CN105330736A - Method for preparing human blood coagulation factors IX and VII subcutaneously from cold-glue-removed blood plasma - Google Patents
Method for preparing human blood coagulation factors IX and VII subcutaneously from cold-glue-removed blood plasma Download PDFInfo
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Abstract
The invention discloses a method for preparing human blood coagulation factors IX and VII subcutaneously from cold-glue-removed blood plasma. The method comprises the following steps: 1, removing cold glue from blood plasma; 2, conducting strong anion-exchange column chromatography the first time; 3, conducting PEG sedimentation for removing impure protein; 4, conducting S/D viral inactivation; 5, conducting strong anion-exchange column chromatography the second time, and obtaining FVII eluent and FIX eluent; 6, conducting weak anion-exchange column chromatography, and concentration for purifying blood coagulation FVII; 7, conducting heparin affinity column chromatography for purifying blood coagulation FIX; 8, conducting ultrafiltration; 9, adding a stabilizing agent, and conducting adjustment; 10, conducting virus-removal filtration through nanofilms; 11, conducting sterilization, filtration and subpackage; 12, conducting freeze-drying; 13, conducting dry-hot viral inactivation. According to the method, PEG sedimentation is adopted for removing the impure protein, the target of preparing high-purity FVII and FIX simultaneously is achieved through combination of an ion-exchange column chromatography technology and an affinity chromatography technology, the process flow is simple, the production cycle is short, a product is subjected to three steps of virus eradicating measures, and use safety is high.
Description
Technical field
The invention belongs to field of biological pharmacy, relate to the preparation of blood products, specifically relate to a kind of method from removing to prepare cold glue blood plasma Human factor IX and VII simultaneously.
Background technology
Human blood coagulation factors VII (FVII) and Human factor IX (FIX) are all thrombin that vitamin K that liver synthesizes relies on, the former is made up of 406 amino-acid residues, molecular weight is about 50KD, iso-electric point 4.9, Plasma is 0.5-2mg/L, the latter is made up of 416 amino-acid residues, and molecular weight is about 57KD, and Plasma is about 5mg/L.Two kinds of thrombin all play very important effect in coagulation process.Wherein FVII is the initiation factor of body external source coagulation pathway, no matter is idiopathic or insecondary F VII shortage, all likely causes hemorrhage, even threat to life, therefore for the patient that F VII lacks, the purifying F VII that infusion is concentrated, its bleeding tendency can be corrected, recover normal coagulation function.
In recent years, restructuring activation people blood coagulation VIIa has gratifying result for the treatment of to thrombocytopenia and dysfunction of platelet, severe trauma, the operating blood of big area, also becomes with platelet cofactor Ⅰ or the congenital A type of Ⅸ inhibition or the alternative medicine of haemophilia B people.The clinical demand of visible human blood coagulation factor VII is very huge.Human body for want of blood coagulation FIX then can cause blood coagulation disorders to be hemophilia B, and this disease is a kind of heredopathia, 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 blood coagulation FIX to reach the object of hemostasis, or regular injections is hemorrhage containing the chemoprophylaxis of blood coagulation FIX.High-purity people's blood coagulation FIX that medicine containing blood coagulation FIX has blood plasma to extract, Human Factor Ⅸ Complex (PCC) and gene recombinant human blood coagulation FIX (" shellfish tax " that Pfizer produces) etc., domestic only have lyophilized prothrombin complex contrates (PCC) kind at present.Clinical practice shows, it is very safe that hemophilia B people injects high-purity people's blood coagulation FIX, and inject Human Factor Ⅸ Complex and may produce severe side effect-thrombosis, so high-purity people's blood coagulation FIX is the choice drug for the treatment of hemophilia B, but traditional high-purity people's blood coagulation FIX production technique, complex procedures, in preparation FIX process, the FVII of preciousness is used as Impurity removal, 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.
For this present situation, this invention exploits a kind of method from removing to prepare cold glue blood plasma high-purity FVII and FIX simultaneously, the present invention adopts ion exchange chromatography to precipitate impurity elimination in conjunction with affinity chromatography technology and PEG, achieve the target of purifying, refining FVII and FIX, flow process is simple, with short production cycle, the yield of FVII and FIX is all more than 50%.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of process stabilizing, good product quality, yield is high, cost is low and production efficiency the is high method simultaneously preparing Human factor IX and VII.
