] S2. After filtration, start stirring to cool the filtrate to -1.0℃. Add a 50% (V / V) ethanol solution pre-cooled to below -5℃ through feed pipe 2, so that the final ethanol content of the liquid in storage chamber 11 is 8% (V / V). The outlet end of the feed pipe 2 extends into the filtrate, and the angle α between the flow direction of the 50% (V / V) ethanol solution at the outlet end and the flow direction of the filtrate through the feed pipe 2 is 10°.

[0041] S3. Adjust the pH to 6.97, stir at 0.5℃ for 1 hour, let stand for 2 hours, and then centrifuge to collect a total of 16.00 kg of precipitate. Among them, "precipitate A" attached to feed pipe 2 is 0.02 kg, and "precipitate B" separated from the liquid in storage chamber 11 is 15.98 kg. The centrifuge is a cup centrifuge with a speed of 3500 rpm, a temperature of -2.0℃, and a centrifugation time of 20 min.

[0042] Precipitates A and B were dissolved 20 times their original volume, and the protein content, polysorbate 80, and tributyl phosphate residues were determined. The residual amount of inactivating agent per milligram of protein was calculated, and the results are shown in Table 1. The detection methods for polysorbate 80 and tributyl phosphate are existing technologies and will not be described in detail here.

[0043] Table 1 Residual amounts of polysorbate 80 and tributyl phosphate

[0044]

[0045] As shown in Table 1, the residual amounts of polysorbate 80 and tributyl phosphate corresponding to fibrinogen in "precipitate A" are about 24% and 38% higher than those in precipitate B, respectively. According to the proportion, the residual amounts of polysorbate 80 and tributyl phosphate per milligram of protein in the total precipitate are 41.5 μg / mg and 13.3 μg / mg, respectively.

[0046] Example 2

[0047] A fibrinogen ethanol precipitation process, comprising:

[0048] S1. Take 24.9 kg of component I, add 3.5 times (V / M) of dissolving solution and dissolve at 25.5℃ for 1.5 hours. Clarify and filter to the fibrinogen precipitation device, push the solution to 300L, add 3000g of polysorbate 80 and 900g of tributyl phosphate, adjust the pH to 6.92, stir at 25℃ for 6 hours to inactivate.

[0049] S2. After filtration, start stirring to cool the filtrate to -1.0℃. Add a 50% (V / V) ethanol solution pre-cooled to below -5℃ through feed pipe 2, so that the final ethanol content of the liquid in storage chamber 11 is 8% (V / V). The outlet end of feed pipe 2 extends into the filtrate, and the angle α between the flow direction of the 50% (V / V) ethanol solution at the outlet end and the flow direction of the filtrate through feed pipe 2 is 60°.

[0050] S3. Adjust the pH to 6.92, stir at 0.5℃ for 1 hour, let stand for 2 hours, and then centrifuge to collect a total of 13.50 kg of precipitate. Among them, 0.2 kg of precipitate A adhered to the feed pipe 2, and 13.30 kg of precipitate B was separated from the solution in the storage chamber 11. The centrifuge was a cup centrifuge with a speed of 3500 rpm and a temperature of -2.0℃ for 20 minutes.

[0051] Precipitates A and B were dissolved in 20 times the amount of dissolving solution, and the protein content, polysorbate 80 and tributyl phosphate residues were detected. The residual amount of inactivating agent per milligram of protein was calculated. The results are shown in Table 2.

[0052] Table 2 Residual amounts of polysorbate 80 and tributyl phosphate

[0053]

[0054] As shown in Table 2, the residual amounts of polysorbate 80 and tributyl phosphate corresponding to fibrinogen in precipitate A are approximately 27% and 36% higher, respectively, than those in precipitate B. Based on these proportions, the residual amounts of polysorbate 80 and tributyl phosphate per milligram of protein in the total precipitate are 42.9 μg / mg and 14.1 μg / mg, respectively.

[0055] Example 3

[0056] A fibrinogen ethanol precipitation process, comprising:

[0057] S1. Take 35.4 kg of the precipitate of component I, add 5 times (V / M) of dissolving solution and dissolve at 25.0℃ for 3.0 h. Filter the solution through a 30 LP filter press with a 1.0 μm filter cartridge to the fibrinogen precipitation device. Push the solution to 350 L, add 3500 g of polysorbate 80 and 1050 g of tributyl phosphate, adjust the pH to 7.15, and inactivate at 25℃ for 6 h.

[0058] S2. After filtration, start stirring to cool the filtrate to -0.5℃. Add a 50% (V / V) ethanol solution pre-cooled to below -5℃ through the feed pipe 2, so that the final ethanol content in the solution is 8% (V / V). The feed pipe 2 includes a connecting part 21 and an extension part 22. The extension part 22 is arc-shaped, and an outlet 24 is provided at the end of the extension part 22 away from the connecting part 21. The outlet 24 is located below the liquid surface of the filtrate. The tangent direction of the circle containing the outlet 24 forms an angle α of 45° with the flow direction of the first liquid flowing through the extension part 22.

