CN104231073B - Preparation method of human coagulation factor VIII - Google Patents
Preparation method of human coagulation factor VIII Download PDFInfo
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- CN104231073B CN104231073B CN201410496187.9A CN201410496187A CN104231073B CN 104231073 B CN104231073 B CN 104231073B CN 201410496187 A CN201410496187 A CN 201410496187A CN 104231073 B CN104231073 B CN 104231073B
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
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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 pharmaceutical technology field is and in particular to a kind of preparation method of human blood coagulation viii.
Background technology
Hemophilia is a kind of hereditary hemorrhagic disease, is because coagulation factor gene mutation leads to the human body intravascular coagulation factor
Level reduces or lacks, thus leading to bleeding.The shortage of thrombin f or deficiency lead to hemophilia a, and thrombin ix lacks
Weary or deficiency leads to hemophilia b.Thrombin replacement therapy is haemophiliachemophiliac primary treatment measure.
With the development of modern chromatographic technique, external blood products enterprise has developed high-purity human blood coagulation product,
Meet the demand of hemophiliac.China considers, national Yao Jian department is not yet approved by blood sources from Viral safety
Thrombin import.Domestic blood products enterprise is limited to technical capability, how based on production human albumin and immunoglobulin,
The market shares such as blood clotting factories are considerably less, wherein thrombin (preventing and treating hemophilia) extremely shortage.Therefore research and develop solidifying
Blood factor class blood products new product, development new technologies research, will be the trends of the times in blood products field.
Due to coagulation factors viii easy in inactivation, lyophilization or dry heat treatment process are easily destroyed the egg of coagulation factors viii
White stability, causes deformation, leads to coagulation factors viii to inactivate, and in the preparation technology flow process of human blood coagulation viii product
The method that inactivation of virus or removal must be comprised, such as pasteurization, the inactivation of s/d method, the inactivation of 20nm nano-film filtration method etc., because
This needs to add certain protective agent or stabilizer before human blood coagulation viii carries out lyophilization or dry heat treatment.
At present, the protective agent that conventional human blood coagulation viii adds in preparation process mainly utilizes albumin or sugar
Alcohol, collaborative aminoacid, inorganic salt etc. play a role, and such as Chinese invention patent 201110240278.2 and 201210468069.8 is public
Introduce human albumin in the protective agent opened, but be introduced into human albumin's not only high cost, and the making of human albumin
With being likely that there are the potential risk propagating other viruses or pathogen；Chinese invention patent 201210060299.0 is using sugar
Alcohol and combination of amino acids are as protective agent, but containing sugar or sugar alcohol in the human blood coagulation viii product being obtained, are not suitable for glycosuria
Patient uses, and limits the range of application of this product, simultaneously after this thermally treated inactivation of viruses of patent product, thrombin
The potency of viii declines serious, blood coagulation activity yield only 33% about after heat treatment, and actual application value is little.
Therefore, in this area, urgently a kind of effective preparation method can make coagulation factors viii keep after inactivation of viruses
Do not introduce other materials such as albumin or sugar alcohol while potency does not decline affects range of application and the preparation cost of product again.
Content of the invention
It is an object of the invention to overcoming above-mentioned the deficiencies in the prior art, provide a kind of preparation side of human blood coagulation viii
Method, does not use human albumin or other animal derived protein, does not use sugar or sugar alcohol yet, prepared product keep higher potency and
Specific activity.
The above-mentioned purpose of the present invention is achieved by following technical solution:
A kind of preparation method of human blood coagulation viii, comprises the steps:
S1. blood plasma cryoprecipitate and cryoprecipitate dissolving are prepared；
S2. aluminium glue absorption；
S3. s/d inactivation of virus；
S4. gel filtration chromatography purification and ultrafiltration concentration；
S5. configuration human blood coagulation viii lyophilizing and dry heat treatment protective agent；
S6. lyophilizing and dry heat treatment after human blood coagulation viii lyophilizing and dry heat treatment protective agent, described people's blood coagulation are added
The lyophilizing of factor viii and the protectant addition of dry heat treatment be concentrated by ultrafiltration after 0.8 ~ 1.5 times of liquor capacity so that mixing
In liquid, the potency of coagulation factors viii is 18 ~ 22 iu/ml；
Described human blood coagulation viii lyophilizing and dry heat treatment protective agent include sodium citrate 1 ~ 25 mmol/l, sodium chloride
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, lysine 10 ~ 150 mmol/l, surfactant 0.01 ~ 0.25 mmol/l；Described surfactant is
Nonionic surfactant.