1., from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: comprise the steps:
(1) producing of cold glue blood plasma is gone
Fresh frozen plasma melted, continuously centrifuged at 0-3 DEG C, remove cold glue, supernatant is cold glue blood plasma, to connect 0.45 μm of filter element filtering with the Supradur50P filter plate that Solution produces afterwards, and what obtain clarifying removes cold glue blood plasma; Before filtration, with damping fluid prewashing filter core; Damping fluid is made up of Trisodium Citrate, sodium-chlor and water, and wherein sodium citrate concentration is 0.01M-0.02M, and sodium chloride concentration is 0.075M-0.15M sodium-chlor, and ph value of buffer solution is 6.50-7.50, temperature 10-15 DEG C;
(2) first time strong anion exchange column chromatography
Strong anion exchange column on cold glue blood plasma is removed in the clarification that step (1) obtains, and pillar balances with level pad 1 in advance; Collect in upper prop process and penetrate liquid in the tank of band low temperature refrigerant chuck, for the processing of follow-up blood product; After upper prop terminates, rinse pillar with level pad 1, then use elution buffer 1 wash-out pillar, collect elutriant, be the solution being rich in FVII and FIX;
(3) PEG precipitates foreigh protein removing
In the elutriant that step (2) is collected, add 50%PEG solution makes final PEG concentration to 3-6%, stirs 0.5-1.5 hour, rear use 1.0 μm of filter element filterings, collects the filtrate of clarification;
(4) S/D inactivation of virus
Tween80 to 1.0%(wt% is added in the filtrate that step (3) is collected), TNBP(tributyl phosphate) to 0.3%(wt%), be warming up to 24-26 DEG C after stirring evenly, rear insulation 6-7 hour;
(5) second time strong anion column chromatography
Solution diluted after above-mentioned S/D is less than 15ms/cm (25 DEG C) to specific conductivity, then goes up strong anion post, pillar balances with level pad 2 in advance; After upper prop terminates, rinse pillar with level pad 2, then use elution buffer 2A wash-out pillar, gained elutriant A is blood coagulation FVII crude product; Use elution buffer 2B wash-out again, collect elutriant B and be blood coagulation FIX crude product;
(6) weak anionic column chromatography purification blood coagulation FVII
By above-mentioned elutriant A diluted to original 2-4 doubly, then go up weak anionic post, pillar balances with level pad 3 in advance; After upper prop terminates, rinse pillar with above level pad 3, then use elution buffer 3 wash-out pillar, gained elutriant is refining blood coagulation FVII solution; Clear filtrate is obtained with 0.45 μm of filter element filtering;
(7) heparin affinity column chromatography purification blood coagulation FIX
Above elutriant B diluted is less than 15ms/cm (25 DEG C) to specific conductivity, upper heparin affinity column, pillar balances with level pad 4 in advance, wash with lavation buffer solution 4 afterwards, use elution buffer 4 wash-out again, collect elutriant, with 0.45 μm of filter element filtering, gained filtrate is refining blood coagulation FIX solution;
(8) ultrafiltration
With the ultra-filtration membrane bag of 5K ~ 10k molecular weight cut-off concentrated above FVII and FIX filtrate respectively, and with dialyzate respectively constant volume dialysis 4-6 doubly, after be concentrated into after required vigor is tired and shift out ultra-filtration membrane bag;
(9) stablizer and adjustment is added
According to required specification respectively mediator's blood coagulation FVII tire and FIX tire to desirable value (being generally 20-30IU/ml), rear add respectively stablizer and respectively adjust pH value to 6.50-7.50;
(10) nanometer film is except virus filtration
Carry out except virus filtration with 15-20 nanometer filter core to above FVII and FIX solution respectively;
(11) Sterile Filtration packing
With 0.22 μm of filter core, above FVII and FIX solution is carried out respectively
(12) freeze-drying
The above blood coagulation FVII of freeze-drying and FIX stoste, obtain blood coagulation FVII and FIX lyophilized powder respectively;
(13) xeothermic except virus filtration
Distinguish or FVII and FIX dried frozen aquatic products be incubated 30 minutes in 100 DEG C of boiling water baths simultaneously, carrying out xeothermic inactivation of virus.
2. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the strong anion exchange column chromatography filler used described in step (2) is CaptoQ, QSepharoseFF, any one in QSepharose4FFQSepharoseHP, QSepharoseXL.
3. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the level pad 1 used of the column chromatography described in step (2) consists of 0.01M-0.02M Trisodium Citrate, 0.075M-0.15M sodium-chlor, PH6.50-7.50; Described elution buffer 1 consists of 0.01M-0.02M Trisodium Citrate, 0.4M-0.8M sodium-chlor, 0.02-0.04M arginine hydrochloride, PH6.50-7.50.
4. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the strong anion exchange column chromatography filler used described in step (6) is CaptoQ, QSepharoseFF, any one in QSepharose4FFQSepharoseHP, QSepharoseXL; Level pad 2 described in step (5) consists of 0.01M-0.02M Trisodium Citrate, 0.075M-0.15M sodium-chlor, PH6.50-7.50; Elution buffer 2A described in step (5) consists of 0.01M-0.02M Trisodium Citrate, 0.175M-0.35M sodium-chlor, 0.02-0.04M arginine hydrochloride, PH6.50-7.50; Elution buffer 2B described in step (5) consists of 0.01M-0.02M Trisodium Citrate, 0.5M-1.0M sodium-chlor, 0.02-0.04M arginine hydrochloride, PH6.50-7.50.
5. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the weak anion exchange column chromatography filler used described in step (6) is DEAE-SepharoseFF or CaptoDEAE; Level pad 3 described in step (6) consists of 0.01M-0.02M Trisodium Citrate, 0.075M-0.1M sodium-chlor, PH6.50-7.50; Described elution buffer 3 consists of 0.01M-0.02M Trisodium Citrate, 0.15M-0.30M sodium-chlor, 0.02-0.04M arginine hydrochloride, PH6.50-7.50.
6. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the heparin affinity column chromatography filler used described in step (7) is HeparinSepharoseFF or HeparinSepharoseCL-6B; Level pad 4 described in step (7) consists of 0.01M-0.02M Trisodium Citrate, 0.1M-0.15M sodium chloride solution, PH6.50-7.50; Described lavation buffer solution 4 consists of 0.01M-0.02M Trisodium Citrate, 0.15-0.25M sodium chloride solution, PH6.50-7.50; Described elution buffer 4 consists of 0.01M-0.02M Trisodium Citrate, 0.02M arginine hydrochloride, 0.4M-1.0M sodium chloride solution, PH6.50-7.50.
7. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the diluent described in step (5) ~ (7) consists of 0.01M-0.02M Trisodium Citrate, 0.02-0.04M arginine hydrochloride, PH6.50-7.50.
8. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the dialyzate described in step (8) consists of 0.01M Trisodium Citrate, 0.075M-0.125M sodium-chlor, PH6.50-7.50; This dialyzate is all applicable to FVII and FIX.
9. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the stablizer described in step (9), for FVIII, stablizer is arginine monohydrochloride and glycine, wherein arginine monohydrochloride adds to 0.5-3%, and glycine adds to 0.5-3%; For FIX, stablizer is Histidine and glycine, and wherein Histidine adds to 0.5-3%, and glycine adds to 0.5-3%.
10. a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously according to claim 1-9, is characterized in that: FVII freeze-dried preparation and FIX freeze-dried preparation all experienced by S/D inactivation of virus, nanometer film except virus filtration and xeothermic inactivation of virus three steps.
superiority of the present invention
1, the present invention adopts ion-exchange packing and the affine filler of heparin from blood plasma, prepare high-purity people's blood coagulation FVII and blood coagulation FIX simultaneously, and flow process is succinct, technology maturation, and blood plasma utilization ratio is high;
2, adopt PEG precipitation to remove foreign protein, significantly reduce the purifying pressure of subsequent columns chromatography, improve the specific activity of finished product;
The introducing of 3, PEG, plays protein protection effect in S/D process, prevents protein denaturation and thrombin inactivation;
4, in column chromatography operation, in elution buffer, all add protein protective agent, greatly reduce the probability that goods are activated;
5, 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, substantially increased the safety in utilization of product.
Accompanying drawing explanation
Fig. 1 is the process flow sheet from removing to prepare cold glue blood plasma Human factor IX and VII simultaneously.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described further; can not think that embodiments of the present invention are only limitted to this; for general technical staff of the technical field of the invention; some simple change made without departing from the inventive concept of the premise or replacement, all should be considered as belonging to the scope of patent protection that claims of the present invention is determined.
