CN107880112A - A kind of human blood coagulation factor VII I preparation method and human blood coagulation factor VII I products - Google Patents
A kind of human blood coagulation factor VII I preparation method and human blood coagulation factor VII I products Download PDFInfo
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Abstract
The invention discloses the preparation method of human blood coagulation factor VII I a kind of and human blood coagulation factor VII I products, it is related to blood product field.For human blood coagulation factor VII I preparation method by the reasonable processing to cryoprecipitate, the tromethamine solution using concentration as 0.015 0.025mol/L is 35 in mass ratio:1 ratio dissolving cryoprecipitate, ion exchange chromatography separating-purifying is combined using polyethylene glycol precipitation, the human blood coagulation factor VII I rate of recovery can be effectively improved and the ratio of finished product is lived, yield reaches 180 240IU/L blood plasma, it is not less than 100IU/mg albumen than work, the vWF factors, the vWF factors and people's coagulation factor VIII ratio are rich in obtained human blood coagulation factor VII I products close to 1:1, except for treating hemophilia A patients', it can also be used to treat von Willebrand disease patient, and human blood coagulation factor VII I has good stability and heat resistance.
Description
Technical field
The present invention relates to blood product field, a kind of preparation method in particular to human blood coagulation factor VII I and
Human blood coagulation factor VII I products.
Background technology
Human blood coagulation factor VII I (Human Cogulation Factor VIII, abbreviation AHF), to lacking human blood coagulation
Coagulation function obstacle caused by VIII has role of correcting, is the rescue of lifelong medication and the emergency treatment sufferer of hemophilia A patients
Medication.In nearly 400,000 hemophilia A patients in the whole world, the patient in China there are about 6~100,000 people, but receive only having for treatment
8000 people or so, from the treatment rate for improving hemophilia A patients and emergency treatment medication meter is ensured, domestic human blood coagulation factor VII I's
Breach is about 20 times of existing yield.The production certification for only having several families and having AHF domestic at present, supplies serious in short supply, nothing
Method meets patient's essential drug demand.Existing AHF is prepared the methods of mainly being adsorbed using polyethylene glycol precipitation, aluminium hydroxide and carried out
Prepare, the shortcomings such as specific activity is low, yield is low be present, in face of domestic AHF market supplies deficiency present situation, develop a kind of high income,
The AHF preparation method higher than work, has positive effect.
In consideration of it, special propose the present invention.
The content of the invention
It is an object of the invention to provide a kind of human blood coagulation factor VII I preparation method, and people is prepared using the preparation method
Blood coagulation factor VIII finished product can improve yield and the ratio of product is lived.
Another object of the present invention is to provide a kind of balance for being used during Purification of Human blood coagulation factor VIII
Liquid.
Another object of the present invention is to provide a kind of washing for being used during Purification of Human blood coagulation factor VIII
Liquid.
Another object of the present invention is to provide a kind of elution for being used during Purification of Human blood coagulation factor VIII
Liquid.
Another object of the present invention is to provide a kind of dialysis for being used during Purification of Human blood coagulation factor VIII
Liquid.
Another object of the present invention is to provide a kind of human blood coagulation factor VII I products, human blood coagulation factor VII I products
It is higher than work, and the vWF factors are rich in, there is good stability and heat resistance.
What the present invention was realized in：
A kind of human blood coagulation factor VII I preparation method, it includes：Cryoprecipitate processing step；
Cryoprecipitate dissolving step includes：Cryoprecipitate isolated from blood plasma is dissolved with tromethamine solution, it is cold heavy to obtain
Shallow lake lysate；
Wherein, tromethamine solution and the mass ratio of cryoprecipitate are 3-5:1, tromethamine solution concentration is 0.015-
0.025mol/L。
Further, in some embodiments of the present invention, preparation method also includes：Polyethylene glycol precipitation step；
Polyethylene glycol precipitation step includes：Polyglycol solution is added into cryoprecipitate lysate, obtains the second mixed solution；
The final concentration control of polyethylene glycol in second mixed solution is 2-5%, the pH of the second mixed solution adjust to
6.3-6.6。
Further, in some embodiments of the present invention, polyglycol solution contains 30-32% polyethylene glycol.