As a kind of preferred version, described human blood coagulation viii lyophilizing and dry heat treatment protective agent by sodium citrate 10 ~
15 mmol/l, sodium chloride 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, lysine 50 ~ 100 mmol/l, surfactant 0.1 ~ 0.2 mmol/l group
Become, described surfactant is nonionic surfactant.
As a kind of further preferred scheme, described human blood coagulation viii lyophilizing and dry heat treatment protective agent are by citric acid
Sodium 13 mmol/l, sodium chloride 80 mmol/l, calcium chloride 6 mmol/l, glycine 80 mmol/l, arginine 80 mmol/l, group
Propylhomoserin 80 mmol/l, lysine 80 mmol/l, surfactant 0.15 mmol/l composition, described surfactant be non-from
Subtype surfactant.
The present invention uses nonionic surfactant, not only can work in coordination with sodium citrate, sodium chloride, calcium chloride, sweet
Propylhomoserin, arginine, histidine, lysine play protective effect, improve the stability of human blood coagulation viii, can also reduce people
Coagulation factors viii freeze-dried products redissolve the dielectric constant in water, it is to avoid protein component leads to flocculation sediment because electrically charged,
Ensure that the quality of last human blood coagulation viii product.In addition it is experimentally confirmed, conventional cationic surfactant is (such as
Hexadecylpyridinium chloride, hexadecyltrimethylammonium chloride etc.) and anion surfactant (as sodium stearate, dodecane
Base sodium sulfate, sulfosuccinate etc.) nonionic surfactant is substantially not so good as to the protective effect of human blood coagulation viii
Superior, cationic surface active agent toxicity is larger simultaneously, and anionic surfactant hemolytic activity is stronger, can reduce people and coagulate
The quality of blood factor viii product.
Preferably, described nonionic surfactant is tween class (Polysorbate, tween), span class (Sorbitan
Alcohol fatty acid ester, span), OPEO class, NPE class, MM class or
Pluronic class (poloxamer class) surfactant.
Specifically, described nonionic surfactant is Tween 80, polysorbate60, polysorbate40, sorbester p17, sorbester p18, department
Disk 20, tritonx-100, neutronyx 600, arlacel a etc..
It is highly preferred that described nonionic surfactant is tween class surfactant, such as Tween 80, polysorbate60, tell
Temperature 40 etc..
Most preferably, described surfactant is Tween 80, and prepared human blood coagulation viii lyophilizing and dry heat treatment are protected
Shield agent effect is optimum.
Preferably, the concretely comprising the following steps of step s6: by human blood coagulation viii lyophilizing and dry heat treatment protective agent and ultrafiltration
Solution after concentration mixes according to volume ratio 1:1 so that the potency of coagulation factors viii is 20 iu/ml in mixed solution, then will
Mixed solution is dispensed in albumen bottle, lyophilization rear seal-cover, then through 80 DEG C of dry heat treatment inactivation of viruses, prepared people's blood coagulation because
Sub- viii finished product.
In the preparation method of human blood coagulation viii of the present invention, step s1 ~ s4 is referred to this area conventional steps,
As a kind of specific embodiments, step s1 ~ s4 concretely comprises the following steps:
S1. blood plasma cryoprecipitate and cryoprecipitate dissolving are prepared: with Fresh Frozen human plasma as raw material, through blood plasma thawing, be centrifuged
Preparation cryoprecipitate, by the cryoprecipitate physiological saline solution containing 8 iu/ml heparin for 15 ~ 20 DEG C of pre-coolings, the use of normal saline
Measure 7 ~ 9 times for Sediment weight, after stirring and dissolving, stand 60min；
S2. aluminium glue absorption: the lysate after standing is warming up to 25 ~ 28 DEG C, adds the hydrogen-oxygen of synthermal mass concentration 3%
Change alumina gel, addition is the 40% of cryoprecipitate weight, added by the way of the lower infiltration of stirring, stir in 80 ~ 100 rpm
Under speed, with latex tubing, gel aluminum hydroxide is pumped into below the liquid level of lysate, adding speed is 80 ~ 100 ml/min, adds
Add and be cooled to 15 ~ 20 DEG C after finishing, 12000 rpm centrifugation remove impurity, take supernatant；
S3. s/d inactivation of virus: supernatant is warming up to 35 DEG C, adds s/d virus inactivating agent insulation 3h to complete disease for the first time
Poison inactivation, obtains inactivation of virus liquid, wherein s/d virus inactivating agent contains 1% polysorbate (tween-80), 0.3% tricresyl phosphate fourth
Ester (tnbp)；
S4. gel filtration chromatography purification and ultrafiltration concentration: gel column washing liquid 1 is balanced, inactivation of virus liquid loading gel column
