CN104231073A - Preparation method of human coagulation factor VIII - Google Patents
Preparation method of human coagulation factor VIII Download PDFInfo
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- CN104231073A CN104231073A CN201410496187.9A CN201410496187A CN104231073A CN 104231073 A CN104231073 A CN 104231073A CN 201410496187 A CN201410496187 A CN 201410496187A CN 104231073 A CN104231073 A CN 104231073A
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Abstract
The invention discloses a preparation method of a human coagulation factor VIII. The human coagulation factor VIII prepared by the method does not contain human serum albumin or other animal-derived protein, does not contain sugar or sugar alcohol, does not have the risk for transmitting other viruses or pathogene, and is wide in applicable crowd scope, and can be used by diabetic patients; the human coagulation factor VIII prepared by the method is fast to redissolve and good in redissolving effect, and still keeps high titer and high specific activity which are respectively larger than 80 percent and 40 IU/mg; in addition, the preparation method is simple, the cost is low, the human coagulation factor VIII is safe and effective, and has a good industrial application prospect.
Description
Technical field
The invention belongs to medical art, be specifically related to the preparation method of a kind of human blood coagulation factor VII I.
Background technology
Hemophilia is a kind of hereditary hemorrhagic disease, is cause human body intravascular coagulation factor level to reduce due to coagulation factor gene sudden change or lack, thus causes hemorrhage.Shortage or the deficiency of thrombin F VIII cause hemophilia A, and shortage or the deficiency of plasma thromboplastin component cause hemophilia B.Thrombin replacement therapy is haemophiliachemophiliac primary treatment measure.
Along with the development of modern chromatographic technique, external blood products enterprise has developed high purity human blood coagulation goods, meets the demand of haemophiliac.China considers from Viral safety, and national Yao Jian department is not yet Approved by the thrombin import of blood sources.Domestic blood products enterprise is limited to technical capacity, and be main mainly with production human serum albumin and immunoglobulin (Ig), the market shares such as blood clotting factors are considerably less, wherein platelet cofactor Ⅰ (preventing and treating hemophilia) extremely shortage.Therefore researching and developing blood clotting factors blood products product innovation, carry out new technologies research, will be the trend of the times in blood products field.
Due to blood coagulation factor VIII easy in inactivation; lyophilize or dry heat treatment process easily destroy the protein stability of blood coagulation factor VIII; cause distortion; cause blood coagulation factor VIII inactivation; and the method for inactivation of virus or removal in preparation technology's flow process of human blood coagulation factor VII I product, must be comprised; such as pasteurization, the deactivation of S/D method, the deactivation of 20nm nano-film filtration method etc., therefore need to add certain protective material or stablizer before human blood coagulation factor VII I carries out lyophilize or dry heat treatment.
At present, the protective material that conventional human blood coagulation factor VII I adds in preparation process mainly utilizes albumin or sugar alcohol, collaborative amino acid, inorganic salt etc. play a role, human serum albumin is introduced in protective material disclosed in Chinese invention patent 201110240278.2 and 201210468069.8, but not only cost is high to introduce human serum albumin, and probably there is the risk of other viruses of potential propagation or pathogenic agent in the use of human serum albumin; Chinese invention patent 201210060299.0 adopts sugar alcohol and combination of amino acids as protective material; but containing sugar or sugar alcohol in obtained human blood coagulation factor VII I product; be not suitable for diabetic subject to use; limit the range of application of these goods; these patent goods are after thermal treatment inactivation of viruses simultaneously; the tiring of blood coagulation factor VIII declines serious, and blood coagulation activity yield only about 33% after thermal treatment, actual application value is little.
Therefore, this area urgently a kind of effective preparation method can make blood coagulation factor VIII keep tiring after inactivation of viruses do not decline while do not introduce again range of application and the preparation cost that other materials such as albumin or sugar alcohol affect product.
Summary of the invention
The object of the invention is to overcome above-mentioned the deficiencies in the prior art, the preparation method of a kind of human blood coagulation factor VII I be provided, not end user's seralbumin or other animal derived protein, do not use sugar or sugar alcohol yet, obtained product keeps higher tiring and specific activity.
Above-mentioned purpose of the present invention is achieved by following technical solution:
A preparation method of human blood coagulation factor VII I, comprises the steps:
S1. blood plasma cryoprecipitate and cryoprecipitate dissolving is prepared;
S2. aluminium glue absorption;
S3. S/D inactivation of virus;
S4. gel filtration chromatography purifying and ultrafiltration and concentration;
S5. human blood coagulation factor VII I freeze-drying and dry heat treatment protective material is configured;
S6. freeze-drying and dry heat treatment after human blood coagulation factor VII I freeze-drying and dry heat treatment protective material is added, described human blood coagulation factor VII I freeze-drying and the protectant add-on of dry heat treatment are 0.8 ~ 1.5 times of liquor capacity after ultrafiltration and concentration, make tiring of blood coagulation factor VIII in mixed solution be 18 ~ 22 IU/ml;
Described human blood coagulation factor VII I freeze-drying and dry heat treatment protective material comprise Trisodium Citrate 1 ~ 25 mmol/L, sodium-chlor 10 ~ 150 mmol/L, calcium chloride 2 ~ 9 mmol/L, glycine 10 ~ 150 mmol/L, arginine 10 ~ 150 mmol/L, Histidine 10 ~ 150 mmol/L, Methionin 10 ~ 150 mmol/L, tensio-active agent 0.01 ~ 0.25 mmol/L; Described tensio-active agent is nonionic surface active agent.
