CN105175486A - Preparation method of high-purity human coagulation factor IX - Google Patents
Preparation method of high-purity human coagulation factor IX Download PDFInfo
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- CN105175486A CN105175486A CN201510683711.8A CN201510683711A CN105175486A CN 105175486 A CN105175486 A CN 105175486A CN 201510683711 A CN201510683711 A CN 201510683711A CN 105175486 A CN105175486 A CN 105175486A
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Abstract
The invention relates to a preparation method of a high-purity human coagulation factor IX, which comprises the following steps: melting refrigerated plasma, and carrying out low-temperature centrifugation; adsorbing with a DEAE (diethylaminoethanol) Sephadex A-50 gel to remove the coagulation factor IX in the cold-glue plasma; removing impure proteins in the solution by using polyethyleneglycol; carrying out S/D virus inactivation; carrying out anion exchange column chromatography to obtain a purified coagulation factor IX solution; passing through a heparin affinity column for further chromatography to obtain a high-purity coagulation factor IX solution; carrying out ultrafiltration, dialysis and concentration, and adding arginine hydrochloride and glycinate as protective agents; filtering through a 20nm filter element to remove viruses; carrying out freeze-drying; and carrying out dry heat virus inactivation. The protein protective agents are added during the gel adsorption, column chromatography and ultrafiltration dialysis, thereby lowering the activation probability of the FIX product thrombin and enhancing the qualification rate of the product. The technique has high product yield; the FIX specific activity can reach 150 IU/mg or so which is much higher than that of the traditional product; and by performing the three-step virus inactivation, the product is safe and reliable to use.
Description
Technical field
The invention belongs to biomedical sector, relate to a kind of Human factor IX, specifically a kind of preparation method of high-purity Human factor IX.
Background technology
Human factor IX (FIX) is the one in human body 13 kinds of thrombin, in human body blood coagulation mechanism, play the part of very important effect.For want of plasma thromboplastin component and cause blood coagulation disorders to be hemophilia B, this disease is a kind of heredopathia, and patient is based on the male sex, and sickness rate is about male sex baby's alive 1/30000, with relevant hemorrhage for feature of spontaneous or wound, bleeding part mainly in joint, soft tissue and muscle.Delay or the deficiency for the treatment of can cause various complication, comprise the relevant joint disease of joint Repeated Hemorrhage and synovitis, can cause joint deformity and dysfunction time serious.In addition, more serious is hemorrhage, especially intracranials hemorrhage and may cause death.
The unique effective means of current treatment hemophilia B is exactly that injection in time contains the medicine of thrombin FIX to reach the object of hemostasis, or regular injection is hemorrhage containing the chemoprophylaxis of plasma thromboplastin component.The Human factor IX etc. of the human blood coagulation FIX that the medicine containing thrombin FIX has blood plasma to extract, Human Factor Ⅸ Complex and gene recombination, have Lyophilized Human plasma thromboplastin component and recombinant human blood coagulation factor IX goods abroad, domestic only have lyophilized prothrombin complex contrates's kind at present.Clinical practice shows, hemophilia B people can the pure Human factor IX of self-injection, usually side effect is not had, safer, but injection Human Factor Ⅸ Complex but may produce severe side effect-thrombosis, so need inject in hospital during general this medicine of hemophilia B people infusion, remain in a hospital under observation in case unexpected.Visible, the human blood coagulation FIX of purifying is the choice drug for the treatment of hemophilia B, but traditional Purification of Human plasma thromboplastin component production technique, operation is more, cost is very high, and easily occur the problem of thrombin activation in process of production and cause product failure, the inactivation of virus means of adding traditional technology are single, the safety of goods can not be ensured completely, and xeothermic inactivation of virus mode often causes larger vigor loss, so, for a long time, though the Human factor IX of purifying fails to replace have side effect but Human Factor Ⅸ Complex with low cost always
Summary of the invention
For above-mentioned technical problem of the prior art, the invention provides a kind of preparation method of high-purity Human factor IX, it is high that the preparation method of described this high-purity Human factor IX solves in prior art method complex procedures, the cost of preparing Human factor IX, the technical problem that Product Safety is not high.