Chromatography, under 280nm length ultraviolet light detection, washes 4 times of column volumes of post with washing liquid 2, with washing liquid 3 eluting coagulation factors viii, and
Collect, then carry out ultrafiltration dealcoholysis and desalination, obtain high concentration and concentrate coagulation factors viii solution, the potency of coagulation factors viii
Control in 40 iu/ml；Wherein gel model deae-saphrose ff, loading and elution flow rate are 220 ~ 250 ml/min,
Washing liquid 1 formula is 110 mm sodium chloride, 10 mm sodium citrate, 1 mm calcium chloride, 16 mm lysines, 120 mm glycine,
ph7.0；The formula of washing liquid 2 is 150 mm sodium chloride, 10 mm sodium citrate, 1 mm calcium chloride, 16 mm lysines, and 120 mm are sweet
Propylhomoserin, ph7.0；The formula of washing liquid 3 is 250 mm sodium chloride, 10 mm sodium citrate, 1 mm calcium chloride, 16 mm lysines, 120
Mm glycine, ph7.0.
The present invention is used heparin to replace conventional use of tris-hcl solution as lysate, can reduce to greatest extent
The loss of c active component in human blood coagulation viii, keeps the biological activity of higher human blood coagulation viii.
The present invention adopts gel aluminum hydroxide absorption method to replace the conventional peg sedimentation method, can effectively remove most of miscellaneous
Albumen, improves the response rate of product further and reduces the protein content in product.
In cryoprecipitate dissolution phase using the pre-cooling physiological saline solution containing heparin, traditional handicraft contains liver with room temperature to the present invention
The dissolving of plain water for injection, to keeping protein active, prevent bacteria breed favourable, protein has in normal saline pre-cooling dissolving water
Stablize thus keeping the activity of thrombin beneficial to holding structure, the time of repose in this technique manufacturing process can make soluble protein
Be sufficiently separated with insoluble protein, be remarkably improved the purity of coagulation factors viii, obtain people's blood coagulation of low albumen high specific acitivity because
Sub- product.
The present invention using cryoprecipitate detached in blood plasma as raw material, after preparing high activity coagulation factors viii, also may be used by clout
For separating preparation human fibrinogen, the natural structure of Fibrinogen can be kept, can remove in blood plasma by a larger margin again
Other thrombins (predominantly,, etc.), improve the purity of product；Effectively remove s/d viral inaction steps simultaneously
The residue of the s/d solvent of middle interpolation, increases the safety of product.(" Chinese Pharmacopoeia " human fibrin primary standard: purity >=
70.0%, this technique develops 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 of dry heat treatment 0.5h.
Lyophilizing of the present invention is the conventional freeze-drying process in this area.
Step s5 of the present invention configures the amino acid classes selected when human blood coagulation viii lyophilizing and dry heat treatment protective agent
With consumption proportion and unconventional selection is it is necessary to simultaneously using the glycine of above-mentioned consumption proportion, arginine, histidine and lysine
Protective agent just enable the work that human blood coagulation viii product keeps high-titer and high specific acitivity through dry heat treatment inactivation of viruses
With.
The human blood coagulation viii product that a kind of preparation method according to human blood coagulation viii of the present invention is obtained.
Compared with prior art, the invention has the following beneficial effects:
(1) according to preparation method of the present invention be obtained human blood coagulation viii product in do not contain human albumin or
Other animal derived protein, do not contain sugar or sugar alcohol yet, be made up of soluble-salt, aminoacid and surfactant, there is not propagation
Other viruses or the risk of pathogen, suitable crowd's scope is wide, can also use including diabeticss.
(2) according in the prepared human blood coagulation viii product of preparation method of the present invention, coagulation factors viii is still
Keep high-titer and high specific acitivity, potency is more than 80%, specific activity and is more than 40 iu/mg.
(3) redissolve soon according to the human blood coagulation viii product that preparation method of the present invention is obtained, during far below redissolving
Between 30min quality standard, and it is good to redissolve effect, redissolves liquid clarification micro-strip opalescence.