As a kind of preferred version; described human blood coagulation factor VII I freeze-drying and dry heat treatment protective material are made up of Trisodium Citrate 10 ~ 15 mmol/L, sodium-chlor 50 ~ 100 mmol/L, calcium chloride 4 ~ 7 mmol/L, glycine 50 ~ 100 mmol/L, arginine 50 ~ 100 mmol/L, Histidine 50 ~ 100 mmol/L, Methionin 50 ~ 100 mmol/L, tensio-active agent 0.1 ~ 0.2 mmol/L, and described tensio-active agent is nonionic surface active agent.
As the further preferred version of one; described human blood coagulation factor VII I freeze-drying and dry heat treatment protective material are made up of Trisodium Citrate 13 mmol/L, sodium-chlor 80 mmol/L, calcium chloride 6 mmol/L, glycine 80 mmol/L, arginine 80 mmol/L, Histidine 80 mmol/L, Methionin 80 mmol/L, tensio-active agent 0.15 mmol/L, and described tensio-active agent is nonionic surface active agent.
The present invention uses nonionic surface active agent; not only can work in coordination with Trisodium Citrate, sodium-chlor, calcium chloride, glycine, arginine, Histidine, Methionin performance provide protection; improve the stability of human blood coagulation factor VII I; the specific inductivity that human blood coagulation factor VII I freeze-dried products is multiple water-soluble can also be reduced; avoid protein component to cause flocculation sediment because of electrically charged, ensure that the quality of last human blood coagulation factor VII I goods.In addition prove by experiment; it is superior that conventional cats product (as hexadecylpyridinium chloride, palmityl trimethyl ammonium chloride etc.) and anion surfactant (as sodium stearate, sodium lauryl sulphate, sulfosuccinic ester etc.) are obviously not so good as nonionic surface active agent to the provide protection of human blood coagulation factor VII I; cationic surfactant toxicity is larger simultaneously; aniorfic surfactant hemolytic activity is comparatively strong, can reduce the quality of human blood coagulation factor VII I product.
Preferably, described nonionic surface active agent is Tween class (polysorbate, tween), Span class (sorbitan fatty acid ester, sapn), polyoxyethylene octylphenol ether class, polyoxyethylene nonylphenol ether class, MM class or Pluronic class (poloxamer class) tensio-active agent.
Particularly, described nonionic surface active agent is tween 80, polysorbate60, polysorbate40, sorbester p17, sorbester p18, span 20, TritonX-100, Neutronyx 600, Arlacel A etc.
More preferably, described nonionic surface active agent is Tween class tensio-active agent, as tween 80, polysorbate60, polysorbate40 etc.
Most preferably, described tensio-active agent is tween 80, obtained human blood coagulation factor VII I freeze-drying and dry heat treatment protective material effect optimum.
Preferably; the concrete steps of step S6 are: human blood coagulation factor VII I freeze-drying and dry heat treatment protective material are mixed according to volume ratio 1:1 with the solution after ultrafiltration and concentration; tiring of blood coagulation factor VIII in mixing solutions is made to be 20 IU/ml; again mixing solutions is dispensed in albumen bottle; lyophilize rear seal-cover; again through 80 DEG C of dry heat treatment inactivation of viruses, obtained human blood coagulation factor VII I finished product.
In the preparation method of human blood coagulation factor VII I of the present invention, step S1 ~ S4 can with reference to this area conventional steps, and as a kind of specific embodiments, the concrete steps of step S1 ~ S4 are:
S1. blood plasma cryoprecipitate and cryoprecipitate dissolving is prepared: with Fresh Frozen human plasma for raw material, through blood plasma thawing, centrifugal preparation cryoprecipitate, by the physiological saline solution containing 8 IU/ml heparin of cryoprecipitate with 15 ~ 20 DEG C of precoolings, the consumption of physiological saline is 7 ~ 9 times of Sediment weight, leaves standstill 60min after stirring and dissolving;
S2. aluminium glue absorption: the lysate after leaving standstill is warming up to 25 ~ 28 DEG C, add the aluminum hydroxide gel of synthermal mass concentration 3%, add-on is 40% of cryoprecipitate weight, adopt and stir the lower mode infiltrated and add, namely under 80 ~ 100 rpm stirring velocitys, below the liquid level with emulsion tube aluminum hydroxide gel being pumped into lysate, interpolation speed is 80 ~ 100 ml/min, be cooled to 15 ~ 20 DEG C after interpolation, the centrifugal removal of impurities of 12000 rpm, gets supernatant liquor;
S3. S/D inactivation of virus: supernatant liquor is warming up to 35 DEG C, add S/D virus inactivating agent insulation 3h and complete first time inactivation of virus, obtain inactivation of virus liquid, wherein S/D virus inactivating agent contains 1% polysorbate (Tween-80), 0.3% tributyl phosphate (TNBP);
S4. gel filtration chromatography purifying and ultrafiltration and concentration: gel column washing lotion 1 is balanced, inactivation of virus liquid loading gel filtration chromatography, under 280nm length ultraviolet light detection, post 4 times of column volumes are washed by washing lotion 2, with washing lotion 3 wash-out blood coagulation factor VIII, and collect, then carry out ultrafiltration dealcoholysis and desalination, obtain high density and concentrate blood coagulation factor VIII solution, tiring of blood coagulation factor VIII controls at 40 IU/ml; Wherein gel model is DEAE-SAPHROSE FF, and loading and elution flow rate are 220 ~ 250 ml/min, and washing lotion 1 formula is 110 mM sodium-chlor, 10 mM Sodium Citrates, 1 mM calcium chloride, 16 mM Methionins, 120 mM glycine, pH7.0; The formula of washing lotion 2 is 150 mM sodium-chlor, 10 mM Sodium Citrates, 1 mM calcium chloride, 16 mM Methionins, 120 mM glycine, pH7.0; The formula of washing lotion 3 is 250 mM sodium-chlor, 10 mM Sodium Citrates, 1 mM calcium chloride, 16 mM Methionins, 120 mM glycine, pH7.0.