The invention provides a kind of preparation method of high-purity Human factor IX, comprise the steps:
1) fresh frozen plasma is melted the rear centrifugal segregation cryoprecipitate of slurry, obtain and remove cold glue blood plasma, cold glue blood plasma then will be gone to be heated to 10-15 DEG C;
2) DEAE-SephadexA-50 gel is placed in a stainless steel resin barrel, first use temperature fully swelling at the water for injection of 75 ~ 85 DEG C, again with the water for injection cooling of 10 ~ 20 DEG C, then balance with damping fluid, described damping fluid is made up of Na-citrate and NaCL, in described damping fluid, the concentration of Na-citrate (Trisodium Citrate) is 0.005M-0.015M, the concentration of NaCL is 0.075M-0.15M, the PH of described damping fluid is 6.50-7.50, the gel that above-mentioned balance is good is joined in cold glue blood plasma, the ratio of going cold glue blood plasma to add 0.5-1.5 gram of xerogel according to often liter adds the gel balanced, stir 0.5-1.5 hour, leave standstill 10-20 minute again,
3) elimination cold glue blood plasma is crossed, collect the DEAE-SephadexA-50 gel having adsorbed plasma thromboplastin component, resin is rinsed 4-6 time afterwards with lavation buffer solution, described lavation buffer solution is made up of Na-citrate and NaCL, in described damping fluid, the concentration of Na-citrate is the concentration of 0.005M-0.015M, NaCL is 0.1M-0.25M, and the PH of described lavation buffer solution is 6.50-7.50; Use elution buffer wash-out resin 2-4 time again, described elution buffer is made up of Na-citrate, ArginineHydrochloride (arginine hydrochloride) and NaCL, in described damping fluid, the concentration of the concentration of Na-citrate to be the concentration of 0.005M-0.015M, ArginineHydrochloride be 0.02M, NaCL is 0.5M-2.0M, the PH of described elution buffer is 6.50-7.50, collect elutriant, then 0.45 μm or 1.0 μm of filter element filtering elutriants are used, collect filtrate, be plasma thromboplastin component crude product solution;
4) add polyoxyethylene glycol in elutriant after filtration, the mass percent concentration of the polyoxyethylene glycol after adding in elutriant is 2-6%, stirs 0.5-1.5 hour, filters;
5) in above-mentioned filtrate, Tween80 is added, the mass percent concentration of Tween80 after adding in above-mentioned filtrate is 1.0%, add tributyl phosphate again, the mass percent concentration of tributyl phosphate after adding in above-mentioned filtrate is 0.3%, 24-26 DEG C is warming up to, rear insulation 6 ~ 7 hours after stirring evenly;
6) then 5-15 DEG C is cooled to, by the first diluted to specific conductivity not higher than 15ms/cm, the first described diluent is 0.01MNa-citrate solution, the PH6.2-7.20 of the first described diluent, then anion-exchange column is gone up, described anion-exchange column fully balances with level pad in advance, described level pad is made up of Na-citrate and NaCL, in described damping fluid, the concentration of Na-citrate is the concentration of 0.005M-0.015M, NaCL is 0.1M-0.15M, and the PH of described level pad is 6.20-7.20, wash with lavation buffer solution again, described lavation buffer solution is made up of Na-citrate and NaCL, in described damping fluid, the concentration of Na-citrate is 0.005M-0.015M, the concentration of NaCL is 0.18-0.3M, the PH of described lavation buffer solution is 6.20-7.20, use elution buffer wash-out again, described elution buffer is by Na-citrate, ArginineHydrochloride and NaCL forms, in described damping fluid, the concentration of Na-citrate is 0.005M-0.015M, the concentration of ArginineHydrochloride is 0.02M, the concentration of NaCL is 0.5M-1.0M, the PH of described elution buffer is 6.80-7.80, collect the plasma thromboplastin component solution that elutriant is purifying,