(4) human albumin, preparation are not contained according to the human blood coagulation viii product that preparation method of the present invention is obtained
Method is simple, with low cost, and product safety effectively, has good prospects for commercial application.
Specific embodiment
With reference to specific embodiment, the present invention is further explained, but specific embodiment is not to the present invention
It is limited in any way.Unless stated otherwise, involved reagent in embodiment, method are reagent commonly used in the art and method.
Embodiment 1
First, prepare human blood coagulation viii solution
(1) cryoprecipitate and cryoprecipitate dissolving are made
With Fresh Frozen human plasma as raw material, through blood plasma thawing, cryoprecipitate is prepared in centrifugation, and cryoprecipitate is pre- with 15 ~ 20 DEG C
The cold physiological saline solution containing 8 iu/ml heparin, the consumption of normal saline is 7 ~ 9 times of Sediment weight, after stirring and dissolving
Stand 60 min.
(2) aluminium glue absorption
Lysate after standing is warming up to 25 ~ 28 DEG C, adds the gel aluminum hydroxide of synthermal mass concentration 3%, add
Measure 40% for cryoprecipitate weight, added by the way of the lower infiltration of stirring, that is, under 80 ~ 100 rpm mixing speeds, use latex
Gel aluminum hydroxide is pumped into below the liquid level of lysate by pipe, and adding speed is 80 ~ 100 ml/min, adds and is cooled to after finishing
15 ~ 20 DEG C, 12000 rpm centrifugation remove impurity, take supernatant.
(3) s/d inactivation of virus
Supernatant is warming up to 35 DEG C, adds s/d virus inactivating agent to be incubated 3 h and completes first time inactivation of virus, obtains virus
Inactivation liquid, wherein s/d virus inactivating agent contains 1% polysorbate (tween-80), 0.3% tributyl phosphate (tnbp).
(4) gel filtration chromatography purification
Gel column washing liquid 1 is balanced, inactivation of virus liquid loading gel filtration chromatography, in 280 nm length ultraviolet light detections
Under, wash 4 times of column volumes of post with washing liquid 2, with washing liquid 3 eluting coagulation factors viii, and collect.Wherein gel model deae-
Saphrose ff, loading and elution flow rate are 220 ~ 250 ml/min, and the formula of washing liquid 1 is 110 mm sodium chloride, 10 mm Chinese hollys
Rafter acid sodium, 1 mm calcium chloride, 16 mm lysines, 120 mm glycine, ph7.0；The formula of washing liquid 2 is 150 mm sodium chloride, 10
Mm sodium citrate, 1 mm calcium chloride, 16 mm lysines, 120 mm glycine, ph7.0；The formula of washing liquid 3 is 250 mm chlorinations
Sodium, 10 mm sodium citrate, 1 mm calcium chloride, 16 mm lysines, 120 mm glycine, ph7.0.
(5) it is concentrated by ultrafiltration
The coagulation factors viii of collection is carried out ultrafiltration dealcoholysis and desalination, obtains high concentration concentration coagulation factors viii molten
Liquid, the potency of coagulation factors viii controls in 40 iu/ml.
2nd, human blood coagulation viii lyophilizing and dry heat treatment protective agent are prepared
Weigh all kinds of inorganic salts, aminoacid and surfactant in proportion, configuration people coagulates using common medical used injection water
Blood factor viii lyophilizing and dry heat treatment protective agent, all kinds of in prepared human blood coagulation viii lyophilizing and dry heat treatment protective agent
Concentration originally is as follows: sodium citrate 13 mmol/l, sodium chloride 80 mmol/l, calcium chloride 6 mmol/l, glycine 80
Mmol/l, arginine 80 mmol/l, histidine 80 mmol/l, lysine 80 mmol/l, Tween 80 0.15 mmol/l.
3rd, human blood coagulation viii lyophilizing and the protectant use of dry heat treatment
The prepared human blood coagulation viii lyophilizing of coagulation factors viii solution and step 2 that step one is obtained and xeothermic
Process protective agent to mix so that the potency of coagulation factors viii controls in 20 iu/ml according to volume ratio 1:1, then by mixed solution
It is dispensed in albumen bottle (10 ml/ bottle), lyophilization rear seal-cover, then through 80 DEG C of dry heat treatment inactivation of viruses, prepared people's blood coagulation
Factor viii finished product.
Embodiment 2
First, prepare human blood coagulation viii solution
Step one with reference to embodiment 1.
