Process for preparing high-purity human blood coagulation factor IX from plasma

Abstract

The invention provides a process for preparing a high-purity human blood coagulation factor IX from plasma. The process comprises the following steps: acquiring plasma; adding anion gel, adsorbing, and collecting an eluted protein solution; adding heparin sodium, filtering, ultra-filtering and dialyzing to obtain dialyzed protein liquid; adding an S / D solution; adding anion gel for secondary adsorption, and collecting eluted protein liquid II; performing filtration, ultrafiltration and dialysis, and collecting eluted protein liquid after dialysis; filtering is performed; carrying out affinity gel chromatography purification, collecting eluent, and carrying out ultrafiltration and dialysis; filtering, and adding a dialysate containing glycine for dialysis to obtain a human blood coagulation factor IX stock solution; adjusting the titer, and adding heparin sodium to obtain a diluted solution; pre-filtering the diluted liquid to obtain a semi-finished product of the human blood coagulation factor IX; sterilizing, filtering, sub-packaging, carrying out vacuum freeze drying, carrying out dry heat virus inactivation, inspecting and packaging. According to the invention, the production process is simplified, the cost is reduced, the activity of the finally obtained human blood coagulation factor IX is greater than 50IU / ml, and the specific activity is higher than 180IU / mg.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a process for preparing high-purity human coagulation factor IX from plasma. Background Art

[0002] Human coagulation factor IX (FIX) is an indispensable vitamin K-dependent glycoprotein involved in the human coagulation process. Its relative molecular mass is about 57kDa. It is synthesized by liver cells in the body and secreted into the blood. The concentration of human coagulation factor IX in plasma is very low, about 5μg / mL in healthy adults. When the content or activity of human coagulation factor IX in plasma drops significantly, the endogenous coagulation pathway is blocked and the human body cannot carry out normal coagulation, thus causing hemophilia B.

[0003] Hemophilia B is mainly inherited. One in 30,000 to 50,000 male babies has hemophilia B, which is a rare genetic disease. Currently, the safest and most effective way to treat hemophilia B is to use purified and concentrated human coagulation factor IX for intravenous injection, but the traditional production process of purified human coagulation factor IX has many steps and is very costly.

[0004] Therefore, there is an urgent need to develop a process for preparing high-purity human coagulation factor IX with relatively simple procedures and lower costs. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a process for preparing high-purity human coagulation factor IX from plasma. The process uses plasma as a raw material and refines the high-purity human coagulation factor IX through centrifugation, anion gel chromatography, ultrafiltration, S / D virus inactivation, and affinity gel chromatography. The process is simple and the cost is low.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A process for preparing high-purity human coagulation factor IX from plasma comprises the following steps:

[0008] (1) Frozen plasma is thawed and centrifuged at 0-4°C to obtain cryoprecipitated plasma;

[0009] (2) adding anionic gel at 1.5-2.0% by weight of plasma to the product obtained in step (1), stirring and adsorbing for 35-65 minutes, and controlling the temperature at 10-20° C. to obtain eluted protein solution I;

[0010] (3) adding the eluted protein solution I obtained in step (2) to sodium heparin injection, filtering, ultrafiltration through an ultrafiltration membrane, and dialyzing to obtain a dialyzed protein solution;

[0011] (4) adding the S / D solution to the dialyzed protein solution obtained in step (3), stirring evenly, controlling the temperature at 24-26° C., and keeping the temperature for 5-7 hours to obtain a S / D protein solution;

[0012] (5) adding 1.5-2.0% by weight of anionic gel to the protein solution after S / D obtained in step (4) for a second adsorption, stirring and adsorbing for 35-65 minutes, controlling the temperature at 10-20° C., and collecting the eluted protein solution II;

[0013] (6) filtering the eluted protein solution II obtained in step (5) through a PP filter element, using an ultrafiltration membrane for ultrafiltration, and dialyzing with a dialysate, and collecting the eluted protein solution II after dialysis;

[0014] (7) filtering the eluted protein solution II obtained after dialysis in step (6) through a PP filter element to obtain a protein solution before affinity chromatography column;

[0015] (8) purifying the protein solution before affinity chromatography obtained in step (7) by affinity gel chromatography, collecting the eluate, and obtaining the protein solution before nanofiltration by ultrafiltration and dialysis;

[0016] (9) After filtering the protein solution before nanofiltration obtained in step (8), adding an equal volume of dialysate containing 1 to 4 g / L glycine and 3 to 9 g / L arginine hydrochloride to perform ultrafiltration and dialyze 1 to 3 times, collecting the target protein solution to obtain a human coagulation factor IX stock solution; adjusting the titer of the human coagulation factor IX stock solution to 55.0 to 65.0 IU / ml with the dialysate, and adding sodium heparin according to the final "0.1 IU / IU human coagulation factor IX titer" to obtain a diluted solution; pre-filtering the diluted solution through a filter element to obtain a human coagulation factor IX semi-finished product;

[0017] (10) The semi-finished product obtained in step (9) is sterilized, filtered, packaged, vacuum freeze-dried, capped, and dry-heat virus inactivated, then sent for inspection and light inspection. After the product is qualified, it is packaged and stored.

[0018] As one of the preferred embodiments of the present invention, in the step (2), the plasma pH is first adjusted to 6.8-7.2 using an acetic acid-sodium acetate buffer, and then 1.5-2.0% of the plasma weight of the balanced anionic gel is added, and the adsorption is stirred for 35-65 minutes, and the temperature is controlled at 10-20°C; then, the adsorbed gel is collected in a gel column, and the gel is washed 3-5 times with a washing solution, each time stirring for 4-6 minutes and then dried; then the gel is eluted with an eluting solution for 2-4 times, each time stirring for 10-15 minutes, and the eluted protein solution I is collected.