The present invention uses heparin to replace the conventional Tris-HCl solution used as lysate, can reduce the loss of C activeconstituents in human blood coagulation factor VII I to greatest extent, keep the biological activity of higher human blood coagulation factor VII I.
The present invention adopts aluminum hydroxide gel absorption method to replace the conventional PEG precipitator method, can effectively remove most of foreign protein, the rate of recovery improving product further and the protein content reduced in product.
The present invention adopts the precooling physiological saline solution containing heparin in cryoprecipitate dissolution phase, traditional technology normal temperature contains heparin injections water dissolution, precooling dissolved water is to keeping protein-active, preventing bacteria breed favourable, protein is conducive to holding structure and stablizes thus the activity keeping thrombin in physiological saline, time of repose in this technique making processes can make soluble protein fully be separated with insoluble protein, the purity of blood coagulation factor VIII can be significantly improved, obtain the human blood coagulation factors VIII goods of low albumen high specific acitivity.
The cryoprecipitate that the present invention is separated in blood plasma is as raw material, after preparing high reactivity blood coagulation factor VIII, clout also can be used for being separated preparation human fibrinogen, fibrinogenic natural structure can be kept, other thrombin in blood plasma (be mainly II, VII, Ⅸ, Ⅹ etc.) can be removed again by a larger margin, improve the purity of goods; Effectively remove the residue of the S/D solvent added in S/D viral inaction steps simultaneously, increase the security of goods.(" Chinese Pharmacopoeia " human fibrin primary standard: purity >=70.0%, this technique development standard: purity >=75.0%).
The condition of dry heat treatment of the present invention is 80 DEG C of dry heat treatment 72h or 100 DEG C dry heat treatment 0.5h.
Freeze-drying of the present invention is the lyophilize process of this area routine.
The amino acid classes that step S5 of the present invention selects when configuring human blood coagulation factor VII I freeze-drying and dry heat treatment protective material and consumption proportion unconventional selection, the effect that must simultaneously use the protective material of the glycine of above-mentioned consumption proportion, arginine, Histidine and Methionin could realize human blood coagulation factor VII I goods to keep high-titer and high specific acitivity through dry heat treatment inactivation of viruses.
A kind of human blood coagulation factor VII I product obtained according to the preparation method of human blood coagulation factor VII I of the present invention.
Compared with prior art, the present invention has following beneficial effect:
(1) in the human blood coagulation factor VII I product obtained according to preparation method of the present invention containing human serum albumin or other animal derived protein, also not containing sugar or sugar alcohol, be made up of soluble salt, amino acid and tensio-active agent, there is not the risk propagating other viruses or pathogenic agent, be applicable to crowd's scope wide, comprise diabetic subject and also can use.
(2), in the human blood coagulation factor VII I product obtained according to preparation method of the present invention, blood coagulation factor VIII still keeps high-titer and high specific acitivity, tire be greater than 80%, specific activity is greater than 40 IU/mg.
(3) the human blood coagulation factor VII I product obtained according to preparation method of the present invention redissolves fast, far below the quality standard of redissolution time 30min, and redissolves effective, and the liquid that redissolves clarifies micro-band opalescence.
(4) the human blood coagulation factor VII I product obtained according to preparation method of the present invention does not contain human serum albumin, and preparation method is simple, with low cost, and product safety is effective, has good prospects for commercial application.
Embodiment
Below in conjunction with specific embodiment, the present invention is further explained, but embodiments of the present invention is not limited in any way.Unless stated otherwise, involved in embodiment reagent, method are the conventional reagent in this area and method.
embodiment 1
One, human blood coagulation factor VII I solution is prepared
(1) cryoprecipitate and cryoprecipitate dissolving is made
With Fresh Frozen human plasma for raw material, through blood plasma thawing, centrifugal preparation cryoprecipitate, by the physiological saline solution containing 8 IU/ml heparin of cryoprecipitate with 15 ~ 20 DEG C of precoolings, the consumption of physiological saline is 7 ~ 9 times of Sediment weight, leaves standstill 60 min after stirring and dissolving.