Embodiment one
1, go the preparation of cold glue blood plasma: get refrigerated plasma 35 bags, with 70% ethanol disinfection and with water for injection rinse after cut bag, in chuck tank, refrigerated plasma is melted, take 20kg, continuously centrifuged at 0-3 DEG C, remove cold glue, collect supernatant, to connect 0.45 μm of filter element filtering with the Supradur50P filter plate that Solution produces afterwards, what obtain clarifying removes cold glue blood plasma; Before filtration, with damping fluid (PH6.90-7.10,0.02M Trisodium Citrate, 0.1M sodium-chlor) prewashing filter core;
2, first time strong anion exchange column chromatography: QSepharose4FF post on cold glue blood plasma is removed in clarification step 1 obtained, and level pad (PH6.90-7.10,0.02M Trisodium Citrate, 0.1M sodium-chlor) balance used in advance by pillar; After upper prop terminates, with above Equilibration buffer wash pillar, then use elution buffer (PH6.90-7.10,0.02M Trisodium Citrate, 0.8M sodium-chlor, 0.04M arginine hydrochloride) wash-out pillar, collect elutriant;
3, PEG precipitates foreigh protein removing: add 50%PEG solution in above elutriant and make final PEG concentration to 3%, stirs 1.5 hours, and rear use 1.0 μm series connection 0.45 μm of filter element filtering, obtains the filtrate of clarifying;
4, S/D inactivation of virus: add Tween80 to 1.0%(wt% in above filtrate), TNBP(tributyl phosphate) to 0.3%(wt%), be warming up to 24-26 DEG C after stirring evenly, rear insulation 6 hours;
5; second time strong anion column chromatography: by the solution diluent (PH6.90-7.10 after above-mentioned S/D; 0.02M Trisodium Citrate; 0.04M arginine hydrochloride) be diluted to specific conductivity and be less than 15ms/cm (25 DEG C); then go up QSepharose4FF post, level pad (PH6.90-7.10,0.02M Trisodium Citrate used in advance by pillar; 0.15M sodium-chlor) balance; After upper prop terminates, with above equilibration buffer solution pillar, then use elution buffer A(PH6.90-7.10,0.02M Trisodium Citrate, 0.25M sodium-chlor, 0.04M arginine hydrochloride) wash-out pillar, collect elutriant A(blood coagulation seven factor crude product); Use elution buffer B (PH6.90-7.10,0.02M Trisodium Citrate, 0.5M sodium-chlor, 0.04M arginine hydrochloride) wash-out again, collect elutriant B(blood coagulation FIX crude product), be placed in 2-8 DEG C;
6; weak anionic column chromatography purification blood coagulation FVII: by above-mentioned elutriant A diluent (PH6.90-7.10; 0.02M Trisodium Citrate; 0.04M arginine hydrochloride) be diluted to original 2.5 times; then go up DEAE-SepharoseFF post, level pad (PH6.90-7.10,0.02M Trisodium Citrate used in advance by pillar; 0.1M sodium-chlor) balance; After upper prop terminates, with above equilibration buffer solution pillar, then use elution buffer (PH6.90-7.10,0.02M Trisodium Citrate, 0.2M sodium-chlor, 0.04M arginine hydrochloride) wash-out pillar, obtain blood coagulation seven factor solutions refined; Obtain clear filtrate with 0.45 μm of filter element filtering, be placed in 2-8 DEG C;
7, heparin affinity column chromatography purification blood coagulation FIX: the elutriant B diluent (PH6.90-7.10 that step 5 is obtained, 0.02M Trisodium Citrate, 0.04M arginine hydrochloride) be diluted to specific conductivity and be less than 15ms/cm (25 DEG C), upper HeparinSepharose6FF post, pillar uses equilibration buffer in advance, level pad is 0.02M Trisodium Citrate, 0.1M sodium chloride solution, PH6.90-7.10), after use lavation buffer solution (0.02M Trisodium Citrate, 0.175M sodium chloride solution, PH6.90-7.10) wash, use elution buffer (0.02M Trisodium Citrate again, 0.04M arginine hydrochloride, 0.5M sodium chloride solution, PH6.90-7.10) wash-out, collect elutriant, with 0.45 μm of above elutriant of filter element filtering, obtain the blood coagulation FIX solution refined,
8; with the ultra-filtration membrane bag enrichment step 6 respectively of 10k molecular weight; the 7 blood coagulation FVII obtained and FIX filtrates, and with dialyzate (0.02M Trisodium Citrate, 0.1M sodium chloride solution; PH6.90-7.10) dialyse respectively; after be concentrated into after required vigor is tired and shift out ultra-filtration membrane bag, obtain blood coagulation FVII and blood coagulation FIX concentrated solution 205 grams respectively, 231 grams; vigor is respectively 39.7IU/ml, 36.4IU/ml;
9, according to specification difference mediator's blood coagulation FVII solution of 30IU/ml and the vigor of FIX solution, rear pH value of adjusting respectively, to 6.90-7.10, adds arginine monohydrochloride to 0.5%, glycine to 3%, as stablizer in FVII solution; Histidine 0.5% is added, glycine to 3%, as stablizer in FIX solution;
10, carry out except virus filtration with 20 nanometer filter cores to above blood coagulation FVII and FIX solution respectively;
11, with 0.22 μm of filter core, Sterile Filtration is carried out and packing to above blood coagulation FVII and FIX solution respectively;
12, by dividing the sample installed to put into the freeze-drying respectively of two Freeze Drying Equipments above, obtain blood coagulation FVII and FIX dried frozen aquatic products;
13, FVII and FIX dried frozen aquatic products is incubated 30 minutes in 100 DEG C of boiling water baths simultaneously, carries out xeothermic inactivation of virus.