7) by the second diluted step 6) elutriant to specific conductivity not higher than 15ms/cm, the second described diluent is by Na-citrate, ArginineHydrochloride forms, in described diluent, the concentration of Na-citrate is 0.01M, the concentration of ArginineHydrochloride is 0.02M, the PH of the second described diluent is 6.50-7.50, then heparin affinity column is gone up, described heparin affinity column fully balances with level pad in advance, described level pad is made up of Na-citrate and NaCL, in described damping fluid, the concentration of Na-citrate is 0.01M-0.02M, the concentration of NaCL is 0.1M-0.15M, described level pad is PH6.50-7.50, wash with lavation buffer solution again, described lavation buffer solution is made up of Na-citrate and NaCL, in described damping fluid, the concentration of Na-citrate is the concentration of 0.01M-0.02M, NaCL is 0.18-0.25M, and the PH of described lavation buffer solution is 6.50-7.50, use elution buffer wash-out again, described elution buffer is made up of Na-citrate, ArginineHydrochloride and NaCL, in described damping fluid, the concentration of the concentration of Na-citrate to be the concentration of 0.01M-0.02M, ArginineHydrochloride be 0.02M, NaCL is 0.4M-1.0M, the PH of described elution buffer is 6.50-7.50, collect elutriant, with 0.45 μm or 0.22 μm of above elutriant of filter element filtering, gained filtrate is high-purity plasma thromboplastin component solution,
8) concentrate above filtrate with the ultra-filtration membrane of 10k molecular weight, then constant volume dialysis, reconcentration higher than 35IU/ml, obtains plasma thromboplastin component concentrated solution to plasma thromboplastin component vigor;
9) mediator's plasma thromboplastin component content is to 20-30IU/ml, then adjust pH value to 6.50-7.50, and add heparin sodium to 0.1-0.4IU/ unit FIX, add arginine hydrochloride and glycine, the mass percent concentration of described arginine hydrochloride in plasma thromboplastin component concentrated solution is 0.5-3%, and the mass percent concentration of described glycine in plasma thromboplastin component concentrated solution is 0.5-3%;
10) carry out except virus filtration with 20 nanometer filter cores;
11) with 0.22 μm of filter core, Sterile Filtration is carried out and packing to product;
12) freeze-drying;
13) in 100 DEG C of boiling water baths, be incubated 30 minutes, carry out xeothermic inactivation of virus, obtain high-purity Human factor IX.
Further, step 6) in, described anion-exchange column is Capto-Q, Q-SepharoseFF, Q-Sepharose4FF, Q-SepharoseHP, Q-SepharoseXL, DEAE-SepharoseFF or Capto-DEAE chromatography column.
Further, described upper heparin affinity column is HeparinSepharose6FF, HeparinSepharoseCL-6B.
The present invention adopts classical DEAESephadexA-50 gel adsorption, and obtain the crude product being rich in thrombin FIX, this gel adsorption is batch processing, to the clarity of blood plasma without particular requirement, without the need to filtering, therefore the treatment time is short, this process operations is simple simultaneously, and plasma thromboplastin component loss is few; After the present invention obtains FIX crude product, add PEG precipitation agent, make foreign protein sedimentation from plasma thromboplastin component solution, filtration, remove most of foreign protein; Meanwhile, PEG is also protein protective agent, can prevent plasma thromboplastin component deactivation in ensuing S/D inactivation of virus operation; The present invention selects the new and effective strong anion filler of Capto-Q, and resolving power is high, and pressure drop is little, and flow velocity is fast, can save a lot of operating time than conventional filler; Obtain the plasma thromboplastin component of purifying thus; The present invention selects the affine filler of HeparinSepharose6FF, refines further the FIX of purifying, obtains the specific activity being up to 153IU/mg; The present invention is in gel adsorption and two follow-up step column chromatography operations, protein protective agent arginine monohydrochloride is all added in elution buffer, also arginine monohydrochloride is added in middle ultrafiltration dialysis operation, greatly reduce the probability of FIX goods thrombin activation, improve the qualification rate of product; Employ S/D inactivation of virus in Production Flow Chart of the present invention altogether, 20 nano-film filtration virus removals and xeothermic inactivation of virus are sick triple goes out except viral measure, carry out effectively going out removing to lipid-coated virus, non-lipid-coated virus and parvovirus, greatly improved the safety in utilization of product.