Further, in some embodiments of the present invention, with acetic acid adjust the second mixed solution pH adjust to
6.3-6.6。
Further, in some embodiments of the present invention, in polyethylene glycol precipitation step, by the second mixed solution
After stirring 05-1h, 1.5-2h is stood.
Further, in some embodiments of the present invention, preparation method also includes：S/D inactivation steps；
S/D inactivation steps include：The supernatant obtained by polyethylene glycol precipitation step is taken, supernatant and S/D viruses are gone out
Solution mixing living, obtains the 3rd mixed solution, is placed under the conditions of 24-26 DEG C, stirring inactivation 6 hours；
Wherein, S/D inactivation of virus solution contains：Polyoxyethylene sorbitan monoleate and tributyl phosphate；
In the 3rd mixed solution：The content control of polyoxyethylene sorbitan monoleate is 1%, and the content control of tributyl phosphate is
0.3%.
Further, in some embodiments of the present invention, after S/D inactivation steps, preparation method also includes：It is pure
Change step；
Purification step comprises the following steps：
Equilibrium step, adsorption step, washing step and elution step；
Wherein, equilibrium step includes：Chromatographic column is balanced with 4-5 times of column volume equilibrium liquid, equilibrium liquid contains：Tromethamine
16-24mmol/L, sodium chloride 80-120mmol/L, glycine are 100-140mmol/L and calcium chloride is 35-45 μm of ol/
L, pH 6.80-7.20.
Adsorption step includes：On the 3rd mixed solution that will handle to obtain through S/D inactivation steps after equilibrium step is handled
Chromatographic column adsorbed.
In purge process, the major function of equilibrium liquid is balanced gel medium, makes its bar with the sample solution of purifying
Part is consistent, avoids it and sample solution condition is inconsistent that the i.e. human blood coagulation factor VII I of destination protein is had an impact, and reduces people and coagulates
Blood factor VIII loss of activity, improve human blood coagulation factor VII I yields.
Further, in some embodiments of the present invention, washing step includes：Washed with 1-2 times of column volume cleaning solution
Chromatographic column is washed, cleaning solution contains：Tromethamine 16-24mmol/L, sodium chloride 140-180mmol/L, glycine 100-
35-45 μm of 140mmol/L, calcium chloride ol/L and polyoxyethylene sorbitan monoleate 40-80mmol/L, pH 6.80-7.20.
Cleaning solution major function is to remove impurity protein to improve destination protein human blood coagulation factor VII I purity, selects cleaning solution
The First Principles of composition are to be eliminated as much as impurity protein on the basis of under guarantor's blood coagulation factor VIII is not washed, cleaning solution
Select the improper yield for either influenceing human blood coagulation factor VII I or influence human blood coagulation factor VII I purity.
The surfactant (polyoxyethylene sorbitan monoleate) of certain content is added in cleaning solution, it is during washing, it is necessary to tight
It lattice control operation condition, slightly can not very cause destination protein human blood coagulation factor VII I denaturation to separate out, influence clean result and coagulate
Glue medium service life, add a certain amount of surfactant i.e. polyoxyethylene sorbitan monoleate) can effectively solve the problem, prevent people's blood coagulation
Factor IX deformation separates out, and improves its yield.
Further, in some embodiments of the present invention, elution step includes：Washed with 1-2 times of column volume eluent
De- chromatographic column, eluent contain：Histidine 20-30mmol/L, calcium chloride 180-220mmol/L, sodium chloride 140-
180mmol/L and glycine 100-140mmol/L, pH 6.80-7.20.
The major function of eluent is on the basis of guarantor's blood coagulation factor VIII purity as far as possible by human blood coagulation
Under VIII elutions, the improper yield and purity for easily influenceing human blood coagulation factor VII I of selection of eluent, serious possibility influences
Human blood coagulation factor VII I molecular structure, cause human blood coagulation factor VII I to be denatured, or cause its bad stability.