(2) aluminium glue absorption
Lysate after leaving standstill is warming up to 25 ~ 28 DEG C, add the aluminum hydroxide gel of synthermal mass concentration 3%, add-on is 40% of cryoprecipitate weight, adopt and stir the lower mode infiltrated and add, namely under 80 ~ 100 rpm stirring velocitys, below the liquid level with emulsion tube aluminum hydroxide gel being pumped into lysate, interpolation speed is 80 ~ 100 ml/min, be cooled to 15 ~ 20 DEG C after interpolation, the centrifugal removal of impurities of 12000 rpm, gets supernatant liquor.
(3) S/D inactivation of virus
Supernatant liquor is warming up to 35 DEG C, add S/D virus inactivating agent be incubated 3 h complete first time inactivation of virus, obtain inactivation of virus liquid, wherein S/D virus inactivating agent contains 1% polysorbate (Tween-80), 0.3% tributyl phosphate (TNBP).
(4) gel filtration chromatography purifying
Gel column washing lotion 1 is balanced, inactivation of virus liquid loading gel filtration chromatography, under 280 nm length ultraviolet light detections, wash post 4 times of column volumes by washing lotion 2, with washing lotion 3 wash-out blood coagulation factor VIII, and collect.Wherein gel model is DEAE-SAPHROSE FF, and loading and elution flow rate are 220 ~ 250 ml/min, and the formula of washing lotion 1 is 110 mM sodium-chlor, 10 mM Sodium Citrates, 1 mM calcium chloride, 16 mM Methionins, 120 mM glycine, pH7.0; The formula of washing lotion 2 is 150 mM sodium-chlor, 10 mM Sodium Citrates, 1 mM calcium chloride, 16 mM Methionins, 120 mM glycine, pH7.0; The formula of washing lotion 3 is 250 mM sodium-chlor, 10 mM Sodium Citrates, 1 mM calcium chloride, 16 mM Methionins, 120 mM glycine, pH7.0.
(5) ultrafiltration and concentration
The blood coagulation factor VIII of collection is carried out ultrafiltration dealcoholysis and desalination, obtains high density and concentrate blood coagulation factor VIII solution, tiring of blood coagulation factor VIII controls at 40 IU/ml.
Two, human blood coagulation factor VII I freeze-drying and dry heat treatment protective material is prepared
Take all kinds of inorganic salt, amino acid and tensio-active agent in proportion; adopt common medical used injection water to configure human blood coagulation factor VII I freeze-drying and dry heat treatment protective material, in obtained human blood coagulation factor VII I freeze-drying and dry heat treatment protective material, all kinds of original concentration is as follows: Trisodium Citrate 13 mmol/L, sodium-chlor 80 mmol/L, calcium chloride 6 mmol/L, glycine 80 mmol/L, arginine 80 mmol/L, Histidine 80 mmol/L, Methionin 80 mmol/L, tween 80 0.15 mmol/L.
Three, human blood coagulation factor VII I freeze-drying and the protectant use of dry heat treatment
Human blood coagulation factor VII I freeze-drying obtained with step 2 for blood coagulation factor VIII solution obtained for step one and dry heat treatment protective material are mixed according to volume ratio 1:1; tiring of blood coagulation factor VIII is made to control at 20 IU/ml; again mixing solutions is dispensed into (10 ml/ bottle) in albumen bottle; lyophilize rear seal-cover; again through 80 DEG C of dry heat treatment inactivation of viruses, obtained human blood coagulation factor VII I finished product.
embodiment 2
One, human blood coagulation factor VII I solution is prepared
With reference to the step one of embodiment 1.
Two, human blood coagulation factor VII I freeze-drying and dry heat treatment protective material is prepared
Take all kinds of inorganic salt, amino acid and tensio-active agent in proportion; adopt common medical used injection water to configure human blood coagulation factor VII I freeze-drying and dry heat treatment protective material, in obtained human blood coagulation factor VII I freeze-drying and dry heat treatment protective material, all kinds of original concentration is as follows: Trisodium Citrate 10 mmol/L, sodium-chlor 100 mmol/L, calcium chloride 4 mmol/L, glycine 50 mmol/L, arginine 50 mmol/L, Histidine 100 mmol/L, Methionin 100 mmol/L, tween 80 0.1 mmol/L.
Three, human blood coagulation factor VII I freeze-drying and the protectant use of dry heat treatment
With reference to the step 3 of embodiment 1.
embodiment 3
One, human blood coagulation factor VII I solution is prepared
With reference to the step one of embodiment 1.
Two, human blood coagulation factor VII I freeze-drying and dry heat treatment protective material is prepared
Take all kinds of inorganic salt, amino acid and tensio-active agent in proportion; adopt common medical used injection water to configure human blood coagulation factor VII I freeze-drying and dry heat treatment protective material, in obtained human blood coagulation factor VII I freeze-drying and dry heat treatment protective material, all kinds of original concentration is as follows: Trisodium Citrate 15 mmol/L, sodium-chlor 50 mmol/L, calcium chloride 7 mmol/L, glycine 100 mmol/L, arginine 100 mmol/L, Histidine 80 mmol/L, Methionin 80 mmol/L, tween 80 0.2 mmol/L.