The present invention compares with prior art, and its technical progress is significant.Of the present invention a kind of from going the method preparing Lyophilized Human thrombin FIX cold glue blood plasma, this technical process is succinct, easy to operate, be easy to realize industrialization, and this technique has product yield is high, specific activity is high advantage, product is through three step inactivation of virus, safe and reliable.
Accompanying drawing explanation
Fig. 1 is a kind of process flow diagram preparing high-purity Human factor IX of the present invention.
Embodiment
Embodiment 1
A preparation method for high-purity Human factor IX, comprises the steps:
1) refrigerated plasma 35 bags is got, surface is with 70% ethanol rinse and with after cold water for injection flushing, cut bag, pouring stainless steel into melts in slurry bucket, melt in 0-3 DEG C of water-bath, accurately take 20kg, centrifugal segregation cryoprecipitate afterwards, cold glue blood plasma 19.8kg is removed in acquisition, is then placed in 15 DEG C of water-bath heating and goes cold glue blood plasma to 13-15 DEG C;
2) take 20 grams of DEAE-SephadexA-50 gels and be placed in gel bucket, first use the hot water for injection of about 2kg (about 80 DEG C) fully swelling, the cold water for injection of about 2kg (about 15 DEG C) is used to rinse cooling again, after use about 1kg damping fluid (PH6.50,0.01MNa-citrate, 0.075MNaCL, 15 DEG C) balance, then be added in cold glue blood plasma, slowly stir 1 hour, latter standing 10 minutes;
3) in gel bucket, cold glue blood plasma is removed with filter-cloth filtering, collect DEAE-SephadexA-50 gel, then in gel bucket, 2 liters of lavation buffer solution (PH6.50 are added, 0.01MNa-citrate, 0.1MNaCL) stir about 10 minutes, at the bottom of bucket, washings is bled off by after filter-cloth filtering, so repeatedly rinse gel 4 times, use elution buffer (PH6.50 again, 0.01MNa-citrate, 0.02MArginineHydrochloride, 0.5MNaCL) wash-out gel 3 times, each 2 liters, collect elutriant and be about 2005g, with 0.45 μm of filter element filtering elutriant, , remove broken glue particulate, obtain filtrate 1950 grams,
4) take PEG (polyoxyethylene glycol) 65 grams, be first dissolved in 65 grams of waters for injection, after join in filtrate, stir after 0.5 hour filter, collect filtrate 2036 grams;
5) add 225 grams, S/D mother liquor (namely Tween8010% (wt%), TNBP (tributyl phosphate) are to 3% (wt%)) in above filtrate, be warming up to 24-26 DEG C after stirring evenly, rear insulation 6 hours;
6) solution after S/D is cooled to 5 DEG C, with diluent (0.01MNa-citrate solution, PH6.20) above-mentioned solution is diluted to 3.5 times of original solution weight, then anion-exchange column Q-SepharoseFF is gone up, chromatography column uses level pad (0.01MNa-citrate in advance, 0.1MNaCL solution, PH6.20) fully balance; Upper prop terminates rear Equilibration buffer wash pillar, after use lavation buffer solution (0.01MNa-citrate, 0.18MNaCL solution, PH6.20) wash, use elution buffer (0.01MNa-citrate, 0.02MArginineHydrochloride, 0.5MNaCL solution again, PH6.80) wash-out, collects elutriant 0.45 μm of filter element filtering;
7) with diluent (0.01MNa-citrate, 0.02MArginineHydrochloride, PH6.50-7.50) above filtrate is diluted, then HeparinSepharose6FF post is gone up, chromatography column fully balances with level pad in advance, level pad is 0.01MNa-citrate, 0.15MNaCL solution (PH6.80), after use lavation buffer solution (0.01MNa-citrate, 0.18MNaCL solution, PH6.80) wash, use elution buffer (0.01MNa-citrate again, 0.02MArginineHydrochloride, 0.5MNaCL solution, PH6.80) wash-out, collect elutriant 810 grams, filtrate 772g is obtained with 0.45 μm of filter element filtering elutriant,