[0019] As one of the preferred embodiments of the present invention, in the step (5), firstly, 1.5-2.0% by weight of the balanced anionic gel is added to the S / D protein solution, and the mixture is stirred for adsorption for 35-65 minutes, with the temperature controlled at 10-20°C; then, the gel is washed 7-9 times with a washing solution, each time stirred for 4-6 minutes and then dried; then, the gel is eluted 2-4 times with an eluting solution, each time stirred for 10-15 minutes, and the eluted protein solution II is collected.

[0020] As one of the preferred embodiments of the present invention, the anionic gel in steps (2) and (5) is NW Rose DEAE (BB) gel produced by Suzhou Nanovita Technology Co., Ltd.; the balancing solution formula during the use of the anionic gel is: 5.51-6.12 g / L sodium citrate, 4.54-5.12 g / L sodium chloride, pH 6.8-7.2; the washing solution formula is: 2.84-3.12 g / L sodium citrate, 10.1-12.5 g / L sodium chloride, pH 6.8-7.2; the eluent formula is: 111-130 g / sodium chloride, 2.84-3.12 g / L sodium citrate, pH 6.8-7.2.

[0021] As one of the preferred embodiments of the present invention, in the step (8), the specific steps of affinity gel chromatography purification are: loading the protein solution before affinity chromatography onto an affinity gel chromatography column equilibrated with an equilibrated solution; after loading, first washing 2 to 4 times the volume of the gel column with the equilibrated solution, then washing 2 to 4 times the volume of the gel column with the washing solution, and then washing the chromatography column with 2 to 4 times the volume of the gel column with the equilibrated solution until a baseline is formed; after washing, eluting with an eluent, and collecting the eluted protein solution III.

[0022] As one of the preferred embodiments of the present invention, in the step (8), the affinity gel adopts NW Rose Plasmid affinity gel produced by Suzhou Nanovita Technology Co., Ltd.; the balance solution formula during the affinity gel chromatography is: 2.0-3.0 g / L tromethamine, 8.0-9.5 g / L sodium chloride, pH 7.0-7.8; the washing solution formula is: 2.42-4.84 g / L tromethamine, 29.25-58.50 g / L sodium chloride, pH 7.2-7.6; the eluent formula is: 7.5-11.3 g / L glycine, pH 6.0-7.0.

[0023] As one of the preferred embodiments of the present invention, in step (8), the specific steps of ultrafiltration and dialysis are: the eluted protein solution collected by affinity gel chromatography purification is concentrated using a 10kD ultrafiltration membrane, and then an equal volume of dialysis fluid is added and dialyzed 5 to 7 times to obtain a protein solution before nanofiltration, and a sample is taken to detect the pH and protein content.

[0024] As one of the preferred embodiments of the present invention, in step (9), the dialysate containing 1-4 g / L glycine and 3-9 g / L arginine hydrochloride has the following formula: 2.9 g / L sodium citrate, 5.8 g / L sodium chloride, 1-4 g / L glycine, 3-9 g / L arginine hydrochloride, pH 6.8-7.2.

[0025] As one of the preferred embodiments of the present invention, in step (10), the vacuum freeze-drying conditions are:

[0026] a. Products are put into the cabinet at room temperature;

[0027] b. The temperature of the plate layer drops to -6°C within 10 minutes and remains at this temperature for 30 minutes;

[0028] c. The temperature of the board layer drops below -45℃ within 60 minutes and remains below this temperature for 180 minutes;

[0029] d. Raise the temperature of the board layer to -15°C within 60 minutes and maintain it for 120 minutes;

[0030] e. The temperature of the board layer drops below -45℃ within 120min and remains below this temperature for 180min;

[0031] f. Turn on the vacuum pump to make the vacuum degree reach 0.3mbar;

[0032] g. The temperature of the board layer is raised to -33°C within 60 minutes and maintained for 180 minutes;

[0033] h. The temperature of the board layer is raised to 0°C within 300 minutes and maintained for 1200 minutes;

[0034] i. The temperature of the board layer rises to 10℃ within 120 minutes and is maintained for 120 minutes;

[0035] j. The temperature of the plate layer rises to 33°C within 120 minutes and is kept warm for 360 minutes;

[0036] k. Pump the ultimate vacuum at 33℃ until the vacuum is qualified;

[0037] l. Press the plug under vacuum.

[0038] As one of the preferred embodiments of the present invention, in step (10), the dry heat virus inactivation process is 80°C

[0039] Heat for 0.5h.

[0040] As one of the preferred embodiments of the present invention, the activity of human coagulation factor IX finally obtained is greater than 50 IU / ml, and the specific activity is higher than 180 IU / mg.

[0041] The advantages of the present invention compared to the prior art are:

[0042] (1) The present invention only adds one step of affinity gel chromatography after anion exchange chromatography, which simplifies the production process of human coagulation factor IX and reduces the cost. At the same time, the affinity chromatography specific adsorption technology is used to greatly improve the overall yield of human coagulation factor IX. The affinity chromatography yield can reach 80-90%;

[0043] (2) The anion gel and affinity gel used in the process of the present invention are both domestically produced gels. The advantages of domestically produced gels include: ① the price is much lower than that of foreign gels, further saving development and production costs; ② the purchase cycle of domestically produced gels is shortened, improving research and development efficiency;

[0044] (3) The specific activity of human coagulation factor IX finally prepared by the present invention is ≥180 IU / mg (European Pharmacopoeia requires ≥50 IU / mg), and the content of impurities such as protein C, protein S, IgA, IgG, fibrinogen, plasminogen, ceruloplasmin, C3, albumin, human coagulation factor II, VII, X, and aluminum residue in the final product is extremely low, which can ensure the safety of the product for clinical use;

[0045] (4) The present invention utilizes affinity gel chromatography to purify human coagulation factor IX. After the loading, washing and elution processes, it can not only remove foreign proteins to the greatest extent, but also maintain the activity of human coagulation factor IX and improve the specific activity of human coagulation factor IX. The human coagulation factor IX activity yield of the eluate is greater than 90%, and the specific activity of human coagulation factor IX is greater than 180 IU / mg.