Eluent can to the greatest extent may be used efficiently against above mentioned problem making one blood coagulation factor VIII used by the preparation method
On the basis of the elution completely of energy ground is lower, improving its yield prevents its structural change, strengthens its stability.
Further, in some embodiments of the present invention, after the purification step, preparation method also includes：Ultrafiltration
Step；
Ultrafiltration step includes：The liquid that flows through obtained by above-mentioned purification step is concentrated, obtains concentrate, concentrate is with thoroughly
Analysis liquid carries out dialysis 7-10 times；
Dialyzate contains：Rafter acid sodium 8-12mmol/L, histidine 20-30mmol/L, glycine 100-140mmol/L and
Calcium chloride 0.8-1.2mmol/L, pH 6.80-7.20.
Small molecule solute in dialyzate is dialyzate composition, because ultrafiltration is to the protein in human blood coagulation factor VII I point
Son is damaging processing, carries out ultrafiltration using above-mentioned dialyzate, the protection ring of stability can be provided for people's blood coagulation factor VIII
Border, while the dialysis formula of liquid selected also is the preparation prescription of people's factor VIII product, as stabilizer and frozen-dried protective
Agent, play a part of safeguarding product stable in physicochemical property, avoid human blood coagulation factor VII I from removing disease in follow-up lyophilized, xeothermic inactivation
Loss of activity in the steps such as poison, improve the human blood coagulation factor VII I rate of recovery and than living.
Further, in some embodiments of the present invention, chromatographic column is ToyopearlDEAE 650M ion exchanges
Gel chromatography column.
A kind of equilibrium liquid, it is suitable to be used to balance chromatographic column during Purification of Human blood coagulation factor VIII, and equilibrium liquid contains
Have：Tromethamine 16-24mmol/L, sodium chloride 80-120mmol/L, glycine 100-140mmol/L and calcium chloride 35-45 μ
Mol/L, pH 6.80-7.20.
Further, in some embodiments of the present invention, equilibrium liquid contains：Tromethamine 18-22mmol/L, chlorination
Sodium is 90-110mmol/L, glycine 110-130mmol/L and 38-42 μm of ol/L of calcium chloride.
A kind of human blood coagulation factor VII I preparation method, it includes purification step；Purification step includes：Chromatograph column equilibration step
Suddenly；
Chromatographic column equilibrium step includes：Chromatographic column is balanced with the equilibrium liquid described in 4-5 times of column volume as above any one.
A kind of cleaning solution, it is suitable to be used to wash chromatographic column during Purification of Human blood coagulation factor VIII, and cleaning solution contains
Have：Tromethamine 16-24mmol/L, sodium chloride 140-180mmol/L, glycine 100-140mmol/L, calcium chloride 35-45 μ
Mol/L and polyoxyethylene sorbitan monoleate 40-80mmol/L, pH 6.80-7.20.
Further, in some embodiments of the present invention, cleaning solution contains：Tromethamine 18-22mmol/L, chlorination
Sodium 150-170mmol/L, glycine 110-130mmol/L, calcium chloride 38-42 μm of ol/L and polyoxyethylene sorbitan monoleate 50-
70mmol/L。
A kind of human blood coagulation factor VII I preparation method, it includes purification step；Purification step includes：Wash chromatographic column step
Suddenly；
Chromatographic column washing step includes：Chromatographic column is washed with the cleaning solution described in 1-2 times of column volume as above any one.
A kind of eluent, it is suitable to be used for elution chromatography post during Purification of Human blood coagulation factor VIII, and eluent contains
Have：Histidine 20-30mmol/L, calcium chloride 180-200mmol/L, sodium chloride 140-180mmol/L and glycine 100-
140mmol/L, pH 6.80-7.20.
Further, in some embodiments of the present invention, eluent contains：Histidine 22-28mmol/L, calcium chloride
For 190-210mmol/L, sodium chloride 150-170mmol/L and glycine 110-130mmol/L.
A kind of human blood coagulation factor VII I preparation method, it includes purification step；Purification step includes：Elution chromatography post walks
Suddenly；
Elution chromatography post step includes：With the elution chromatographic column described in 1-2 times of column volume as above any one.