8) with the ultra-filtration membrane (0.1M of 10k molecular weight
2) concentrate eluant is to about 300ml, rear use 1.5 liters of dialyzates (0.01MNa-citrate, 0.1MNaCL solution, PH7.00) constant volume is dialysed, and again concentrates and shifts out ultra-filtration membrane bag, obtaining concentrated solution 245 grams, recording FIX vigor 35.1IU/ml;
9) mediator's plasma thromboplastin component content is to 30IU/ml, according to calculating, take arginine hydrochloride 1.5 grams, glycine 8.3 grams, add dialyzate described in step 9 23 grams, arginine hydrochloride, glycine added dialyzate and suspend, after be added in concentrated solution described in step 9, then adjust pH value to 7.00, and add heparin sodium to 0.15IU/ unit nine factor;
10) carry out except virus filtration with 20 nanometer filter cores;
11) Sterile Filtration is carried out and packing to product, 10ml/ bottle with 0.22 μm of filter core;
12) freeze-drying;
13) in 100 DEG C of boiling water baths, be incubated 30 minutes, carry out xeothermic inactivation of virus; Survey FIX vigor, 28.1IU/ml;
Embodiment 2
A preparation method of high-purity human blood coagulation FIX, comprises the steps:
1) with embodiment 1;
2) DEAE-SephadexA-50 gel consumption, swelling with cooling with embodiment 1, rear use about 1kg damping fluid (PH7.35,0.01MNa-citrate, 0.15MNaCL, 15 DEG C) balance, be then added in cold glue blood plasma, slow stirring 1 hour, latter standing 20 minutes;
3) in gel bucket, cold glue blood plasma is removed with filter-cloth filtering, collect DEAE-SephadexA-50 gel, then in gel bucket, 2 liters of lavation buffer solution (PH7.35 are added, 0.015MNa-citrate, 0.2MNaCL) stir about 5 minutes, at the bottom of bucket, bleed off washings by after filter-cloth filtering, so repeatedly rinse gel 6 times; Use elution buffer (PH7.35,0.015MNa-citrate, 0.02MArginineHydrochloride again, 1.0MNaCL) wash-out gel 4 times, collects elutriant and is about 3988g by each 1 liter, with 0.45 μm of filter element filtering elutriant, obtain filtrate 3936 grams;
4) take PEG (polyoxyethylene glycol) 260 grams, be first dissolved in 260 grams of waters for injection, after join in filtrate, stir after 1.5 hours filter, collect filtrate 4402 grams;
5) add 500 grams, S/D mother liquor (namely Tween8010% (wt%), TNBP (tributyl phosphate) are to 3% (wt%)) in above filtrate, be warming up to 24-26 DEG C after stirring evenly, rear insulation 6 hours;
6) solution after S/D is cooled to 15 DEG C, with diluent (0.01MNa-citrate solution, PH7.20) above-mentioned solution is diluted to specific conductivity 13.8ms/cm, then DEAE-SepharoseFF chromatography column is gone up, chromatography column uses level pad (0.01MNa-citrate in advance, 0.15MNaCL solution, PH7.20) fully balance; Upper prop terminates rear Equilibration buffer wash, after use lavation buffer solution (0.01MNa-citrate, 0.3MNaCL solution, PH7.20) wash, then use elution buffer (0.01MNa-citrate, 0.02MArginineHydrochloride, 1.0MNaCL solution, PH7.80) wash-out, collects elutriant, with 0.45 μm of filter element filtering;
7) with diluent (0.01MNa-citrate, 0.02MArginineHydrochloride, PH7.50) above filtrate is diluted, then HeparinSepharoseCL-6B post is gone up, chromatography column fully balances with level pad in advance, level pad is 0.01MNa-citrate, 0.15MNaCL solution (PH7.50), after use lavation buffer solution (0.01MNa-citrate, 0.2MNaCL, PH7.50) wash, use elution buffer (0.01MNa-citrate again, 0.02MArginineHydrochloride, 1.0MNaCL, PH7.50) wash-out, collect elutriant 764 grams, filtrate 713g is obtained with 0.45 μm of filter element filtering elutriant,
8) with the ultra-filtration membrane (0.1M of 10k molecular weight