[0046] (5) The present invention adopts S / D, nano-membrane filtration, and dry heat three-step virus inactivation to ensure the safety of the drug. Among them, the dry heat inactivation process is heating at 80°C for 0.5h, which can inactivate viruses such as HBV, HCV, HIV and HAV;

[0047] (6) During the dilution process of the present invention, 1-4 g / L glycine and 3-9 g / L arginine hydrochloride are used as protective agents (added in the form of dialysate), and sodium heparin is added at a titer of 0.1 IU / IU human coagulation factor IX to ensure that the active ingredients are not affected during the subsequent freeze-drying and dry-heating processes. At the same time, the addition of sodium heparin ensures the safety of subsequent medication.

[0048] (7) The freeze-drying process of the present invention includes an annealing process, so that the crystal size, shape, and pore distribution of the freeze-dried product are more uniform, and the quality of the same batch of products is consistent. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a process flow chart for preparing high-purity human coagulation factor IX from plasma in Examples 1 to 3. DETAILED DESCRIPTION

[0050] The embodiments of the present invention are described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the reagent products and experimental methods used in the following embodiments, unless otherwise specified, are conventional reagents or methods in the art and will not be repeated.

[0051] Example 1

[0052] A process for preparing high-purity human coagulation factor IX from plasma in this embodiment comprises the following steps:

[0053] (1) After receiving 3000L of human plasma that has passed the quarantine period, the bags are opened under the protection of laminar flow in a Class D environment; the plasma is combined into a slurry melting tank and thawed with circulating water at 30°C, and the plasma temperature is controlled at 0°C; after the plasma is melted, it is centrifuged with a centrifuge with a centrifugal force of 6000g, an outlet temperature of 0°C, and an outlet flow rate of 2kg / min / unit, and the cryoprecipitated plasma is collected; the cryoprecipitated plasma is transferred to an adsorption tank, and the plasma temperature is controlled at 10°C.

[0054] (2) First, the pH value of the plasma in step (1) was adjusted to 6.8 using acetic acid-sodium acetate buffer, and then 1.5% of the weight of the plasma was added to an anionic gel (NW Rose DEAE (BB) gel produced by Suzhou Nanotech Co., Ltd.) with equilibrium (equilibrium solution: 5.51 g / L sodium citrate, 4.54 g / L sodium chloride, pH 6.8), and the adsorption was stirred for 35 minutes, and the temperature was controlled at 10°C; then, the adsorbed gel was collected in a gel column, and the gel was washed 3 times with a washing solution (2.84 g / L sodium citrate, 10.1 g / L sodium chloride, pH 6.8), each time stirred for 4 minutes and then dried; then the gel was eluted twice with an elution solution (111 g / sodium chloride, 2.84 g / L sodium citrate, pH 6.8), each time stirred for 10 minutes, and the eluted protein solution I was collected.

[0055] (3) The eluted protein solution I obtained in step (2) was added with 140 IU of heparin sodium injection, filtered through a PP filter element with a pore size of 0.45 μm, concentrated to 50 L using a 10 KD ultrafiltration membrane, and dialyzed using 200 L of dialysate (4.0 g / L arginine hydrochloride, 5.4 g / L sodium citrate, pH 7.2) until the conductivity of the dialyzed solution was less than 15 ms / cm, and the dialyzed protein solution was collected.

[0056] (4) Add 13 kg of S / D solution (32 g / L tributyl phosphate, 105 g / L polysorbate 80) to the dialyzed protein solution obtained in step (3), and the liquid feeding rate should be 2 kg / min; after the addition of the S / D solution is completed, set the stirring speed to 20 r / min, and keep warm at 24°C for 5 h to obtain the S / D protein solution.

[0057] (5) Add 1.5-2.0% by weight of the equilibrated anionic gel (same as step 2) to the S / D protein solution obtained in step (4), and stir for adsorption for 35 minutes while controlling the temperature at 10°C; then, wash the gel 7 times with 50 L of washing solution, stirring for 4 minutes each time and then letting it dry; then, elute the gel 2 times with 45 L of eluting solution, stirring for 10 minutes each time, and collect the eluted protein solution II.

[0058] (6) The eluted protein solution II obtained in step (5) was filtered through a PP filter element with a pore size of 0.45 μm, and the filtered eluted protein solution II was concentrated to 50 L using a 10KD ultrafiltration membrane. It was then dialyzed using 350 L of dialysis fluid (4.0 g / L arginine hydrochloride, 5.4 g / L sodium citrate, pH 7.2), and the dialyzed eluted protein solution II was collected.

[0059] (7) Filter the eluted protein solution II obtained in step (6) through a PP filter element with a pore size of 0.45 μm to obtain the protein solution before affinity chromatography column.