A kind of dialyzate, it is suitable to be used for the I containing human blood coagulation factor VII that dialyses during Purification of Human blood coagulation factor VIII
Solution, dialyzate contains：Rafter acid sodium 8-12mmol/L, histidine 20-30mmol/L, glycine 100-140mmol/L and
Calcium chloride 0.8-1.2mmol/L, pH 6.80-7.20.
Further, in some embodiments of the present invention, dialyzate contains：Rafter acid sodium 9-11mmol/L, histidine
22-28mmol/L, glycine 110-130mmol/L and calcium chloride 0.9-1.1mmol/L.
A kind of human blood coagulation factor VII I preparation method, it includes ultrafiltration step：
Ultrafiltration step includes：With the solution of dialyzate dialysis I containing human blood coagulation factor VII described in as above any one.
A kind of human blood coagulation factor VII I products, its preparation method system as the human blood coagulation factor VII I described in as above any one
Prepare.
Further, in some embodiments of the present invention, press《Pharmacopoeia of People's Republic of China》2015 editions three " people
Blood coagulation factor VIII " method detects, in above-mentioned human blood coagulation factor VII I products：Polyethylene glycol residual quantity is 0.03g/L, poly- sorb
The residual quantity of ester 80 is 17 μ g/ml, residual quantity of tributyl phosphate is 3 μ g/ml, vWF factor actives are 15.7IU/ml and people's blood coagulation
Factor IX specific activity is 104.8IU/mg.
Certainly, it is necessary to which explanation, is pressed《Pharmacopoeia of People's Republic of China》2015 editions three " human blood coagulation factor VII I " sides
Method detects, and there is the human blood coagulation factor VII I products in above-mentioned point value error range to belong to protection scope of the present invention.
The invention has the advantages that：
Human blood coagulation factor VII I provided by the invention preparation method by the reasonable processing to cryoprecipitate, using concentration as
0.015-0.025mol/L tromethamine solution is 3-5 in mass ratio:1 ratio dissolving cryoprecipitate, by cryoprecipitate
Rationally processing, it is 0.015-0.025mol/L tromethamine solution in mass ratio for 3-5 using concentration:1 ratio dissolving is cold heavy
Form sediment, ion exchange chromatography separating-purifying is combined using polyethylene glycol precipitation, can effectively improve human blood coagulation factor VII I's
The ratio of the rate of recovery and finished product is lived, and yield reaches 180-240IU/L blood plasma, is not less than 100IU/mg albumen, the preparation side than work
The vWF factors, the vWF factors and people's coagulation factor VIII ratio are rich in human blood coagulation factor VII I products obtained by method close to 1:1,
Except the treatment for hemophilia A patients, the treatment of von Willebrand disease patient can be used for.And the preparation prescription used
Make product that there is good stability and heat resistance, through it is xeothermic inactivation (such as 100 DEG C, 30min) virus treated after, people's blood coagulation because
Sub- VIII activity is not lost substantially.
Equilibrium liquid provided by the invention contains tromethamine 16-24mmol/L, sodium chloride 80-120mmol/L, glycine
100-140mmol/L and calcium chloride 35-45 μm of ol/L, pH 6.80-7.20.Layer is caused using equilibrium liquid balance chromatographic column
The consistent of gel media and the loading sample on post is analysed, avoids condition is inconsistent from being had an impact to sample, is advantageous to improve
The human blood coagulation factor VII I rate of recovery and the ratio of finished product are lived.
Cleaning solution provided by the invention contains tromethamine 16-24mmol/L, sodium chloride 140-180mmol/L, glycine
35-45 μm of 100-140mmol/L, calcium chloride ol/L and polyoxyethylene sorbitan monoleate 40-80mmol/L, pH 6.80-7.20, using this
Cleaning solution can remove impurity protein as much as possible, and ensure that destination protein i.e. human blood coagulation is firmly bonded to chromatographic column
On, and prevent destination protein i.e. human blood coagulation factor VII I denaturation from separating out, avoid human blood coagulation factor VII I from being lost in, be advantageous to improve people
The rate of recovery and ratio of blood coagulation factor VIII are lived.