2) concentrate eluant is to about 300ml, rear use 1.5 liters of dialyzates (0.01MNa-citrate, 0.1MNaCL solution, PH7.50) constant volume is dialysed, and again concentrates and shifts out ultra-filtration membrane bag, obtaining concentrated solution 237 grams, recording FIX vigor 36IU/ml;
9) mediator's thrombin FIX content is to 30IU/ml, according to calculating, take arginine hydrochloride 8.6 grams, glycine 1.5 grams, add dialyzate described in step 9 48 grams, arginine hydrochloride, glycine added dialyzate and dissolve, after be added in concentrated solution described in step 9, then adjust pH value to 7.00, and add heparin sodium to 0.3IU/ unit nine factor;
10) carry out except virus filtration with 20 nanometer filter cores;
11) Sterile Filtration is carried out and packing to product, 10ml/ bottle with 0.22 μm of filter core;
12) freeze-drying;
13) in 100 DEG C of boiling water baths, be incubated 30 minutes, carry out xeothermic inactivation of virus; Survey FIX vigor, 27.8IU/ml.
Embodiment 3
A preparation method for high-purity Human factor IX, comprises the steps:
1) with embodiment 1;
2) DEAE-SephadexA-50 gel consumption, swelling with cooling with embodiment 1, rear use about 1kg damping fluid (PH7.05,0.01MNa-citrate, 0.10MNaCL, 15 DEG C) balance, be then added in cold glue blood plasma, slow stirring 1 hour, latter standing 20 minutes;
3) in gel bucket, cold glue blood plasma is removed with filter-cloth filtering, collect DEAE-SephadexA-50 gel, then in gel bucket, 2 liters of lavation buffer solution (PH7.05 are added, 0.01MNa-citrate, 0.15MNaCL) stir about 8 minutes, at the bottom of bucket, washings is bled off by after filter-cloth filtering, so repeatedly rinse gel 5 times, use elution buffer (PH7.05 again, 0.01MNa-citrate, 0.02MArginineHydrochloride, 0.65MNaCL) wash-out gel 3 times, each 1 liter, collect elutriant and be about 2955g, with 0.45 μm of filter element filtering elutriant, obtain filtrate 2904 grams,
4) take PEG (polyoxyethylene glycol) 130 grams, be first dissolved in 130 grams of waters for injection, after join in filtrate, stir after 1 hour filter, collect filtrate 3108 grams;
5) add 350 grams, S/D mother liquor (namely Tween8010% (wt%), TNBP (tributyl phosphate) are to 3% (wt%)) in above filtrate, be warming up to 24-26 DEG C after stirring evenly, rear insulation 6 hours;
6) solution after S/D is cooled to 10 DEG C, with diluent (0.01MNa-citrate solution, PH7.05) above-mentioned solution is diluted, then Capto-Q chromatography column is gone up, chromatography column uses level pad (0.01MNa-citrate in advance, 0.15MNaCL solution, PH7.05) fully balance; Upper prop terminates rear Equilibration buffer wash, after use lavation buffer solution (0.01MNa-citrate, 0.25MNaCL solution, PH6.20) wash, then use elution buffer (0.01MNa-citrate, 0.02MArginineHydrochloride, 0.6MNaCL solution, PH7.05) wash-out, collects elutriant, with 0.45 μm of filter element filtering;
7) with diluent (0.01MNa-citrate, 0.02MArginineHydrochloride, PH6.50-7.50) above elutriant is diluted, then HeparinSepharose6FF post is gone up, chromatography column fully balances with level pad in advance, level pad is 0.01MNa-citrate, 0.15MNaCL solution (PH7.15), after use lavation buffer solution (0.02MNa-citrate, 0.25MNaCL solution, PH7.15) wash, use elution buffer (0.02MNa-citrate again, 0.02MArginineHydrochloride, 0.65MNaCL solution, PH7.15) wash-out, collect elutriant 739 grams, filtrate 692g is obtained with 0.45 μm of filter element filtering elutriant,
8) with the ultra-filtration membrane (0.1M of 10k molecular weight
2) concentrate eluant is to about 300ml, rear use 1.5 liters of dialyzates (0.01MNa-citrate, 0.1MNaCL solution, PH7.15) constant volume is dialysed, and again concentrates and shifts out ultra-filtration membrane bag, obtaining concentrated solution 218 grams, recording FIX vigor 38IU/ml;