2nd, human blood coagulation viii lyophilizing and dry heat treatment protective agent are prepared
Weigh all kinds of inorganic salts, aminoacid and surfactant in proportion, configuration people coagulates using common medical used injection water
Blood factor viii lyophilizing and dry heat treatment protective agent, all kinds of in prepared human blood coagulation viii lyophilizing and dry heat treatment protective agent
Concentration originally is as follows: sodium citrate 10 mmol/l, sodium chloride 100 mmol/l, calcium chloride 4 mmol/l, glycine 50
Mmol/l, arginine 50 mmol/l, histidine 100 mmol/l, lysine 100 mmol/l, Tween 80 0.1 mmol/l.
3rd, human blood coagulation viii lyophilizing and the protectant use of dry heat treatment
Step 3 with reference to embodiment 1.
Embodiment 3
First, prepare human blood coagulation viii solution
Step one with reference to embodiment 1.
2nd, human blood coagulation viii lyophilizing and dry heat treatment protective agent are prepared
Weigh all kinds of inorganic salts, aminoacid and surfactant in proportion, configuration people coagulates using common medical used injection water
Blood factor viii lyophilizing and dry heat treatment protective agent, all kinds of in prepared human blood coagulation viii lyophilizing and dry heat treatment protective agent
Concentration originally is as follows: sodium citrate 15 mmol/l, sodium chloride 50 mmol/l, calcium chloride 7 mmol/l, glycine 100
Mmol/l, arginine 100 mmol/l, histidine 80 mmol/l, lysine 80 mmol/l, Tween 80 0.2 mmol/l.
3rd, human blood coagulation viii lyophilizing and the protectant use of dry heat treatment
Step 3 with reference to embodiment 1.
Embodiment 4
Key step is same as Example 1, and distinctive points are in step 2 to prepare human blood coagulation viii lyophilizing and xeothermic
When processing protective agent, Tween 80 is replaced with polysorbate60.
Embodiment 5
Key step is same as Example 1, and distinctive points are in step 2 to prepare human blood coagulation viii lyophilizing and xeothermic
When processing protective agent, Tween 80 is replaced with polysorbate40.
Embodiment 6
Key step is same as Example 1, and distinctive points are in step 2 to prepare human blood coagulation viii lyophilizing and xeothermic
When processing protective agent, Tween 80 is replaced with sorbester p17.
Embodiment 7
Key step is same as Example 1, and distinctive points are in step 2 to prepare human blood coagulation viii lyophilizing and xeothermic
When processing protective agent, Tween 80 is replaced with tritonx-100.
Embodiment 8
Key step is same as Example 1, and distinctive points are in step 2 to prepare human blood coagulation viii lyophilizing and xeothermic
When processing protective agent, Tween 80 is replaced with neutronyx 600.
Embodiment 9
Key step is same as Example 1, and distinctive points are in step 2 to prepare human blood coagulation viii lyophilizing and xeothermic
When processing protective agent, Tween 80 is replaced with arlacel a(anhydromannitol monoleate).
Comparative example 1
Key step is same as Example 1, and distinctive points are the human blood coagulation viii lyophilizing being obtained in step 2 and do
Heat treatment protective agent does not contain Tween 80 composition.
Comparative example 2
Key step is same as Example 1, and distinctive points are the human blood coagulation viii lyophilizing being obtained in step 2 and do
In heat treatment protective agent, the content of Tween 80 is 0.5 mmol/l.
Comparative example 3
Key step is same as Example 1, and distinctive points are the human blood coagulation viii lyophilizing being obtained in step 2 and do
Glycine is not contained in heat treatment protective agent.
Comparative example 4
Key step is same as Example 1, and distinctive points are the human blood coagulation viii lyophilizing being obtained in step 2 and do
Arginine is not contained in heat treatment protective agent.
Comparative example 5
Key step is same as Example 1, and distinctive points are the human blood coagulation viii lyophilizing being obtained in step 2 and do
Histidine is not contained in heat treatment protective agent.
Comparative example 6
Key step is same as Example 1, and distinctive points are the human blood coagulation viii lyophilizing being obtained in step 2 and do
Lysine is not contained in heat treatment protective agent.
Comparative example 7
Key step is same as Example 1, and distinctive points are that step 2 prepares human blood coagulation viii lyophilizing and xeothermic place
During reason protective agent, Tween 80 is replaced with hexadecylpyridinium chloride.