Three, human blood coagulation factor VII I freeze-drying and the protectant use of dry heat treatment
With reference to the step 3 of embodiment 1.
embodiment 4
Key step is identical with embodiment 1, and distinctive points is, when preparing human blood coagulation factor VII I freeze-drying and dry heat treatment protective material in step 2, tween 80 is replaced with polysorbate60.
embodiment 5
Key step is identical with embodiment 1, and distinctive points is, when preparing human blood coagulation factor VII I freeze-drying and dry heat treatment protective material in step 2, tween 80 is replaced with polysorbate40.
embodiment 6
Key step is identical with embodiment 1, and distinctive points is, when preparing human blood coagulation factor VII I freeze-drying and dry heat treatment protective material in step 2, tween 80 is replaced with sorbester p17.
embodiment 7
Key step is identical with embodiment 1, and distinctive points is, when preparing human blood coagulation factor VII I freeze-drying and dry heat treatment protective material in step 2, tween 80 is replaced with TritonX-100.
embodiment 8
Key step is identical with embodiment 1, and distinctive points is, when preparing human blood coagulation factor VII I freeze-drying and dry heat treatment protective material in step 2, tween 80 is replaced with Neutronyx 600.
embodiment 9
Key step is identical with embodiment 1, tween 80 is replaced with Arlacel A(anhydromannitol monoleate when distinctive points is to prepare human blood coagulation factor VII I freeze-drying and dry heat treatment protective material in step 2).
comparative example 1
Key step is identical with embodiment 1, and distinctive points is human blood coagulation factor VII I freeze-drying obtained in step 2 and dry heat treatment protective material not containing tween 80 composition.
comparative example 2
Key step is identical with embodiment 1, and distinctive points is that the content of tween 80 in human blood coagulation factor VII I freeze-drying obtained in step 2 and dry heat treatment protective material is 0.5 mmol/L.
comparative example 3
Key step is identical with embodiment 1, and distinctive points is in human blood coagulation factor VII I freeze-drying obtained in step 2 and dry heat treatment protective material not containing glycine.
comparative example 4
Key step is identical with embodiment 1, and distinctive points is in human blood coagulation factor VII I freeze-drying obtained in step 2 and dry heat treatment protective material not containing arginine.
comparative example 5
Key step is identical with embodiment 1, and distinctive points is in human blood coagulation factor VII I freeze-drying obtained in step 2 and dry heat treatment protective material not containing Histidine.
comparative example 6
Key step is identical with embodiment 1, and distinctive points is in human blood coagulation factor VII I freeze-drying obtained in step 2 and dry heat treatment protective material not containing Methionin.
comparative example 7
Key step is identical with embodiment 1, when distinctive points is that step 2 prepares human blood coagulation factor VII I freeze-drying and dry heat treatment protective material, tween 80 is replaced with hexadecylpyridinium chloride.
comparative example 8
Key step is identical with embodiment 1, and distinctive points is, in human blood coagulation factor VII I freeze-drying obtained in step 2 and dry heat treatment protective material, tween 80 is replaced with sodium lauryl sulphate.
correlation detection
(1) above-described embodiment and comparative example are obtained human blood coagulation factor VII I finished product to test with reference to the detection method of Chinese Pharmacopoeia quality standard, adopt following index to carry out product quality and analyze:
1, product xeothermic after tire.
2, the xeothermic rear specific activity of product (pharmacopoeial quality standard is >=1.0 IU/mg protein).
3, (it is qualified for meeting States Pharmacopoeia specifications, and it is defective for not meeting States Pharmacopoeia specifications product appearance, visible foreign matters and redissolution time.States Pharmacopoeia specifications: outward appearance should be oyster white loosening body, after redissolving, solution should be achromaticity and clarification liquid, can be with slight opalescence; Visible foreign matters, except having allowed micro-tiny protein grain, all the other should conform with the regulations; The redissolution time answers≤30 min).