9) mediator's plasma thromboplastin component content is to 30IU/ml, according to calculating, take arginine hydrochloride 4.2 grams, glycine 4.2 grams, add dialyzate described in step 9 50 grams, arginine hydrochloride, glycine added dialyzate and dissolve, after be added in concentrated solution described in step 9, then adjust pH value to 7.15, and add heparin sodium to 0.2IU/ unit nine factor;
10) carry out except virus filtration with 20 nanometer filter cores;
11) Sterile Filtration is carried out and packing to product, 10ml/ bottle with 0.22 μm of filter core;
12) freeze-drying;
13) in 100 DEG C of boiling water baths, be incubated 30 minutes, carry out xeothermic inactivation of virus; Survey FIX vigor, 27.1IU/ml.
Preproduction FIX yield and specific activity table look-up
Claims (3)
1. a preparation method for high-purity Human factor IX, is characterized in that comprising the steps:
1) fresh frozen plasma is melted the rear centrifugal segregation cryoprecipitate of slurry, obtain and remove cold glue blood plasma, cold glue blood plasma then will be gone to be heated to 10-15 DEG C;
2) DEAE-SephadexA-50 gel is placed in a stainless steel resin barrel, first use temperature fully swelling at the water for injection of 75 ~ 85 DEG C, again with the water for injection cooling of 10 ~ 20 DEG C, then balance with damping fluid, described damping fluid is made up of Na-citrate and NaCL, in described damping fluid, the concentration of Na-citrate is 0.005M-0.015M, the concentration of NaCL is 0.075M-0.15M, the PH of described damping fluid is 6.50-7.50, the gel that above-mentioned balance is good is joined in cold glue blood plasma, the ratio of going cold glue blood plasma to add 0.5-1.5 gram of xerogel according to often liter adds the gel balanced, stir 0.5-1.5 hour, leave standstill 10-20 minute again,
3) elimination cold glue blood plasma is crossed, collect the DEAESephadexA-50 gel having adsorbed plasma thromboplastin component, resin is rinsed 4-6 time afterwards with lavation buffer solution, described lavation buffer solution is made up of Na-citrate and NaCL, in described lavation buffer solution, the concentration of Na-citrate is 0.005M-0.015M, the concentration of NaCL is 0.1M-0.25M, the PH of described lavation buffer solution is 6.50-7.50, use elution buffer wash-out resin 2-4 time again, described elution buffer is by Na-citrate, ArginineHydrochloride and NaCL forms, in described elution buffer, the concentration of Na-citrate is 0.005M-0.015M, the concentration of ArginineHydrochloride is 0.02M, the concentration of NaCL is 0.5M-2.0M, the PH of described elution buffer is 6.50-7.50, collect elutriant, then 0.45 μm or 1.0 μm of filter element filtering elutriants are used, collect filtrate, be plasma thromboplastin component crude product solution,
4) add polyoxyethylene glycol in elutriant after filtration, the mass percent concentration of the polyoxyethylene glycol after adding in elutriant is 2-6%, stirs 0.5-1.5 hour, filters;
5) in above-mentioned filtrate, Tween80 is added, the mass percent concentration of Tween80 after adding in above-mentioned filtrate is 1.0%, add tributyl phosphate again, the mass percent concentration of tributyl phosphate after adding in above-mentioned filtrate is 0.3%, 24-26 DEG C is warming up to, rear insulation 6 ~ 7 hours after stirring evenly;