Eluent provided by the invention contains histidine 22-28mmol/L, calcium chloride 190-210mmol/L, sodium chloride
150-170mmol/L and glycine 110-130mmol/L.The eluent may insure destination protein i.e. human blood coagulation factor VII I
It is eluted as much as possible from chromatographic column, and avoids human blood coagulation factor VII I denaturation or its stability from reducing, raising is advantageous to improve
The human blood coagulation factor VII I rate of recovery and ratio is lived.
Dialyzate provided by the invention contains：Rafter acid sodium 8-12mmol/L, histidine 20-30mmol/L, glycine 100-
140mmol/L and calcium chloride 0.8-1.2mmol/L, pH 6.80-7.20.Because ultrafiltration is to the egg in human blood coagulation factor VII I
White matter molecule is damaging processing, carries out ultrafiltration using above-mentioned dialyzate, can provide stability for people's blood coagulation factor VIII
Environmental protection, while the dialysis formula of liquid selected also is the preparation prescription of people's factor VIII product, as stabilizer and jelly
Dry protective agent, play a part of safeguarding product stable in physicochemical property, avoid human blood coagulation factor VII I in follow-up lyophilized, xeothermic inactivation
The loss of activity in the steps such as virus is removed, improves the human blood coagulation factor VII I rate of recovery and than living.
Embodiment
, below will be in the embodiment of the present invention to make the purpose, technical scheme and advantage of the embodiment of the present invention clearer
Technical scheme be clearly and completely described.Unreceipted actual conditions person, builds according to normal condition or manufacturer in embodiment
The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, it is the conventional production that can be obtained by commercially available purchase
Product.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
The preparation method for the human blood coagulation factor VII I that the present embodiment provides, it comprises the following steps:
1st, raw blood plasma acquisition step：
The collection of 1.1 blood plasma and quality should meet current edition《Pharmacopoeia of People's Republic of China》" blood product is given birth in three
The regulation of production human plasma ", it should freeze after collection within 4 hours, -30 DEG C preserved no more than 1 year.
1.2 blood plasma should without grumeleuse, without fibrin deposition, non-piarhemia, no haemolysis.
2nd, cryoprecipitate preparation process：
2097kg refrigerated plasmas (3495 person-portion blood plasma of meter) are weighed, (need ethanol solution control to exist through 70% ethanol solution
After 70%-75%) soaking or wiping surface sterilization processing, broken bag, after being merged into tank, melt in less than 37 DEG C water-baths, control is melted
Slurry temperature degree is 4 DEG C (melting slurry temperature control at 0-4 DEG C).
Cryoprecipitate separation is carried out to the blood plasma after fusion using low-temperature and high-speed centrifuge, controls and is centrifuged out liquid per desk centrifuge
Speed is no more than 3000ml/min, goes out liquid temperature degree in 4 DEG C (in other embodiments, going out liquid temperature control at 0-4 DEG C), centrifugation knot
26kg cryoprecipitates are collected after beam, are frozen in -35.0 DEG C of freezers.
3rd, cryoprecipitate dissolving step：
Cryoprecipitate is from after -35.0 DEG C of freezer outbounds, after being thawed in advance 6 hours in 10-30 DEG C of environment, using Philip
After HR7629 pulverizers crush cryoprecipitate, pour into the 0.02mol/L tromethamines solution (78L) of 3 times of precipitation capacities, stirring is molten
Solution 2 hours, molten rear volume is 104L.
4th, polyethylene glycol (PEG) settling step：
Precipitation dissolving terminate after, add prepare 31.56%PEG solution (in other embodiments, PEG solution concentrations
Control is in 30-32%) 12.97kg, make in product PEG ultimate densities be 3.5% (in other embodiments, PEG ultimate density controls
System is in 2-5%)；Add PEG after with 0.1mol/L acetums adjust pH to 6.53, by the temperature control of mixed solution 20.0
After being stirred 0.5 hour at DEG C (in other examples, the temperature control of mixed solution is at 20-30 DEG C), 1.5 hours are stood
Afterwards, centrifuge.