Embodiment two
1, go the preparation of cold glue blood plasma: with embodiment one;
2, first time strong anion exchange column chromatography: Capto-Q post on cold glue blood plasma is removed in clarification step 1 obtained, and level pad (PH7.30-7.40,0.02M Trisodium Citrate, 0.1M sodium-chlor) balance used in advance by pillar; After upper prop terminates, with above Equilibration buffer wash pillar, then use elution buffer (PH7.30-7.40,0.02M Trisodium Citrate, 0.8M sodium-chlor, 0.02M arginine hydrochloride) wash-out pillar, collect elutriant (being rich in blood coagulation FVII and FIX);
3, PEG precipitates foreigh protein removing: add 50%PEG solution in above elutriant and make final PEG concentration to 6%, stirs 0.5 hour, and rear use 1.0 μm series connection 0.45 μm of filter element filtering, obtains the filtrate of clarifying;
4, S/D inactivation of virus: with embodiment one;
5; second time strong anion column chromatography: by the solution diluent (PH7.30-7.40 after above-mentioned S/D; 0.02M Trisodium Citrate; 0.02M arginine hydrochloride) be diluted to specific conductivity and be less than 15ms/cm (25 DEG C); then go up Capto-Q post, level pad (PH7.30-7.40,0.02M Trisodium Citrate used in advance by pillar; 0.15M sodium-chlor) balance; After upper prop terminates, with above equilibration buffer solution pillar, then use elution buffer A(PH7.30-7.40,0.02M Trisodium Citrate, 0.3M sodium-chlor, 0.02M arginine hydrochloride) wash-out pillar, collect elutriant A(blood coagulation seven factor crude product); Use elution buffer B (PH7.30-7.40,0.02M Trisodium Citrate, 1.0M sodium-chlor, 0.02M arginine hydrochloride) wash-out again, collect elutriant B(blood coagulation nine factor crude product); Obtain blood coagulation FIX crude product, be placed in 2-8 DEG C;
6; weak anionic column chromatography purification blood coagulation FVII: by above-mentioned elutriant A diluent (PH7.30-7.40; 0.02M Trisodium Citrate; 0.02M arginine hydrochloride) be diluted to original 3 times; then go up DEAE-SepharoseFF post, level pad (PH7.30-7.40,0.02M Trisodium Citrate used in advance by pillar; 0.1M sodium-chlor) balance; After upper prop terminates, with above equilibration buffer solution pillar, then use elution buffer (PH7.30-7.40,0.02M Trisodium Citrate, 0.3M sodium-chlor, 0.02M arginine hydrochloride) wash-out pillar, obtain blood coagulation seven factor solutions refined; Obtain clear filtrate with 0.45 μm of filter element filtering, be placed in 2-8 DEG C;
7, heparin affinity column chromatography purification blood coagulation FIX: the elutriant B diluent (PH7.30-7.40 that step 5 is obtained, 0.02M Trisodium Citrate, 0.02M arginine hydrochloride) be diluted to specific conductivity and be less than 15ms/cm (25 DEG C), upper HeparinSepharoseCL-6B post, level pad (0.02M Trisodium Citrate used in advance by pillar, 0.15M sodium chloride solution, PH7.30-7.40) balance, , after use lavation buffer solution (0.02M Trisodium Citrate, 0.25M sodium chloride solution, PH7.30-7.40) wash, use elution buffer (0.02M Trisodium Citrate again, 0.04M arginine hydrochloride, 1.0M sodium chloride solution, PH7.30-7.40) wash-out, collect elutriant, the blood coagulation FIX solution refined is obtained with 0.45 μm of above elutriant of filter element filtering,
8; with the ultra-filtration membrane bag enrichment step 6 respectively of 10k molecular weight; the 7 blood coagulation FVII obtained and FIX filtrates, and with dialyzate (0.02M Trisodium Citrate, 0.1M sodium chloride solution; PH6.90-7.10) dialyse respectively; after be concentrated into after required vigor is tired and shift out ultra-filtration membrane bag, obtain blood coagulation FVII and blood coagulation FIX concentrated solution 196 grams respectively, 219 grams; vigor is respectively 41.1IU/ml, 38.2IU/ml;
9 ~ 13, with embodiment one.