[0059] S3. Adjust the pH to 6.95, stir at 0.2℃ for 1 hour, let stand for 2 hours, and then centrifuge to collect a total of 18.50 kg of precipitate. Among them, 0.07 kg of precipitate A adhered to the feed pipe 2, and 18.43 kg of precipitate B was separated from the solution in the storage chamber 11. The centrifuge was a cup centrifuge with a speed of 3500 rpm and a temperature of -2.0℃ for 20 minutes.

[0060] Precipitates A and B were dissolved 20 times each, and the protein content, polysorbate 80, and tributyl phosphate residues were detected. The residual amount of inactivating agent per milligram of protein was calculated, and the results are shown in Table 3.

[0061] Table 3 Residual amounts of polysorbate 80 and tributyl phosphate

[0062]

[0063]

[0064] As shown in Table 3, the residual amounts of polysorbate 80 and tributyl phosphate per milligram of protein in precipitate A are approximately 29% and 35% higher, respectively, than those in precipitate B. Based on these proportions, the residual amounts of polysorbate 80 and tributyl phosphate per milligram of protein in the total precipitate are 41.9 μg / mg and 13.9 μg / mg, respectively.

[0065] Example 4

[0066] A fibrinogen ethanol precipitation process, comprising:

[0067] S1. Take 33.9 kg of the precipitate of component I, add 5 times (V / M) of the first dissolving solution and dissolve at 25.0℃ for 3.0 h. Filter the solution through a 30 L filter press with a 1.0 μm filter element to the fibrinogen precipitation device. Push the solution to 350 L, add 3500 g of polysorbate 80 and 1050 g of tributyl phosphate, adjust the pH to 7.08, and inactivate at 25℃ for 6 h.

[0068] S2. After filtration, start stirring to cool the filtrate to -0.5℃. Add a 50% (V / V) ethanol solution pre-cooled to below -5℃ through feed pipe 2, so that the final ethanol content in the solution is 8% (V / V). The feed pipe 2 is made of elastic silicone and is vertically arranged. Its outlet 24 is located below the surface of the filtrate. When the filtrate flows, the feed pipe 2 forms an angle of 0-60° with the flow direction of the filtrate due to the impact of the filtrate.

[0069] S3. Adjust the pH to 6.90, stir at 0.2℃ for 1 hour, let stand for 2 hours, and then centrifuge to collect a total of 18.00 kg of precipitate. Of this, 0 kg of precipitate A attached to the ethanol pipe and 18.00 kg of precipitate B separated from the solution were collected. The centrifugation was performed using a cup centrifuge at a speed of 3500 rpm and a temperature of -2.0℃ for 20 minutes.

[0070] Precipitates A and B were dissolved 20 times each, and the protein content, polysorbate 80, and tributyl phosphate residues were detected. The residual amount of inactivating agent per milligram of protein was calculated, and the results are shown in Table 4.

[0071] Table 4 Residual amounts of polysorbate 80 and tributyl phosphate

[0072]

[0073] As shown in Table 4, the amount of precipitate A at this time is 0 kg, and the purification effect is good.

[0074] Example 5

[0075] A fibrinogen ethanol precipitation process, comprising:

[0076] S1. Take 30.6 kg of the precipitate of component I, add 5 times (V / M) of dissolving solution and dissolve at 25.0℃ for 3.0 h. Filter the solution through a 30 LP filter press with a 1.0 μm filter cartridge to a fibrinogen precipitation device. Purge the solution to 300 L, add 3000 g of polysorbate 80 and 900 g of tributyl phosphate, adjust the pH to 7.01, and inactivate at 25℃ for 6 h.

[0077] S2. After filtration, start stirring and cool the filtrate to -0.5℃. Add a 50% (V / V) ethanol solution pre-cooled to below -5℃ through feed pipe 2 to make the final ethanol content in the solution 8% (V / V).

[0078] The feed pipe 2 includes a connecting part 21 and an extension part 22. A rotating part 23 is provided between the extension part 22 and the connecting part 21, which can tilt downward after the first liquid flows. The rotating part 23 is a flexible hose, which is detachably connected to the connecting part 21 and the extension part 22. This ensures that after the agitator rotates, the outlet 23 automatically swings under the impact force of the flowing filtrate, with the direction generally consistent with the flow direction of the filtrate.

[0079] S3. Adjust the pH to 6.86, stir at 0℃ for 1 hour, let stand for 2 hours, and then centrifuge to collect a total of 16.60 kg of precipitate. Among them, 0.03 kg of precipitate A adhered to the feed pipe 2, and 16.57 kg of precipitate B was separated from the solution in the storage chamber 11. The centrifuge was a cup centrifuge with a speed of 3500 rpm and a temperature of -2.0℃ for 20 minutes.