[0060] (8) The protein solution before affinity chromatography obtained in step (7) is loaded onto an affinity gel chromatography column (the affinity gel uses NW Rose Plasmid affinity gel produced by Suzhou Nanotech Co., Ltd.) equilibrated with an equilibration solution (2.0 g / L tromethamine, 8.0 g / L sodium chloride, pH 7.0); after loading, the equilibration solution is used to wash the gel column twice by volume, and then the washing solution (2.43 g / L tromethamine, 29.25 g / L sodium chloride, pH 7.2) is used to wash the gel column twice by volume, and then the equilibration solution is used to wash the chromatography column by washing the gel column twice by volume until a baseline is formed; after washing, the elution is performed with an eluent (7.5 g / L glycine, pH 6.0), and the eluted protein solution III is collected.

[0061] The eluted protein solution collected by affinity gel chromatography purification was concentrated with a 10kD ultrafiltration membrane, and then dialyzed five times with equal volumes of dialysis fluid (2.9 g / L sodium citrate, 5.8 g / L sodium chloride and 1 g / L glycine, pH 6.8) to obtain the protein solution before nanofiltration, and samples were taken to detect pH and protein content.

[0062] (9) After filtering the protein solution before nanofiltration obtained in step (8) through a 0.1 μm series 20 nm filter element, add a dialysate (2.9 g / L sodium citrate, 5.8 g / L sodium chloride, 1 g / L glycine, 3 g / L arginine hydrochloride, pH 6.8) of equal volume for ultrafiltration and dialysis once, collect the target protein solution, and obtain a human coagulation factor IX stock solution; adjust the titer of the human coagulation factor IX stock solution to 55.0 IU / ml with the dialysate, and add sodium heparin according to the final "0.1 IU / IU human coagulation factor IX titer" to obtain a diluted solution; pre-filter the diluted solution through a 0.2 μm filter element to obtain a human coagulation factor IX semi-finished product.

[0063] (10) The semi-finished product obtained in step (9) is sterilized and filtered through a 0.2 μm filter element, packaged, vacuum freeze-dried, capped, and dry-heat virus inactivated (80° C., 0.5 h), then sent for inspection and light inspection. After the product passes the inspection, it is packaged and stored.

[0064] Wherein, vacuum freeze drying conditions are:

[0065] a. Products are put into the cabinet at room temperature;

[0066] b. The temperature of the plate layer drops to -6°C within 10 minutes and remains at this temperature for 30 minutes;

[0067] c. The temperature of the board layer drops below -45℃ within 60 minutes and remains below this temperature for 180 minutes;

[0068] d. Raise the temperature of the board layer to -15°C within 60 minutes and maintain it for 120 minutes;

[0069] e. The temperature of the board layer drops below -45℃ within 120min and remains below this temperature for 180min;

[0070] f. Turn on the vacuum pump to make the vacuum degree reach 0.3mbar;

[0071] g. The temperature of the board layer is raised to -33°C within 60 minutes and maintained for 180 minutes;

[0072] h. The temperature of the board layer is raised to 0°C within 300 minutes and maintained for 1200 minutes;

[0073] i. The temperature of the board layer rises to 10℃ within 120 minutes and is maintained for 120 minutes;

[0074] j. The temperature of the plate layer rises to 33°C within 120 minutes and is kept warm for 360 minutes;

[0075] k. Pump the ultimate vacuum at 33℃ until the vacuum is qualified;

[0076] l. Press the plug under vacuum.

[0077] Example 2

[0078] A process for preparing high-purity human coagulation factor IX from plasma in this embodiment comprises the following steps:

[0079] (1) After receiving 3000L of human plasma that has passed the quarantine period, the bags are opened under the protection of laminar flow in a Class D environment; the plasma is combined into a slurry melting tank and thawed with circulating water at 35°C, and the plasma temperature is controlled at 4°C; after the plasma is thawed, it is centrifuged with a centrifuge with a centrifugal force of 10,000g, an outlet temperature of 4°C, and an outlet flow rate of 3kg / min / unit, and the cryoprecipitated plasma is collected; the cryoprecipitated plasma is transferred to an adsorption tank, and the plasma temperature is controlled at 15°C.

[0080] (2) First, the pH value of the plasma in step (1) was adjusted to 7.0 using acetic acid-sodium acetate buffer, and then 1.8% of the plasma weight of anionic gel (NW Rose DEAE (BB) gel produced by Suzhou Nanotech Co., Ltd.) was added, and the adsorption was stirred for 50 minutes, and the temperature was controlled at 15°C; then, the adsorbed gel was collected in a gel column, and the gel was washed 4 times with a washing solution (3.10 g / L sodium citrate, 11.7 g / L sodium chloride, pH7.0), each time stirred for 5 minutes and then dried; then the gel was eluted 3 times with an elution solution (117 g / sodium chloride, 3.10 g / L sodium citrate, pH7.0), each time stirred for 12 minutes, and the eluted protein solution I was collected.

[0081] (3) The eluted protein solution I obtained in step (2) was added with 140 IU of heparin sodium injection, filtered through a PP filter element with a pore size of 0.45 μm, concentrated to 50 L using a 10 KD ultrafiltration membrane, and dialyzed using 200 L of dialysate (4.25 g / L arginine hydrochloride, 6.0 g / L sodium citrate, pH 7.5) until the conductivity of the dialyzed solution was less than 15 ms / cm, and the dialyzed protein solution was collected.

[0082] (4) Add 13 kg of S / D solution (33 g / L tributyl phosphate, 110 g / L polysorbate 80) to the dialyzed protein solution obtained in step (3), and the liquid feeding rate should be 2.0 kg / min; after the addition of the S / D solution is completed, set the stirring speed to 100 r / min, and keep warm at 25°C for 6 h to obtain the S / D protein solution.