5th, S/D inactivation steps：
Supernatant made from collection step 4, supernatant volume 108L after accurate metering filtering, by 1:10 ratios add S/D
(polyoxyethylene sorbitan monoleate content is 11% to inactivation of virus solution, and tributyl phosphate content is 3.3%), to make polyoxyethylene sorbitan monoleate in product
Content reaches 1%, and the content of tributyl phosphate reaches 0.3%, when warming-in-water to mixed solution is 24.0 DEG C, starts timing and goes out
It is living, the temperature of mixed solution is kept under the conditions of 25 DEG C, stirring inactivation 6 hours.
6th, ion-exchange chromatography：
After inactivation after the clarified filtering of solution, liquor capacity is 119L, upper Toyopearl DEAE after accurate measurement filtering
650M ionexchange gel chromatographies post adsorbs；
Then, with 4 times of bed volume equilibrium liquid balanced gels；
Wherein, equilibrium liquid contains：Tromethamine 20mmol/L, sodium chloride 120mmol/L, glycine 120mmol/L and
Calcium chloride 40 μm of ol/L, pH 7.0；
With 2 times of bed volume cleaning solution detergent gels；
Wherein, cleaning solution contains：Tromethamine 20mmol/L, sodium chloride 160mmol/L, glycine 120mmol/L, chlorination
Calcium 40 μm of ol/L and polyoxyethylene sorbitan monoleate 60mmol/L, pH 7.0；
With 2 times of bed volume elution gels；
Wherein, eluent contains：Histidine is 25mmol/L, calcium chloride 200mmol/L, sodium chloride 160mmol/L,
Glycine is 120mmol/L, pH 7.0.
And collected according to peak shape and flow through liquid 11L (having eluted peak to start to collect, returning to baseline to peak stops collecting).
7th, ultrafiltration step：The liquid liquid that flows through being collected into first is concentrated into 4L, then dialysed with dialyzate；
Wherein, dialyzate contains：Sodium citrate 10mmol/L, histidine 25mmol/L, glycine 120mmol/L and chlorine
Change calcium is 1mmol/L, pH 7.0；
20-30 DEG C of solution temperature during dialysis；
Dialysed 8 times using equimultiple volume ultrafiltration dialysis liquid, liquor capacity is 5.2L after ultrafiltration dialysis.
8th, dilution is prepared and aseptic filtration dispenses：
Solution surveys potency (its testing result is shown in Table 3) after taking ultrafiltration, and 19L is diluted to by packing specification plus ultrafiltration dialysis liquid,
Human blood coagulation factor VII I potency is 23IU/ml, aseptic filtration, is dispensed by 10.5ml/ bottles, is divided into and fills 1790 bottles,
9th, freeze：
After aseptic filtration packing, adjustment refrigerant temperature extremely freezes 3 hours for less than -40 DEG C, 20pa or so is evacuated to, by 5
DEG C/hr heating baffle temperatures to -10 DEG C, -10 DEG C of refrigerant is kept for 15 hours, heats up refrigerant to 34 DEG C by 10 DEG C/hr, 34 DEG C of refrigerant
Kept for 10 hours, vacuum seal, roll lid；
10th, xeothermic inactivation：
Freeze-dried products are moved into water-bath sterilization cabinet, temperature control time 30min, xeothermic end, turns at 100 ± 0.5 DEG C
Enter storehouse to be checked, sample censorship, that is, obtain human blood coagulation factor VII I finished products.
Final product quality detection project reference《Pharmacopoeia of People's Republic of China》2015 editions three " human blood coagulation factor VII I "
(P272-273 pages) is detected, and testing result is as shown in table 1.
The human blood coagulation factor VII I final product quality testing results of table 1
As seen from Table 1, human blood coagulation factor VII I finished products indices meet the requirements, especially human blood coagulation factor VII I into
Product reach 104.8IU/mg than work, far above 10.0IU/mg protein as defined in standard；Polyethylene glycol residual quantity is only 0.03g/L,
Far below content as defined in standard；In addition, it should be noted that human blood coagulation factor VII I finished products produced by the present invention also have
VWF factor actives, its activity reach 15.7IU/ml.