Comparative example 8
Key step is same as Example 1, and distinctive points are the human blood coagulation viii lyophilizing being obtained in step 2 and do
In heat treatment protective agent, Tween 80 is replaced with sodium lauryl sulphate.
Coherent detection
(1) above-described embodiment and comparative example are obtained human blood coagulation viii finished product with reference to Chinese Pharmacopoeia quality standard
Detection method is tested, and carries out product quality using following index and analyzes:
1st, the xeothermic rear potency of product.
2nd, the xeothermic rear specific activity (pharmacopoeial quality standard is >=1.0 iu/mg protein) of product.
3rd, product appearance, visible foreign matters and redissolve the time (meet States Pharmacopoeia specifications be qualified, do not meet States Pharmacopoeia specifications be do not conform to
Lattice.States Pharmacopoeia specifications: outward appearance should be milky loosening body, and after redissolution, solution should be colorless clear liquid, can carry slight opalescence；It can be seen that
Foreign body, in addition to having allowed micro tiny protein body, remaining should meet regulation；The redissolution time answers≤30 min).
Testing result is as shown in the table:
| Xeothermic rear potency (%) | 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 body | ≥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 body | ≥30 |
| Comparative example 8 | 67.4 | 27.21 | Poor | More protein body | ≥30 |
The every testing result that can be seen that embodiment 1 ~ 9 from upper table result all meets pharmacopoeial quality standard, and comparative example
1 ~ 8, xeothermic after potency and specific activity be remarkably decreased, simultaneously not using the comparative example 1,7 and 8 of nonionic surfactant
Outward appearance, visible foreign matters and redissolution time do not reach pharmacopoeial quality standard yet, and explanation must be simultaneously using non-ionic surfactant
Agent, glycine, arginine, histidine and lysine, and consumption is also required to control and just enables institute of the present invention in particular range
The beneficial effect stated.
(2) the detailed examining report result of embodiment 1 human blood coagulation viii finished product is as follows, and the inspection of this batch product is closed
Lattice:
| Inspection project | Quality standard | Assay |
| Discrimination test | Only produce precipitation line with AHS or blood plasma, with anti-horse, anti-cattle, Anti- pig, anti-sheep blood serum or blood plasma do not produce precipitation line. | Qualified |
| Outward appearance | Should be milky loosening body, after redissolution, should be colourless clear liquid, Slight opalescence can be carried. | White powder, redissolves clear Clearly, micro-strip opalescence |
| Vacuum | Bluish violet aura should occur | Light blue |
| The redissolution time (minute) | ≤30 | 5 |
| Visible foreign matters | Allow micro tiny protein body | Accidental protein body |
| Content uniformity | Regulation should be met | Meet regulation |
| Moisture (%) | ≤3.0 | 1.28 |
| Ph value | 6.5~7.5 | 6.95 |
| Sodium ion (mmol/l) | ≤160 | 142 |
| Citric acid ion concentration (mmol/l) | ≤25 | 12 |
| Potency (%) | 86.6 | |
| Specific activity (iu/mg albumen Matter) | ≥1.0 | 44.41 |
| Anti- a hemagglutinin | ≤ 1:64 | Qualified |
| Anti- b hemagglutinin | ≤ 1:64 | Qualified |
| hbsag | Negative | Negative |
| Anti- hiv | Negative | Negative |
| Anti- hcv | Negative | Negative |
| Sterility test | Asepsis growth | Qualified |
| Abnormal toxicity test (Cavia porcelluss Test, mouse test) | Cavia porcelluss should be good for and be deposited, body weight increase；Mice should be good for and be deposited, body weight increase | Qualified |
| Pyrogen test | Regulation should be met | Do not examine |
| Tributyl phosphate content (μ g/ml) | ≤10 | 6.85 |
| Polyoxyethylene sorbitan monoleate content (μ g/ml) | ≤100 | 20.04 |
| Glycine Levels (mg/ bottle) | ≤130 | 92.6 |
| Calcium ion content (mg/ bottle) | ≤9 | 1.30 |
(3) the inactivation of virus effect to embodiment 1 ~ 9 human blood coagulation viii finished product for the dry heating method inactivation
The human blood coagulation viii finished product of Example 1 ~ 9 carries out inactivation of virus effect detection, and result is as follows:
| Indicator virus | Prv(Pseudorabies viruses) | Sindbis(sindbis alphaviruses) |
| Embodiment 1 inactivating efficacy | >4.5 logtcid50/0.1ml | >4.5 logpfu/ml |
| Embodiment 2 inactivating efficacy | >4.5 logtcid50/0.1ml | >4.5 logpfu/ml |