[0083] (5) Add 1.8% by weight of the equilibrated anionic gel (same as step 2) to the S / D protein solution obtained in step (4), and stir for 50 min while controlling the temperature at 15°C; then, wash the gel 8 times with 50 L of washing solution, stirring for 5 min each time and then letting it dry; then, elute the gel 3 times with 45 L of eluting solution, stirring for 12 min each time, and collect the eluted protein solution II.

[0084] (6) The eluted protein solution II obtained in step (5) was filtered through a PP filter element with a pore size of 0.45 μm, and the filtered eluted protein solution II was concentrated to 50 L using a 10KD ultrafiltration membrane. It was then dialyzed using 350 L of dialysis fluid (4.25 g / L arginine hydrochloride, 6.0 g / L sodium citrate, pH 7.5), and the dialyzed eluted protein solution II was collected.

[0085] (7) Filter the eluted protein solution II obtained in step (6) through a PP filter element with a pore size of 0.45 μm to obtain the protein solution before affinity chromatography column.

[0086] (8) The protein solution before affinity chromatography obtained in step (7) is loaded onto an affinity gel chromatography column (the affinity gel uses NW Rose Plasmid affinity gel produced by Suzhou Nanotech Co., Ltd.) equilibrated with an equilibration solution (2.42 g / L tromethamine, 8.78 g / L sodium chloride, pH 7.5); after loading, the equilibration solution is used to wash 3 times the volume of the gel column, and then the washing solution (2.42 g / L tromethamine, 43.88 g / L sodium chloride, pH 7.5) is used to wash 3 times the volume of the gel column, and then the equilibration solution is used to wash the chromatography column for another 3 times the volume of the gel column until a baseline is formed; after washing, the elution is performed with an eluent (9.4 g / L glycine, pH 6.5), and the eluted protein solution III is collected.

[0087] The eluted protein solution collected by affinity gel chromatography purification was concentrated with a 10kD ultrafiltration membrane, and then dialyzed six times with equal volumes of dialysis fluid (2.9 g / L sodium citrate, 5.8 g / L sodium chloride and 2 g / L glycine, pH 7.0) to obtain the protein solution before nanofiltration, and samples were taken to detect pH and protein content.

[0088] (9) The protein solution before nanofiltration obtained in step (8) is filtered through a 0.1 μm series 20 nm filter element, and then an equal volume of dialysate (2.9 g / L sodium citrate, 5.8 g / L sodium chloride, 2 g / L glycine, 7 g / L arginine hydrochloride, pH 7.0) (2.9 g / L sodium citrate, 5.8 g / L sodium chloride and 2 g / L glycine, pH 7.0) is added for ultrafiltration and dialyzation twice, and the target protein solution is collected to obtain a human coagulation factor IX stock solution; the titer of the human coagulation factor IX stock solution is adjusted to 60 IU / ml with the dialysate, and sodium heparin is added according to the final "0.1 IU / IU human coagulation factor IX titer" to obtain a diluted solution; the diluted solution is pre-filtered through a 0.2 μm filter element to obtain a human coagulation factor IX semi-finished product.

[0089] (10) The semi-finished product obtained in step (9) is sterilized and filtered through a 0.2 μm filter element, packaged, vacuum freeze-dried, capped, and dry-heat virus inactivated (80° C., 0.5 h), then sent for inspection and light inspection. After the product passes the inspection, it is packaged and stored.

[0090] Wherein, vacuum freeze drying conditions are:

[0091] a. Products are put into the cabinet at room temperature;

[0092] b. The temperature of the plate layer drops to -6°C within 10 minutes and remains at this temperature for 30 minutes;

[0093] c. The temperature of the board layer drops below -45℃ within 60 minutes and remains below this temperature for 180 minutes;

[0094] d. Raise the temperature of the board layer to -15°C within 60 minutes and maintain it for 120 minutes;

[0095] e. The temperature of the board layer drops below -45℃ within 120min and remains below this temperature for 180min;

[0096] f. Turn on the vacuum pump to make the vacuum degree reach 0.3mbar;

[0097] g. The temperature of the board layer is raised to -33°C within 60 minutes and maintained for 180 minutes;

[0098] h. The temperature of the board layer is raised to 0°C within 300 minutes and maintained for 1200 minutes;

[0099] i. The temperature of the board layer rises to 10℃ within 120 minutes and is maintained for 120 minutes;

[0100] j. The temperature of the plate layer rises to 33°C within 120 minutes and is kept warm for 360 minutes;

[0101] k. Pump the ultimate vacuum at 33℃ until the vacuum is qualified;

[0102] l. Press the plug under vacuum.

[0103] Example 3

[0104] A process for preparing high-purity human coagulation factor IX from plasma in this embodiment comprises the following steps:

[0105] (1) After receiving 3000L of human plasma that has passed the quarantine period, the bags are opened under the protection of laminar flow in a Class D environment; the plasma is combined into a slurry melting tank and thawed with circulating water at 33°C, and the plasma temperature is controlled at 2°C; after the plasma is melted, it is centrifuged with a centrifuge with a centrifugal force of 20,000g, an outlet temperature of 2°C, and an outlet flow rate of 4kg / min / unit, and the cryoprecipitated plasma is collected; the cryoprecipitated plasma is transferred to an adsorption tank, and the plasma temperature is controlled at 20°C.

[0106] (2) First, the pH value of the plasma in step (1) was adjusted to 7.2 using acetic acid-sodium acetate buffer, and then an anionic gel (NW Rose DEAE (BB) gel produced by Suzhou Nanotech Co., Ltd.) with a balance of 2.0% by weight of the plasma (balance solution: 6.12 g / L sodium citrate, 5.12 g / L sodium chloride, pH 7.2) was added, and the adsorption was stirred for 65 minutes, and the temperature was controlled at 20°C; then, the adsorbed gel was collected in a gel column, and the gel was washed 5 times with a washing solution (3.12 g / L sodium citrate, 12.5 g / L sodium chloride, pH 7.2), each time stirred for 6 minutes and then dried; then the gel was eluted 4 times with an elution solution (130 g / sodium chloride, 3.12 g / L sodium citrate, pH 7.2), each time stirred for 15 minutes, and the eluted protein solution I was collected.