Embodiment three
1, go the preparation of cold glue blood plasma: with embodiment one;
2, first time strong anion exchange column chromatography: Capto-Q post on cold glue blood plasma is removed in clarification step 1 obtained, and level pad (PH6.50-6.60,0.02M Trisodium Citrate, 0.15M sodium-chlor) balance used in advance by pillar; After upper prop terminates, with above Equilibration buffer wash pillar, then use elution buffer (PH7.30-7.40,0.02M Trisodium Citrate, 0.6M sodium-chlor, 0.02M arginine hydrochloride) wash-out pillar, collect elutriant (being rich in blood coagulation FVII and FIX);
3, PEG precipitates foreigh protein removing: add 50%PEG solution in above elutriant and make final PEG concentration to 4%, stirs 1 hour, and rear use 1.0 μm series connection 0.45 μm of filter element filtering, obtains the filtrate of clarifying;
4, S/D inactivation of virus: with embodiment one;
5; second time strong anion column chromatography: by the solution diluent (PH6.50-6.60 after above-mentioned S/D; 0.02M Trisodium Citrate; 0.02M arginine hydrochloride) be diluted to specific conductivity and be less than 15ms/cm (25 DEG C); then go up Capto-Q post, level pad (PH6.50-6.60,0.02M Trisodium Citrate used in advance by pillar; 0.15M sodium-chlor) balance; After upper prop terminates, with above equilibration buffer solution pillar, then use elution buffer A(PH7.30-7.40,0.02M Trisodium Citrate, 0.25M sodium-chlor, 0.02M arginine hydrochloride) wash-out pillar, collect elutriant A(blood coagulation seven factor crude product); Use elution buffer B (PH7.30-7.40,0.02M Trisodium Citrate, 0.7M sodium-chlor, 0.02M arginine hydrochloride) wash-out again, collect elutriant B(blood coagulation FIX crude product), be placed in 2-8 DEG C;
6; weak anionic column chromatography purification blood coagulation FVII: by above-mentioned elutriant A diluent (PH6.50-6.60; 0.02M Trisodium Citrate; 0.02M arginine hydrochloride) be diluted to original 2 times; then go up DEAE-SepharoseFF post, level pad (PH6.50-6.60,0.02M Trisodium Citrate used in advance by pillar; 0.1M sodium-chlor) balance; After upper prop terminates, with above equilibration buffer solution pillar, then use elution buffer (PH6.50-6.60,0.02M Trisodium Citrate, 0.25M sodium-chlor, 0.02M arginine hydrochloride) wash-out pillar, obtain blood coagulation seven factor solutions refined; Obtain clear filtrate with 0.45 μm of filter element filtering, be placed in 2-8 DEG C;
7, heparin affinity column chromatography purification blood coagulation FIX: the elutriant B diluent (PH6.90-7.10 that step 5 is obtained, 0.02M Trisodium Citrate, 0.02M arginine hydrochloride) be diluted to specific conductivity and be less than 15ms/cm (25 DEG C), upper HeparinSepharose6FF post, level pad (0.02M Trisodium Citrate used in advance by pillar, 0.15M sodium chloride solution, PH6.90-7.10) balance, , after use lavation buffer solution (0.02M Trisodium Citrate, 0.25M sodium chloride solution, PH6.90-7.10) wash, use elution buffer (0.02M Trisodium Citrate again, 0.04M arginine hydrochloride, 0.6M sodium chloride solution, PH6.90-7.10) wash-out, collect elutriant, the blood coagulation FIX solution refined is obtained with 0.45 μm of above elutriant of filter element filtering,
8, with embodiment one, finally obtain blood coagulation FVII and blood coagulation FIX concentrated solution 219 grams respectively, 242 grams, vigor is respectively 37.8IU/ml, 36.7IU/ml;
9 ~ 13, with embodiment one.