| Embodiment 3 inactivating efficacy | >4.5 logtcid50/0.1ml | >4.5 logpfu/ml |
| Embodiment 4 inactivating efficacy | >4.5 logtcid50/0.1ml | >4.5 logpfu/ml |
| Embodiment 5 inactivating efficacy | >4.5 logtcid50/0.1ml | >4.5 logpfu/ml |
| Embodiment 6 inactivating efficacy | >4.5 logtcid50/0.1ml | >4.5 logpfu/ml |
| Embodiment 7 inactivating efficacy | >4.5 logtcid50/0.1ml | >4.5 logpfu/ml |
| Embodiment 8 inactivating efficacy | >4.5 logtcid50/0.1ml | >4.5 logpfu/ml |
| Embodiment 9 inactivating efficacy | >4.5 logtcid50/0.1ml | >4.5 logpfu/ml |
Can be seen that the inactivation of virus effect of the human blood coagulation viii finished product of the embodiment of the present invention 1 ~ 9 from upper table result
State specified standards (States Pharmacopoeia specifications value is more than 4.0 log) all can be reached.The present inventor's coagulation factors viii lyophilizing and dry
Heat treatment protective agent does not interfere with its dry heat treatment to viral deactivation while protecting quality of item yet.
Claims (9)
1. a kind of preparation method of human blood coagulation viii is it is characterised in that comprise the steps:
S1. blood plasma cryoprecipitate and cryoprecipitate dissolving are prepared；
S2. aluminium glue absorption；
S3. s/d inactivation of virus；
S4. gel filtration chromatography purification and ultrafiltration concentration；
S5. configuration human blood coagulation viii lyophilizing and dry heat treatment protective agent；
S6. lyophilizing and dry heat treatment after human blood coagulation viii lyophilizing and dry heat treatment protective agent, described human blood coagulation are added
Viii lyophilizing and the protectant addition of dry heat treatment be concentrated by ultrafiltration after 0.8 ~ 1.5 times of liquor capacity so that in mixed liquor
The potency of coagulation factors viii is 18 ~ 22 iu/ml；
Described human blood coagulation viii lyophilizing and dry heat treatment protective agent include sodium citrate 1 ~ 25 mmol/l, sodium chloride 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, lysine 10 ~ 150 mmol/l, surfactant 0.01 ~ 0.25 mmol/l；Described surfactant is non-
Ionic surfactant.
2. according to claim 1 the preparation method of human blood coagulation viii it is characterised in that described human blood coagulation viii
Lyophilizing and dry heat treatment protective agent are by sodium citrate 10 ~ 15 mmol/l, sodium chloride 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, lysine 50 ~
100 mmol/l, surfactant 0.1 ~ 0.2 mmol/l form, and described surfactant is nonionic surfactant.
3. according to claim 2 the preparation method of human blood coagulation viii it is characterised in that described human blood coagulation viii
Lyophilizing and dry heat treatment protective agent are by sodium citrate 13 mmol/l, sodium chloride 80 mmol/l, calcium chloride 6 mmol/l, glycine
80 mmol/l, arginine 80 mmol/l, histidine 80 mmol/l, lysine 80 mmol/l, surfactant 0.15
Mmol/l forms, and described surfactant is nonionic surfactant.
4. according to any one of claim 1 ~ 3 preparation method of human blood coagulation viii it is characterised in that described nonionic
Type surfactant is tween class, span class, OPEO class, NPE class, mannide
Monoleate class or pluronic class surfactant.
5. according to any one of claim 1 ~ 3 preparation method of human blood coagulation viii it is characterised in that described nonionic
Type surfactant is Tween 80, polysorbate60, polysorbate40, sorbester p17, sorbester p18, span 20, tritonx-100, neutronyx
600 or arlacel a.
6. according to claim 4 human blood coagulation viii preparation method it is characterised in that described non-ionic surface live
Property agent be tween class surfactant.
7. according to claim 6 human blood coagulation viii preparation method it is characterised in that described tween class surface live
Property agent be Tween 80.
8. according to any one of claim 1 ~ 3 preparation method of human blood coagulation viii it is characterised in that the tool of step s6
Body step is: by the solution after human blood coagulation viii lyophilizing and dry heat treatment protective agent and ultrafiltration concentration according to volume ratio 1:1
Mix so that the potency of coagulation factors viii is 20 iu/ml in mixed solution, then mixed solution is dispensed in albumen bottle, cold
The dry rear seal-cover of lyophilizing, then through 80 DEG C of dry heat treatment inactivation of viruses, prepared human blood coagulation viii finished product.