[0107] (3) The eluted protein solution I obtained in step (2) was added with 140 IU of heparin sodium injection, filtered through a PP filter element with a pore size of 0.45 μm, and concentrated to 50 L using a 10KD ultrafiltration membrane. The eluted protein solution I was dialyzed using 200 L of dialysate (4.5 g / L arginine hydrochloride, 6.5 g / L sodium citrate, pH 7.6) until the conductivity of the dialyzed solution was less than 15 ms / cm, and the dialyzed protein solution was collected.

[0108] (4) Add 13 kg of S / D solution (34 g / L tributyl phosphate, 115 g / L polysorbate 80) to the dialyzed protein solution obtained in step (3), and the liquid feeding rate should be 2.5 kg / min; after the addition of the S / D solution is completed, set the stirring speed to 120 r / min, and keep warm at 26°C for 7 h to obtain the S / D protein solution.

[0109] (5) Add 1.5-2.0% by weight of the equilibrated anionic gel (same as step 2) to the S / D protein solution obtained in step (4), and stir and adsorb for 65 minutes with the temperature controlled at 20°C; then, wash the gel 9 times with 50 L of washing solution, stirring for 6 minutes each time and then letting it dry; then, elute the gel 4 times with 45 L of eluting solution, stirring for 15 minutes each time, and collect the eluted protein solution II.

[0110] (6) The eluted protein solution II obtained in step (5) was filtered through a PP filter element with a pore size of 0.45 μm, and the filtered eluted protein solution II was concentrated to 50 L using a 10KD ultrafiltration membrane. It was then dialyzed using 350 L of dialysate (4.5 g / L arginine hydrochloride, 6.5 g / L sodium citrate, pH 7.6), and the dialyzed eluted protein solution II was collected.

[0111] (7) Filter the eluted protein solution II obtained in step (6) through a PP filter element with a pore size of 0.45 μm to obtain the protein solution before affinity chromatography column.

[0112] (8) The protein solution before affinity chromatography obtained in step (7) is loaded onto an affinity gel chromatography column (the affinity gel uses NW Rose Plasmid affinity gel produced by Suzhou Nanotech Co., Ltd.) equilibrated with an equilibration solution (3.0 g / L tromethamine, 9.5 g / L sodium chloride, pH 7.8); after loading, the equilibration solution is used to wash 4 times the volume of the gel column, and then the washing solution (4.84 g / L tromethamine, 58.50 g / L sodium chloride, pH 7.6) is used to wash 4 times the volume of the gel column, and then the equilibration solution is used to wash the chromatography column for another 4 times the volume of the gel column until a baseline is formed; after washing, the elution is performed with an eluent (11.3 g / L glycine, pH 7.0), and the eluted protein solution III is collected.

[0113] The eluted protein solution collected by affinity gel chromatography purification was concentrated with a 10kD ultrafiltration membrane, and then dialyzed seven times with equal volumes of dialysis fluid (2.9 g / L sodium citrate, 5.8 g / L sodium chloride and 4 g / L glycine, pH 7.2) to obtain the protein solution before nanofiltration, and samples were taken to detect pH and protein content.

[0114] (9) After filtering the protein solution before nanofiltration obtained in step (8) through a 0.1 μm series 20 nm filter element, add a dialysate (2.9 g / L sodium citrate, 5.8 g / L sodium chloride, 4 g / L glycine, 9 g / L arginine hydrochloride, pH 7.2) of equal volume for ultrafiltration and dialyzation three times, collect the target protein solution, and obtain a human coagulation factor IX stock solution; adjust the titer of the human coagulation factor IX stock solution to 65.0 IU / ml with the dialysate, and add sodium heparin according to the final "0.1 IU / IU human coagulation factor IX titer" to obtain a diluted solution; pre-filter the diluted solution through a 0.2 μm filter element to obtain a human coagulation factor IX semi-finished product.

[0115] (10) The semi-finished product obtained in step (9) is sterilized and filtered through a 0.2 μm filter element, packaged, vacuum freeze-dried, capped, and dry-heat virus inactivated (80° C., 0.5 h), then sent for inspection and light inspection. After the product passes the inspection, it is packaged and stored.

[0116] Wherein, vacuum freeze drying conditions are:

[0117] a. Products are put into the cabinet at room temperature;

[0118] b. The temperature of the plate layer drops to -6°C within 10 minutes and remains at this temperature for 30 minutes;

[0119] c. The temperature of the board layer drops below -45℃ within 60 minutes and remains below this temperature for 180 minutes;

[0120] d. Raise the temperature of the board layer to -15°C within 60 minutes and maintain it for 120 minutes;

[0121] e. The temperature of the board layer drops below -45℃ within 120min and remains below this temperature for 180min;

[0122] f. Turn on the vacuum pump to make the vacuum degree reach 0.3mbar;

[0123] g. The temperature of the board layer is raised to -33°C within 60 minutes and maintained for 180 minutes;

[0124] h. The temperature of the board layer is raised to 0°C within 300 minutes and maintained for 1200 minutes;

[0125] i. The temperature of the board layer rises to 10℃ within 120 minutes and is maintained for 120 minutes;

[0126] j. The temperature of the plate layer rises to 33°C within 120 minutes and is kept warm for 360 minutes;

[0127] k. Pump the ultimate vacuum at 33℃ until the vacuum is qualified;

[0128] l. Press the plug under vacuum.