Detected result is as shown in the table:
| ? | Tire after xeothermic (%) | Xeothermic rear specific activity (IU/mg protein) | Outward appearance | Visible foreign matters | The redissolution time (min) |
| Embodiment 1 | 86.6 | 44.41 | Qualified | Qualified | ≤5 |
| Embodiment 2 | 85.9 | 42.12 | Qualified | Qualified | ≤5 |
| Embodiment 3 | 86.2 | 42.19 | Qualified | Qualified | ≤5 |
| Embodiment 4 | 84.5 | 40.03 | Qualified | Qualified | ≤5 |
| Embodiment 5 | 84.1 | 40.01 | Qualified | Qualified | ≤5 |
| Embodiment 6 | 82.7 | 38.35 | Qualified | Qualified | ≤5 |
| Embodiment 7 | 81.8 | 37.69 | Qualified | Qualified | ≤5 |
| Embodiment 8 | 82.2 | 38.81 | Qualified | Qualified | ≤5 |
| Embodiment 9 | 81.6 | 37.23 | Qualified | Qualified | ≤5 |
| Comparative example 1 | 65.2 | 26.05 | Poor | More protein grain | ≥30 |
| Comparative example 2 | 72.8 | 29.40 | Qualified | Qualified | ≤5 |
| Comparative example 3 | 73.6 | 29.81 | Qualified | Qualified | ≤5 |
| Comparative example 4 | 74.4 | 30.97 | Qualified | Qualified | ≤5 |
| Comparative example 5 | 75.2 | 30.68 | Qualified | Qualified | ≤5 |
| Comparative example 6 | 75.1 | 31.04 | Qualified | Qualified | ≤5 |
| Comparative example 7 | 67.2 | 27.13 | Poor | More protein grain | ≥30 |
| Comparative example 8 | 67.4 | 27.21 | Poor | More protein grain | ≥30 |
As can be seen from upper table result, every detected result of embodiment 1 ~ 9 all meets pharmacopoeial quality standard, and comparative example 1 ~ 8, after xeothermic tire and specific activity significantly declines, do not use the outward appearance of the comparative example 1,7 and 8 of nonionic surface active agent, visible foreign matters and redissolution time not to reach pharmacopoeial quality standard simultaneously yet, explanation must use nonionic surface active agent, glycine, arginine, Histidine and Methionin simultaneously, and consumption also needs to control could realize beneficial effect of the present invention at specified range.
(2) the detailed examining report result of embodiment 1 human blood coagulation factor VII I finished product is as follows, and this batch of goods are up to the standards:
| interventions Requested | quality standard | assay |
| telling test | only produce precipitation line with AHS or blood plasma, do not produce precipitation line with anti-horse, anti-ox, anti-pig, anti-sheep blood serum or blood plasma. | qualified |
| outward appearance | should be oyster white loosening body, after redissolution, should be colourless clear liquid, slight opalescence can be with. | white powder, redissolves and clarifies, micro-band opalescence |
| vacuum tightness | bluish voilet aura should be there is | light blue |
| the redissolution time (minute) | ≤ 30 | 5 |
| visible foreign matters | allow micro-tiny protein grain | accidental protein grain |
| content uniformity | should conform with the regulations | conform with the regulations |
| moisture (%) | ≤ 3.0 | 1.28 |
| pH value | 6.5 ~ 7.5 | 6.95 |
| sodium ion (mmol/L) | ≤ 160 | 142 |
| citric Acid ion content (mmol/L) | ≤ 25 | 12 |
| tire (%) | ? | 86.6 |
| specific activity (IU/mg protein) | >=1.0 | 44.41 |
| anti-A hemagglutinin | ≤ 1:64 | qualified |
| anti-B hemagglutinin | ≤ 1:64 | qualified |
| hBsAg | negative | negative |
| anti-HIV | negative | negative |
| whose anti-HCV | negative | negative |
| sterility test | asepsis growth | qualified |
| abnormal toxicity test (cavy test, mouse test) | cavy should be good for and be deposited, and body weight increases; Mouse should be good for and be deposited, and body weight increases | qualified |
| pyrogen test | should conform with the regulations | do not examine |
| tributyl phosphate content (μ g/ml) | ≤ 10 | 6.85 |
| polysorbate 80 content (μ g/ml) | ≤ 100 | 20.04 |
| glycine Levels (mg/ bottle) | ≤ 130 | 92.6 |
| calcium ion content (mg/ bottle) | ≤ 9 | 1.30 |
(3) dry heating method deactivation is to the inactivation of virus effect of embodiment 1 ~ 9 human blood coagulation factor VII I finished product
The human blood coagulation factor VII I finished product of Example 1 ~ 9 carries out inactivation of virus effect detection, and result is as follows:
| Indicator virus | PRV(Pseudorabies virus) | Sindbis(sindbis alphavirus) |
| Embodiment 1 inactivating efficacy | >4.5 LogTCID 50/0.1ml | >4.5 LogPFU/ml |
| Embodiment 2 inactivating efficacy | >4.5 LogTCID 50/0.1ml | >4.5 LogPFU/ml |
| Embodiment 3 inactivating efficacy | >4.5 LogTCID 50/0.1ml | >4.5 LogPFU/ml |
| Embodiment 4 inactivating efficacy | >4.5 LogTCID 50/0.1ml | >4.5 LogPFU/ml |
| Embodiment 5 inactivating efficacy | >4.5 LogTCID 50/0.1ml | >4.5 LogPFU/ml |
| Embodiment 6 inactivating efficacy | >4.5 LogTCID 50/0.1ml | >4.5 LogPFU/ml |
| Embodiment 7 inactivating efficacy | >4.5 LogTCID 50/0.1ml | >4.5 LogPFU/ml |
| Embodiment 8 inactivating efficacy | >4.5 LogTCID 50/0.1ml | >4.5 LogPFU/ml |
| Embodiment 9 inactivating efficacy | >4.5 LogTCID 50/0.1ml | >4.5 LogPFU/ml |
As can be seen from upper table result, the inactivation of virus effect of the human blood coagulation factor VII I finished product of the embodiment of the present invention 1 ~ 9 all can reach state specified standards (States Pharmacopoeia specifications value is for being greater than 4.0 Log).The freeze-drying of the present inventor's blood coagulation factor VIII and dry heat treatment protective material also can not affect the deactivation of its dry heat treatment to virus while protection quality of item.