9. according to any one of claim 1 ~ 3 preparation method of human blood coagulation viii it is characterised in that step s1 ~ s4
Concretely comprise the following steps:
S1. blood plasma cryoprecipitate and cryoprecipitate dissolving are prepared: with Fresh Frozen human plasma as raw material, through blood plasma thawing, centrifugation preparation
Cryoprecipitate, by the cryoprecipitate physiological saline solution containing 8 iu/ml heparin for 15 ~ 20 DEG C of pre-coolings, the consumption of normal saline is
7 ~ 9 times of Sediment weight, stand 60min after stirring and dissolving；
S2. aluminium glue absorption: the lysate after standing is warming up to 25 ~ 28 DEG C, adds the aluminium hydroxide of synthermal mass concentration 3%
Gel, addition is the 40% of cryoprecipitate weight, is added, that is, in 80 ~ 100 rpm mixing speeds by the way of the lower infiltration of stirring
Under, with latex tubing, gel aluminum hydroxide is pumped into below the liquid level of lysate, adding speed is 80 ~ 100 ml/min, has added
It is cooled to 15 ~ 20 DEG C after finishing, 12000 rpm centrifugation remove impurity, take supernatant；
S3. s/d inactivation of virus: supernatant is warming up to 35 DEG C, adds s/d virus inactivating agent insulation 3h to complete first time virus and goes out
Live, obtain inactivation of virus liquid, wherein s/d virus inactivating agent contains 1% polysorbate, 0.3% tributyl phosphate；
S4. gel filtration chromatography purification and ultrafiltration concentration: gel column washing liquid 1 is balanced, inactivation of virus liquid loading gel column layer
Analysis, under 280nm length ultraviolet light detection, washes 4 times of column volumes of post with washing liquid 2, with washing liquid 3 eluting coagulation factors viii, and receives
Collection, then carries out ultrafiltration dealcoholysis and desalination, obtains high concentration and concentrates coagulation factors viii solution, the potency control of coagulation factors viii
System is in 40 iu/ml；Wherein gel model deae-saphrose ff, loading and elution flow rate are 220 ~ 250ml/min, wash
Liquid 1 formula is 110 mm sodium chloride, 10 mm sodium citrate, 1 mm calcium chloride, 16 mm lysines, 120 mm glycine,
ph7.0；The formula of washing liquid 2 is 150 mm sodium chloride, 10 mm sodium citrate, 1 mm calcium chloride, 16 mm lysines, and 120 mm are sweet
Propylhomoserin, ph7.0；The formula of washing liquid 3 is 250 mm sodium chloride, 10 mm sodium citrate, 1 mm calcium chloride, 16 mm lysines, 120
Mm glycine, ph7.0.
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| 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 |
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| CN105348382B (en) * | 2015-12-05 | 2020-09-11 | 上海洲跃生物科技有限公司 | Preparation method of high-purity human coagulation factor VIII |
| 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 |
| 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 |
| CN114787185B (en) * | 2019-12-30 | 2023-08-08 | 四川远大蜀阳药业有限责任公司 | Freezing method of blood coagulation factor VIII intermediate |
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5679776A (en) * | 1989-09-05 | 1997-10-21 | Centre Regional De Transfusion Sanguine De Lille | Process for preparing a concentrate of blood coagulation factor VIII-von willebrand factor complex from total plasma |
| CN101967188A (en) * | 2010-11-08 | 2011-02-09 | 江西博雅生物制药股份有限公司 | Process for preparing human antihemophilic factor VIII |
-
2014
- 2014-09-25 CN CN201410496187.9A patent/CN104231073B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5679776A (en) * | 1989-09-05 | 1997-10-21 | Centre Regional De Transfusion Sanguine De Lille | Process for preparing a concentrate of blood coagulation factor VIII-von willebrand factor complex from total plasma |
| CN101967188A (en) * | 2010-11-08 | 2011-02-09 | 江西博雅生物制药股份有限公司 | Process for preparing human antihemophilic factor VIII |
Non-Patent Citations (1)
| Title |
|---|
| Coagulation factor VIII: structure and stability;Wei Wang et al.;《International Journal of Pharmaceutics》;20030618;第259卷(第1-2期);第7页右栏第2段至第11页左栏第2段 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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