subordinate list preproduction FVII and FIX yield and specific activity table look-up
Claims (10)
1., from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: comprise the steps:
(1) producing of cold glue blood plasma is gone
Fresh frozen plasma melted, continuously centrifuged at 0-3 DEG C, remove cold glue, supernatant is cold glue blood plasma, to connect 0.45 μm of filter element filtering with the Supradur50P filter plate that Solution produces afterwards, and what obtain clarifying removes cold glue blood plasma; Before filtration, with damping fluid prewashing filter core; Damping fluid is made up of Trisodium Citrate, sodium-chlor and water, and wherein sodium citrate concentration is 0.01M-0.02M, and sodium chloride concentration is 0.075M-0.15M sodium-chlor, and ph value of buffer solution is 6.50-7.50, temperature 10-15 DEG C;
(2) first time strong anion exchange column chromatography
Strong anion exchange column on cold glue blood plasma is removed in the clarification that step (1) obtains, and pillar balances with level pad 1 in advance; Collect in upper prop process and penetrate liquid in the tank of band low temperature refrigerant chuck, for the processing of follow-up blood product; After upper prop terminates, rinse pillar with level pad 1, then use elution buffer 1 wash-out pillar, collect elutriant, be the solution being rich in FVII and FIX;
(3) PEG precipitates foreigh protein removing
In the elutriant that step (2) is collected, add 50%PEG solution makes final PEG concentration to 3-6%, stirs 0.5-1.5 hour, rear use 1.0 μm of filter element filterings, collects the filtrate of clarification;
(4) S/D inactivation of virus
Tween80 to 1.0%(wt% is added in the filtrate that step (3) is collected), TNBP(tributyl phosphate) to 0.3%(wt%), be warming up to 24-26 DEG C after stirring evenly, rear insulation 6-7 hour;
(5) second time strong anion column chromatography
Solution diluted after above-mentioned S/D is less than 15ms/cm (25 DEG C) to specific conductivity, then goes up strong anion post, pillar balances with level pad 2 in advance; After upper prop terminates, rinse pillar with level pad 2, then use elution buffer 2A wash-out pillar, gained elutriant A is blood coagulation FVII crude product; Use elution buffer 2B wash-out again, collect elutriant B and be blood coagulation FIX crude product;
(6) weak anionic column chromatography purification blood coagulation FVII
By above-mentioned elutriant A diluted to original 2-4 doubly, then go up weak anionic post, pillar balances with level pad 3 in advance; After upper prop terminates, rinse pillar with above level pad 3, then use elution buffer 3 wash-out pillar, gained elutriant is refining blood coagulation FVII solution; Clear filtrate is obtained with 0.45 μm of filter element filtering;
(7) heparin affinity column chromatography purification blood coagulation FIX
Above elutriant B diluted is less than 15ms/cm (25 DEG C) to specific conductivity, upper heparin affinity column, pillar balances with level pad 4 in advance, wash with lavation buffer solution 4 afterwards, use elution buffer 4 wash-out again, collect elutriant, with 0.45 μm of filter element filtering, gained filtrate is refining blood coagulation FIX solution;
(8) ultrafiltration
With the ultra-filtration membrane bag of 5K ~ 10k molecular weight cut-off concentrated above FVII and FIX filtrate respectively, and with dialyzate respectively constant volume dialysis 4-6 doubly, after be concentrated into after required vigor is tired and shift out ultra-filtration membrane bag;
(9) stablizer and adjustment is added
According to required specification respectively mediator's blood coagulation FVII tire and FIX tire to desirable value (being generally 20-30IU/ml), rear add respectively stablizer and respectively adjust pH value to 6.50-7.50;
(10) nanometer film is except virus filtration
Carry out except virus filtration with 15-20 nanometer filter core to above FVII and FIX solution respectively;
(11) Sterile Filtration packing
With 0.22 μm of filter core, above FVII and FIX solution is carried out respectively
(12) freeze-drying
The above blood coagulation FVII of freeze-drying and FIX stoste, obtain blood coagulation FVII and FIX lyophilized powder respectively;
(13) xeothermic except virus filtration
Distinguish or FVII and FIX dried frozen aquatic products be incubated 30 minutes in 100 DEG C of boiling water baths simultaneously, carrying out xeothermic inactivation of virus.
2. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the strong anion exchange column chromatography filler used described in step (2) is CaptoQ, QSepharoseFF, any one in QSepharose4FFQSepharoseHP, QSepharoseXL.
3. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the level pad 1 used of the column chromatography described in step (2) consists of 0.01M-0.02M Trisodium Citrate, 0.075M-0.15M sodium-chlor, PH6.50-7.50; Described elution buffer 1 consists of 0.01M-0.02M Trisodium Citrate, 0.4M-0.8M sodium-chlor, 0.02-0.04M arginine hydrochloride, PH6.50-7.50.
4. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the strong anion exchange column chromatography filler used described in step (6) is CaptoQ, QSepharoseFF, any one in QSepharose4FFQSepharoseHP, QSepharoseXL; Level pad 2 described in step (5) consists of 0.01M-0.02M Trisodium Citrate, 0.075M-0.15M sodium-chlor, PH6.50-7.50; Elution buffer 2A described in step (5) consists of 0.01M-0.02M Trisodium Citrate, 0.175M-0.35M sodium-chlor, 0.02-0.04M arginine hydrochloride, PH6.50-7.50; Elution buffer 2B described in step (5) consists of 0.01M-0.02M Trisodium Citrate, 0.5M-1.0M sodium-chlor, 0.02-0.04M arginine hydrochloride, PH6.50-7.50.
5. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the weak anion exchange column chromatography filler used described in step (6) is DEAE-SepharoseFF or CaptoDEAE; Level pad 3 described in step (6) consists of 0.01M-0.02M Trisodium Citrate, 0.075M-0.1M sodium-chlor, PH6.50-7.50; Described elution buffer 3 consists of 0.01M-0.02M Trisodium Citrate, 0.15M-0.30M sodium-chlor, 0.02-0.04M arginine hydrochloride, PH6.50-7.50.
6. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the heparin affinity column chromatography filler used described in step (7) is HeparinSepharoseFF or HeparinSepharoseCL-6B; Level pad 4 described in step (7) consists of 0.01M-0.02M Trisodium Citrate, 0.1M-0.15M sodium chloride solution, PH6.50-7.50; Described lavation buffer solution 4 consists of 0.01M-0.02M Trisodium Citrate, 0.15-0.25M sodium chloride solution, PH6.50-7.50; Described elution buffer 4 consists of 0.01M-0.02M Trisodium Citrate, 0.02M arginine hydrochloride, 0.4M-1.0M sodium chloride solution, PH6.50-7.50.
7. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the diluent described in step (5) ~ (7) consists of 0.01M-0.02M Trisodium Citrate, 0.02-0.04M arginine hydrochloride, PH6.50-7.50.
8. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the dialyzate described in step (8) consists of 0.01M Trisodium Citrate, 0.075M-0.125M sodium-chlor, PH6.50-7.50; This dialyzate is all applicable to FVII and FIX.
9. according to claim 1 a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously, it is characterized in that: the stablizer described in step (9), for FVIII, stablizer is arginine monohydrochloride and glycine, wherein arginine monohydrochloride adds to 0.5-3%, and glycine adds to 0.5-3%; For FIX, stablizer is Histidine and glycine, and wherein Histidine adds to 0.5-3%, and glycine adds to 0.5-3%.
10. a kind of from going the method preparing Human factor IX and VII cold glue blood plasma simultaneously according to claim 1-9, is characterized in that: FVII freeze-dried preparation and FIX freeze-dried preparation all experienced by S/D inactivation of virus, nanometer film except virus filtration and xeothermic inactivation of virus three steps.
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| CN111378029B (en) * | 2018-12-29 | 2023-05-05 | 四川远大蜀阳药业有限责任公司 | Preparation method of human coagulation factor IX |
| CN109651502A (en) * | 2019-01-29 | 2019-04-19 | 华兰生物工程股份有限公司 | A method of isolating and purifying plasma thromboplastin component, X and VII simultaneously from human plasma |
| CN111849945A (en) * | 2019-04-25 | 2020-10-30 | 正大天晴药业集团股份有限公司 | Method for purifying human blood coagulation factor VIIa |
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| CN114829371A (en) * | 2019-12-12 | 2022-07-29 | Emd密理博公司 | Enhanced virus filtration using diafiltration buffer |
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| CN114306578A (en) * | 2020-10-12 | 2022-04-12 | 华兰生物工程股份有限公司 | A pharmaceutical composition containing human blood coagulation factor IX and its preparation method |
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