6) then 5-15 DEG C is cooled to, by the first diluted to specific conductivity not higher than 15ms/cm, the first described diluent is 0.01MNa-citrate solution, the PH6.2-7.20 of the first described diluent, then anion-exchange column is gone up, described anion-exchange column fully balances with level pad in advance, described level pad is made up of Na-citrate and NaCL, in described damping fluid, the concentration of Na-citrate is the concentration of 0.005M-0.015M, NaCL is 0.1M-0.15M, and the PH of described level pad is 6.20-7.20, wash with lavation buffer solution again, described washing lavation buffer solution is made up of Na-citrate and NaCL, in described lavation buffer solution, the concentration of Na-citrate is 0.005M-0.015M, the concentration of NaCL is 0.18-0.3M, the PH of described lavation buffer solution is 6.20-7.20, use elution buffer wash-out again, described elution buffer is by Na-citrate, ArginineHydrochloride, form with NaCL, in described damping fluid, the concentration of Na-citrate is 0.005M-0.015M, the concentration of ArginineHydrochloride is 0.02M, the concentration of NaCL is 0.5M-1.0M, the PH of described elution buffer is 6.80-7.80, collect the plasma thromboplastin component solution that elutriant is purifying,
7) by the second diluted step 6) elutriant to specific conductivity not higher than 15ms/cm, the second described dilution is by Na-citrate, ArginineHydrochloride forms, in described diluent, the concentration of Na-citrate is 0.01M, the concentration of ArginineHydrochloride is 0.02M, the PH of the second described diluent is 6.50-7.50, then heparin affinity column is gone up, described heparin affinity column fully balances with level pad in advance, described level pad is made up of Na-citrate and NaCL, in described damping fluid, the concentration of Na-citrate is 0.01M-0.02M, the concentration of NaCL is 0.1M-0.15M, described level pad is PH6.50-7.50, wash with lavation buffer solution again, described lavation buffer solution is made up of Na-citrate and NaCL, in described damping fluid, the concentration of Na-citrate is 0.01M-0.02M, the concentration of NaCL is 0.18-0.25M, the PH of described lavation buffer solution is 6.50-7.50, use elution buffer wash-out again, described elution buffer is by Na-citrate, ArginineHydrochloride and NaCL forms, in described damping fluid, the concentration of Na-citrate is 0.01M-0.02M, the concentration of ArginineHydrochloride is 0.02M, the concentration of NaCL is 0.4M-1.0M, the PH of described elution buffer is 6.50-7.50, collect elutriant, with 0.45 μm or 0.22 μm of above elutriant of filter element filtering, gained filtrate is high-purity plasma thromboplastin component solution,
8) concentrate above filtrate with the ultra-filtration membrane of 10k molecular weight, then constant volume dialysis, reconcentration higher than 35IU/ml, obtains plasma thromboplastin component concentrated solution to plasma thromboplastin component vigor;
9) mediator's plasma thromboplastin component content is to 20-30IU/ml, then adjust pH value to 6.50-7.50, and add heparin sodium to 0.1-0.4IU/ unit FIX, add arginine hydrochloride and glycine, the mass percent concentration of described arginine hydrochloride in plasma thromboplastin component concentrated solution is 0.5-3%, and the mass percent concentration of described glycine in plasma thromboplastin component concentrated solution is 0.5-3%;
10) carry out except virus filtration with 20 nanometer filter cores;
11) with 0.22 μm of filter core, Sterile Filtration is carried out and packing to product;
12) freeze-drying;
13) in 100 DEG C of boiling water baths, be incubated 30 minutes, carry out xeothermic inactivation of virus, obtain high-purity Human factor IX.
2. the preparation method of a kind of high-purity Human factor IX according to claim 1, it is characterized in that: step 6) in, described anion-exchange column is Capto-Q, Q-SepharoseFF, Q-Sepharose4FF, Q-SepharoseHP, Q-SepharoseXL, DEAE-SepharoseFF or Capto-DEAE chromatography column.
3. the preparation method of a kind of high-purity Human factor IX according to claim 1, is characterized in that: described heparin affinity column is HeparinSepharose6FF, HeparinSepharoseCL-6B.
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