Claims (10)
1. a preparation method of human blood coagulation factor VII I, is characterized in that, comprises the steps:
S1. blood plasma cryoprecipitate and cryoprecipitate dissolving is prepared;
S2. aluminium glue absorption;
S3. S/D inactivation of virus;
S4. gel filtration chromatography purifying and ultrafiltration and concentration;
S5. human blood coagulation factor VII I freeze-drying and dry heat treatment protective material is configured;
S6. freeze-drying and dry heat treatment after human blood coagulation factor VII I freeze-drying and dry heat treatment protective material is added, described human blood coagulation factor VII I freeze-drying and the protectant add-on of dry heat treatment are 0.8 ~ 1.5 times of liquor capacity after ultrafiltration and concentration, make tiring of blood coagulation factor VIII in mixed solution be 18 ~ 22 IU/ml;
Described human blood coagulation factor VII I freeze-drying and dry heat treatment protective material comprise Trisodium Citrate 1 ~ 25 mmol/L, sodium-chlor 10 ~ 150 mmol/L, calcium chloride 2 ~ 9 mmol/L, glycine 10 ~ 150 mmol/L, arginine 10 ~ 150 mmol/L, Histidine 10 ~ 150 mmol/L, Methionin 10 ~ 150 mmol/L, tensio-active agent 0.01 ~ 0.25 mmol/L; Described tensio-active agent is nonionic surface active agent.
2. the preparation method of human blood coagulation factor VII I according to claim 1; it is characterized in that; described human blood coagulation factor VII I freeze-drying and dry heat treatment protective material are made up of Trisodium Citrate 10 ~ 15 mmol/L, sodium-chlor 50 ~ 100 mmol/L, calcium chloride 4 ~ 7 mmol/L, glycine 50 ~ 100 mmol/L, arginine 50 ~ 100 mmol/L, Histidine 50 ~ 100 mmol/L, Methionin 50 ~ 100 mmol/L, tensio-active agent 0.1 ~ 0.2 mmol/L, and described tensio-active agent is nonionic surface active agent.
3. the preparation method of human blood coagulation factor VII I according to claim 2; it is characterized in that; described human blood coagulation factor VII I freeze-drying and dry heat treatment protective material are made up of Trisodium Citrate 13 mmol/L, sodium-chlor 80 mmol/L, calcium chloride 6 mmol/L, glycine 80 mmol/L, arginine 80 mmol/L, Histidine 80 mmol/L, Methionin 80 mmol/L, tensio-active agent 0.15 mmol/L, and described tensio-active agent is nonionic surface active agent.
4. the preparation method of human blood coagulation factor VII I according to any one of claim 1 ~ 3, it is characterized in that, described nonionic surface active agent is Tween class, Span class, polyoxyethylene octylphenol ether class, polyoxyethylene nonylphenol ether class, MM class or Pluronic class tensio-active agent.
5. the preparation method of human blood coagulation factor VII I according to any one of claim 1 ~ 3, it is characterized in that, described nonionic surface active agent is tween 80, polysorbate60, polysorbate40, sorbester p17, sorbester p18, span 20, TritonX-100, Neutronyx 600 or Arlacel A.
6. the preparation method of human blood coagulation factor VII I according to claim 4, it is characterized in that, described nonionic surface active agent is Tween class tensio-active agent.
7. the preparation method of human blood coagulation factor VII I according to claim 6, it is characterized in that, described Tween class tensio-active agent is tween 80.
8. the preparation method of human blood coagulation factor VII I according to any one of claim 1 ~ 3; it is characterized in that; the concrete steps of step S6 are: human blood coagulation factor VII I freeze-drying and dry heat treatment protective material are mixed according to volume ratio 1:1 with the solution after ultrafiltration and concentration; tiring of blood coagulation factor VIII in mixing solutions is made to be 20 IU/ml; again mixing solutions is dispensed in albumen bottle; lyophilize rear seal-cover, then through 80 DEG C of dry heat treatment inactivation of viruses, obtained human blood coagulation factor VII I finished product.