[0080] Precipitates A and B were dissolved 20 times each, and the protein content, polysorbate 80, and tributyl phosphate residues were detected. The residual amount of inactivating agent per milligram of protein was calculated, and the results are shown in Table 5.

[0081] Table 5 Residual amounts of polysorbate 80 and tributyl phosphate

[0082]

[0083] As shown in Table 5, the residual amounts of polysorbate 80 and tributyl phosphate per milligram of protein in precipitate A are approximately 29% and 36% higher, respectively, than those in precipitate B. Based on these proportions, the residual amounts of polysorbate 80 and tributyl phosphate per milligram of protein in the total precipitate are 41.6 μg / mg and 13.5 μg / mg, respectively.

[0084] Comparative Example 1

[0085] Ethanol precipitation was performed using the same method as in Example 1, except that the feed pipe 2 extended vertically below the liquid surface and was made of metal. The outlet direction of the feed pipe 2 was always perpendicular to the flow direction of the filtrate. Other process parameters were the same as in Example 1. The experiment was repeated three times, and the results are shown in Table 6.

[0086] Table 6. Residual analysis of inactivating agents in different groups

[0087]

[0088] As shown in Table 6, when fibrinogen is separated by the low-temperature ethanol method, the proportion of clump-shaped and block-shaped precipitate A is significantly reduced when using the fibrinogen precipitation equipment of the present invention, which is beneficial to reduce protein denaturation and improve product stability; the residual amount of inactivating agent in the precipitate is significantly reduced, which is beneficial to the control of process impurities in the product.

[0089] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A fibrinogen purification process, characterized in that, Ethanol precipitation is performed using a fibrinogen precipitation device, which includes a shell (1), a drive device (3), and a feed pipe (2). The shell (1) is provided with a liquid storage chamber (11) for containing a first liquid containing fibrinogen. The drive device (3) is connected to the shell (1) to ensure that the first liquid is in a flowing state. The feed pipe (2) is provided through the shell (1) for conveying a second liquid. The feed pipe (2) includes an inclined extension (22) that partially extends into the first liquid. The direction of the extension (22) forms an angle α with the flow direction of the first liquid flowing through the feed pipe (2), where 0 < α < 90°. The process includes the following steps: S1. Take the precipitate of component I, add 2-6 times (V / M) of dissolving solution to dissolve for 1-3 hours, filter to the fibrinogen precipitation device; add polysorbate 80 and / or tributyl phosphate, adjust the pH to 7.15 and inactivate at room temperature for 3-6 hours; S2. Cool down to 0-2℃, and add the pre-cooled ethanol solution through feed pipe 2 to make the final ethanol content in the solution 8% (V / V). S3. Adjust the pH to 6.95, stir at 0.2℃ for 1 hour, let stand for 2 hours, and then centrifuge to collect the precipitate.

2. The fibrinogen purification process according to claim 1, characterized in that, Steps S2 and S3 are repeated 1-3 times.

3. The fibrinogen purification process according to claim 1, characterized in that, The driving device (3) is a stirring assembly, which includes a stirring paddle. The stirring paddle is vertically disposed inside the housing (1). The stirring paddle is driven by a motor and can make the liquid flow clockwise or counterclockwise along the inner wall of the housing (1).

4. The fibrinogen purification process according to claim 3, characterized in that, The feed pipe (2) also includes a connecting part (21) connected to the extension (22). The extension (22) is straight and inclined downward. The inclination direction of the extension (22) forms an angle α with the flow direction of the first liquid through the extension (22), where 0 < α < 60°.

5. The fibrinogen purification process according to claim 3, characterized in that, The feed pipe (2) includes a connecting part (21) connected to the extension (22). The extension (22) has an outlet (24) at one end away from the connecting part (21). The extension (22) is arc-shaped. The tangent direction of the circle containing the extension (22) at the outlet (24) forms an angle α with the flow direction of the first liquid flowing through the extension (22), where 0 < α < 60°.

6. The fibrinogen purification process according to any one of claims 4 or 5, characterized in that, The connecting part (21) is disposed near the inner wall surface of the housing (1), and the connecting part (21) and the extension part (22) are integrally formed.

7. The fibrinogen purification process according to claim 1, characterized in that, The material of the feed pipe (2) is silicone.

8. The fibrinogen purification process according to claim 1, characterized in that, The feed pipe (2) includes a connecting part (21) and an extension part (22). A rotating part (23) is provided between the extension part (22) and the connecting part (21), which can make the extension part (22) tilt downward after the first liquid flows.

9. The fibrinogen purification process according to claim 8, characterized in that, The rotating part (23) is a connecting hose.

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