[0129] Experimental Example 1

[0130] This experimental example is used to verify the quality of human coagulation factor IX prepared by the present invention.

[0131] Taking Example 2 as an example, the human coagulation factor IX product prepared by the process of the present invention was compared with the key quality indicators of human coagulation factor IX included in the European Pharmacopoeia. The results are shown in Table 1.

[0132] Table 1 Comparison of key quality indicators of human coagulation factor IX

[0133]

[0134]

[0135] It can be seen from the results in Table 1 that the human coagulation factor IX product prepared by the process of the present invention has excellent quality, an activity greater than 50 IU / ml, and a specific activity higher than 180 IU / mg.

[0136] Experimental Example 2

[0137] This experimental example is used to verify the effect of "affinity gel chromatography" on the quality of the present invention's coagulation factor IX.

[0138] Taking the preparation process of human coagulation factor IX in Example 2 as an example, the components of the protein solution before the affinity gel chromatography were analyzed, and the main impurities were human coagulation factors II, VII, and X. According to the salt concentration gradient of the washing liquid and the eluent, the washing liquid includes 2.43g / L tromethamine and 29.25g / L sodium chloride; the eluent includes 9.4g / L glycine. Under these conditions, other impurity factors can be removed, and the activity yield of human coagulation factor IX can reach more than 90%. Table 2 is the affinity gel chromatography results.

[0139] Table 2 Affinity gel chromatography results

[0140]

[0141]

[0142] Experimental Example 3

[0143] This experimental example is used to verify the effect of "different amino acids as protective agents" on the quality of the present invention's coagulation factor IX.

[0144] Take human coagulation factor IX intermediate (human coagulation factor IX titer 54IU / ml, containing about 2.9g / L sodium citrate), use different amino acids as protective agents, add lyophilization protective agents, and each experimental group has about 60ml of semi-finished products. Each sample is divided into 10ml bottles and filled into 5 bottles. After lyophilization, the appearance, reconstitution time, visible foreign matter and potency index are tested. The results are shown in Table 3.

[0145] Table 3 Results of freeze-drying with different amino acids as protective agents

[0146]

[0147] From the results in Table 1, it can be seen that glycine as a protective agent, adding a certain amount of sodium chloride at the same time has a good appearance and re-dissolution time. Arginine hydrochloride, glycine, and lysine hydrochloride alone as protective agents have basically lost their potency after freeze-drying. Subsequently, different amino acids were considered to be added with sodium chloride as protective agents, and 6 formulas were designed (see Table 4). After freeze-drying, the appearance, re-dissolution time, visible foreign matter, and potency index tests were performed. The results are shown in Table 5.

[0148] Table 4 Grouping of different amino acids combined with sodium chloride

[0149]

[0150] Table 5 Results of freeze-dried combination of different amino acids and sodium chloride

[0151]

[0152]

[0153] From the results in Table 5, it can be seen that the use of "Group 4 (2.0 g / L glycine + 7.0 g / L arginine hydrochloride + 5.8 g / L sodium chloride)" as a protective agent has a good appearance and re-dissolution time, no foreign matter visible to the naked eye, and less potency loss after freeze-drying. The use of "Groups 2, 3, 5, and 6" as a protective agent has a good appearance and re-dissolution time after freeze-drying, protein spots are precipitated, and the potency loss after freeze-drying is less than that of "Group 1".

[0154] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A process for preparing high-purity human coagulation factor IX from plasma, characterized in that: The steps include: (1) Frozen plasma is thawed and centrifuged at 0-4°C to obtain cryoprecipitated plasma; (2) adding anionic gel at 1.5-2.0% by weight of plasma to the product obtained in step (1), stirring and adsorbing for 35-65 minutes, and controlling the temperature at 10-20° C. to obtain eluted protein solution I; (3) adding the eluted protein solution I obtained in step (2) to sodium heparin injection, filtering, ultrafiltration through an ultrafiltration membrane, and dialyzing to obtain a dialyzed protein solution; (4) adding the S / D solution to the dialyzed protein solution obtained in step (3), stirring evenly, controlling the temperature at 24-26° C., and keeping the temperature for 5-7 hours to obtain a S / D protein solution; (5) adding 1.5-2.0% by weight of anionic gel to the protein solution after S / D obtained in step (4) for a second adsorption, stirring and adsorbing for 35-65 minutes, controlling the temperature at 10-20° C., and collecting the eluted protein solution II; (6) filtering the eluted protein solution II obtained in step (5) through a PP filter element, using an ultrafiltration membrane for ultrafiltration, and dialyzing with a dialysate, and collecting the eluted protein solution II after dialysis; (7) filtering the eluted protein solution II obtained after dialysis in step (6) through a PP filter element to obtain a protein solution before affinity chromatography column; (8) purifying the protein solution before affinity chromatography obtained in step (7) by affinity gel chromatography, collecting the eluate, and obtaining the protein solution before nanofiltration by ultrafiltration and dialysis; (9) After filtering the protein solution before nanofiltration obtained in step (8), adding an equal volume of dialysate containing 1 to 4 g / L glycine and 3 to 9 g / L arginine hydrochloride, ultrafiltration and dialysis are performed 1 to 3 times, and the target protein solution is collected to obtain a human coagulation factor IX stock solution; The titer of the human coagulation factor IX stock solution is adjusted to 55.0-65.0 IU / ml with dialysate, and sodium heparin is added according to the final "0.1 IU / IU human coagulation factor IX titer" to obtain a diluted solution; the diluted solution is pre-filtered through a filter element to obtain a human coagulation factor IX semi-finished product; (10) The semi-finished product obtained in step (9) is sterilized, filtered, packaged, vacuum freeze-dried, capped, and dry-heat virus inactivated, then sent for inspection and light inspection. After the product is qualified, it is packaged and stored.