9. the preparation method of human blood coagulation factor VII I according to any one of claim 1 ~ 3, it is characterized in that, the concrete steps of step S1 ~ S4 are:
S1. blood plasma cryoprecipitate and cryoprecipitate dissolving is prepared: with Fresh Frozen human plasma for raw material, through blood plasma thawing, centrifugal preparation cryoprecipitate, by the physiological saline solution containing 8 IU/ml heparin of cryoprecipitate with 15 ~ 20 DEG C of precoolings, the consumption of physiological saline is 7 ~ 9 times of Sediment weight, leaves standstill 60min after stirring and dissolving;
S2. aluminium glue absorption: the lysate after leaving standstill is warming up to 25 ~ 28 DEG C, add the aluminum hydroxide gel of synthermal mass concentration 3%, add-on is 40% of cryoprecipitate weight, adopt and stir the lower mode infiltrated and add, namely under 80 ~ 100 rpm stirring velocitys, below the liquid level with emulsion tube aluminum hydroxide gel being pumped into lysate, interpolation speed is 80 ~ 100 ml/min, be cooled to 15 ~ 20 DEG C after interpolation, the centrifugal removal of impurities of 12000 rpm, gets supernatant liquor;
S3. S/D inactivation of virus: supernatant liquor is warming up to 35 DEG C, add S/D virus inactivating agent insulation 3h and complete first time inactivation of virus, obtain inactivation of virus liquid, wherein S/D virus inactivating agent contains 1% polysorbate, 0.3% tributyl phosphate;
S4. gel filtration chromatography purifying and ultrafiltration and concentration: gel column washing lotion 1 is balanced, inactivation of virus liquid loading gel filtration chromatography, under 280nm length ultraviolet light detection, post 4 times of column volumes are washed by washing lotion 2, with washing lotion 3 wash-out blood coagulation factor VIII, and collect, then carry out ultrafiltration dealcoholysis and desalination, obtain high density and concentrate blood coagulation factor VIII solution, tiring of blood coagulation factor VIII controls at 40 IU/ml; Wherein gel model is DEAE-SAPHROSE FF, and loading and elution flow rate are 220 ~ 250ml/min, and washing lotion 1 formula is 110 mM sodium-chlor, 10 mM Sodium Citrates, 1 mM calcium chloride, 16 mM Methionins, 120 mM glycine, pH7.0; The formula of washing lotion 2 is 150 mM sodium-chlor, 10 mM Sodium Citrates, 1 mM calcium chloride, 16 mM Methionins, 120 mM glycine, pH7.0; The formula of washing lotion 3 is 250 mM sodium-chlor, 10 mM Sodium Citrates, 1 mM calcium chloride, 16 mM Methionins, 120 mM glycine, pH7.0.
10. the human blood coagulation factor VII I product that obtains of the preparation method of a human blood coagulation factor VII I according to any one of claim 1 ~ 9.
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Cited By (9)
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| CN105294858A (en) * | 2015-12-05 | 2016-02-03 | 上海洲跃生物科技有限公司 | Method for preparing freeze-dried human blood coagulation factor VIII |
| CN105348382A (en) * | 2015-12-05 | 2016-02-24 | 上海洲跃生物科技有限公司 | Method for preparing high-purity human coagulation factor VIII |
| CN106139127A (en) * | 2016-08-05 | 2016-11-23 | 无锡药明康德生物技术股份有限公司 | Factor Ⅷ,rDNA lyophilized formulations |
| CN107880112A (en) * | 2017-12-28 | 2018-04-06 | 华兰生物工程股份有限公司 | A kind of human blood coagulation factor VII I preparation method and human blood coagulation factor VII I products |
| WO2018102960A1 (en) * | 2016-12-05 | 2018-06-14 | Chung Chin Sun | Novel method for cryoprecipitate activity preservation |
| WO2018112780A1 (en) * | 2016-12-21 | 2018-06-28 | Chung Chin Sun | Novel method for blood plasma protein activity preservation |
| WO2021134180A1 (en) * | 2019-12-30 | 2021-07-08 | 四川远大蜀阳药业有限责任公司 | Method for cryopreserving blood coagulation factor viii intermediate product |
| WO2023020914A1 (en) | 2021-08-18 | 2023-02-23 | Biotest Ag | Dry heat treatment of plasma-derived factor viii |
| CN116410258A (en) * | 2023-04-13 | 2023-07-11 | 上海太阳生物技术有限公司 | Factor XI deficiency plasma protective agent and application thereof |
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| CN105294858A (en) * | 2015-12-05 | 2016-02-03 | 上海洲跃生物科技有限公司 | Method for preparing freeze-dried human blood coagulation factor VIII |
| CN105348382A (en) * | 2015-12-05 | 2016-02-24 | 上海洲跃生物科技有限公司 | Method for preparing high-purity human coagulation factor VIII |
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| CN106139127B (en) * | 2016-08-05 | 2020-04-07 | 无锡药明生物技术股份有限公司 | Recombinant blood coagulation factor VIII freeze-dried preparation |
| WO2018102960A1 (en) * | 2016-12-05 | 2018-06-14 | Chung Chin Sun | Novel method for cryoprecipitate activity preservation |
| WO2018112780A1 (en) * | 2016-12-21 | 2018-06-28 | Chung Chin Sun | Novel method for blood plasma protein activity preservation |
| US11872315B2 (en) | 2016-12-21 | 2024-01-16 | Sun, Chung Chin | Method for blood plasma protein activity preservation |
| CN107880112A (en) * | 2017-12-28 | 2018-04-06 | 华兰生物工程股份有限公司 | A kind of human blood coagulation factor VII I preparation method and human blood coagulation factor VII I products |
| WO2021134180A1 (en) * | 2019-12-30 | 2021-07-08 | 四川远大蜀阳药业有限责任公司 | Method for cryopreserving blood coagulation factor viii intermediate product |
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| CN116410258A (en) * | 2023-04-13 | 2023-07-11 | 上海太阳生物技术有限公司 | Factor XI deficiency plasma protective agent and application thereof |
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