2. The process for preparing high-purity human coagulation factor IX from plasma according to claim 1, characterized in that: In the step (2), the plasma pH is first adjusted to 6.8-7.2 using an acetic acid-sodium acetate buffer, and then 1.5-2.0% of the plasma weight of the balanced anionic gel is added, and the adsorption is carried out for 35-65 minutes with stirring, and the temperature is controlled at 10-20° C.; then, the adsorbed gel is collected in a gel column, and the gel is washed 3-5 times with a washing solution, each time stirring for 4-6 minutes and then drained; The gel was then eluted with the eluent for 2 to 4 times, with each stirring for 10 to 15 minutes, and the eluted protein solution I was collected.

3. The process for preparing high-purity human coagulation factor IX from plasma according to claim 1, characterized in that: In the step (5), firstly, 1.5-2.0% by weight of the balanced anionic gel is added to the S / D protein solution, and the mixture is stirred for adsorption for 35-65 minutes, with the temperature controlled at 10-20° C.; then, the gel is washed 7-9 times with a washing solution, each time stirred for 4-6 minutes and then dried; then, the gel is eluted 2-4 times with an eluting solution, each time stirred for 10-15 minutes, and the eluted protein solution II is collected.

4. The process for preparing high-purity human coagulation factor IX from plasma according to claim 1, characterized in that: The anionic gel in steps (2) and (5) is NW Rose DEAE gel produced by Suzhou Nanotech Co., Ltd.; the balancing solution formula during the use of the anionic gel is: 5.51-6.12 g / L sodium citrate, 4.54-5.12 g / L sodium chloride, pH 6.8-7.2; the washing solution formula is: 2.84-3.12 g / L sodium citrate, 10.1-12.5 g / L sodium chloride, pH 6.8-7.2; the eluent formula is: 111-130 g / sodium chloride, 2.84-3.12 g / L sodium citrate, pH 6.8-7.

2.

5. The process for preparing high-purity human coagulation factor IX from plasma according to claim 1, characterized in that: In the step (8), the specific steps of affinity gel chromatography purification are: loading the protein solution before affinity chromatography onto an affinity gel chromatography column equilibrated with an equilibrated solution; after loading, first washing 2 to 4 times the volume of the gel column with the equilibrated solution, then washing 2 to 4 times the volume of the gel column with the washing solution, and then washing the chromatography column with 2 to 4 times the volume of the gel column with the equilibrated solution until a baseline is formed; after washing, eluting with an eluent, and collecting the eluted protein solution III.

6. The process for preparing high-purity human coagulation factor IX from plasma according to claim 1, characterized in that: In the step (8), the affinity gel adopts NW Rose Plasmid affinity gel produced by Suzhou Nanovita Technology Co., Ltd.; the balance solution formula during the affinity gel chromatography is: 2.0-3.0 g / L tromethamine, 8.0-9.5 g / L sodium chloride, pH 7.0-7.8; the washing solution formula is: 2.42-4.84 g / L tromethamine, 29.25-58.50 g / L sodium chloride, pH 7.2-7.6; the eluent formula is: 7.5-11.3 g / L glycine, pH 6.0-7.

0.

7. The process for preparing high-purity human coagulation factor IX from plasma according to claim 1, characterized in that: In the step (8), the specific steps of ultrafiltration and dialysis are: the eluted protein solution collected by affinity gel chromatography purification is concentrated with a 10kD ultrafiltration membrane, and then an equal volume of dialysis fluid is added to dialyze 5 to 7 times to obtain a protein solution before nanofiltration, and a sample is taken to detect the pH and protein content.

8. The process for preparing high-purity human coagulation factor IX from plasma according to claim 1, characterized in that: In the step (9), the dialysate containing 1-4 g / L glycine and 3-9 g / L arginine hydrochloride has the following formula: 2.9 g / L sodium citrate, 5.8 g / L sodium chloride, 1-4 g / L glycine, 3-9 g / L arginine hydrochloride, pH 6.8-7.

2.

9. The process for preparing high-purity human coagulation factor IX from plasma according to claim 1, characterized in that: In the step (10), the vacuum freeze-drying conditions are: a. Products are put into the cabinet at room temperature; b. The temperature of the plate layer drops to -6°C within 10 minutes and remains at this temperature for 30 minutes; c. The temperature of the board layer drops below -45℃ within 60 minutes and remains below this temperature for 180 minutes; d. Raise the temperature of the board layer to -15°C within 60 minutes and maintain it for 120 minutes; e. The temperature of the board layer drops below -45℃ within 120min and remains below this temperature for 180min; f. Turn on the vacuum pump to make the vacuum degree reach 0.3mbar; g. The temperature of the board layer is raised to -33°C within 60 minutes and maintained for 180 minutes; h. The temperature of the board layer is raised to 0°C within 300 minutes and maintained for 1200 minutes; i. The temperature of the board layer rises to 10℃ within 120min and is maintained for 120min; j. The temperature of the plate layer rises to 33°C within 120 minutes and is kept warm for 360 minutes; k. Pump the ultimate vacuum at 33℃ until the vacuum is qualified; l. Press the plug under vacuum.

10. The process for preparing high-purity human coagulation factor IX from plasma according to any one of claims 1 to 9, characterized in that: The activity of the human coagulation factor IX finally obtained is greater than 50 IU / ml, and the specific activity is higher than 180 IU / mg.

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