Embodiment 2
The preparation method for the human blood coagulation factor VII I that the present embodiment provides is substantially the same manner as Example 1, unlike, at this
In embodiment：
Equilibrium liquid contains：Tromethamine 18mmol/L, sodium chloride 90mmol/L, glycine 110mmol/L and calcium chloride
38μmol/L。
Dialyzate contains：Rafter acid sodium 11mmol/L, histidine 28mmol/L, glycine 130mmol/L and calcium chloride
1.1mmol/L。
Embodiment 3
The preparation method for the human blood coagulation factor VII I that the present embodiment provides is substantially the same manner as Example 1, unlike, at this
In embodiment：
Equilibrium liquid contains：Tromethamine 22mmol/L, sodium chloride 110mmol/L, glycine 130mmol/L and calcium chloride
42μmol/L。
Embodiment 4
The preparation method for the human blood coagulation factor VII I that the present embodiment provides is substantially the same manner as Example 1, unlike, at this
In embodiment：
Cleaning solution contains：Tromethamine 18mmol/L, sodium chloride 150mmol/L, glycine 110mmol/L, the μ of calcium chloride 38
Mol/L and polyoxyethylene sorbitan monoleate 50mmol/L.
Eluent contains：Histidine 28mmol/L, calcium chloride 210mmol/L, sodium chloride 170mmol/L and glycine
130mmol/L。
Embodiment 5
The preparation method for the human blood coagulation factor VII I that the present embodiment provides is substantially the same manner as Example 1, unlike, at this
In embodiment：
Cleaning solution contains：Tromethamine 22mmol/L, sodium chloride 170mmol/L, glycine 130mmol/L, the μ of calcium chloride 42
Mol/L and polyoxyethylene sorbitan monoleate 70mmol/L.
Dialyzate contains：Rafter acid sodium 9mmol/L, histidine 22mmol/L, glycine 110mmol/L and calcium chloride
0.9mmol/L。
Embodiment 6
The preparation method for the human blood coagulation factor VII I that the present embodiment provides is substantially the same manner as Example 1, unlike, at this
In embodiment：
Eluent contains：Histidine 22mmol/L, calcium chloride 190mmol/L, sodium chloride 150mmol/L and glycine
110mmol/L。
Experimental example 1
To determine vWF factor contents in the preparation technology product, the production of the preparation method preparation to embodiment 1- embodiments 6
VWF factor contents in product are investigated, using vWF:Ag detection kits (SIEMENS, the term of validity 2017/09/30)；
Stand Plasma (SSC/ISTH, the term of validity 2020/12), are determined using immunoturbidimetry.Normal coagulation Quality Control blood plasma is with dilute
Release liquid and make 2：1、5：4、3：5、2：5、5：38 gradient dilution, sample are diluted to according to human blood coagulation factor VII I active marker's numerical value
1IU/ml, mixed respectively with latex reagent, reaction buffer, when solidification is determined on Sysmex CA-1500 Automatic coagulometers
Between, by the result measured compared with the standard curve of normal reference plasma assay results, you can calculate vWF:Ag contents.As a result
It is shown in Table 2.
The testing result of vWF factor contents in the human blood coagulation factor VII I finished products of table 2
As can be known from Table 2, the people's blood coagulation for the human blood coagulation factor VII I preparation methods production that 1-6 of the embodiment of the present invention is provided
VWF in Factor IX:Ag contents and VIII content ratios between 0.69-0.79, the A enterprises that have listed and B enterprises
0.46 compares with 0.14, and the vWF factors and VIII factors ratio are close to 1:1, it removes the treatment for hemophilia A patients, also may be used
For the treatment of von Willebrand disease (VonWille brand, abbreviation vWD) patient.
At present, the Main Means for treating von Willebrand disease are that the cryoprecipitate for being transfused human plasma source carries out replacement therapy,
But cryoprecipitate has great viral transmission danger, its application is very limited due to a lack of effective virus inactivation technology.
And by human blood coagulation factor VII I preparation methods provided by the invention produce human blood coagulation factor VII I contain vWF because
Son, its application can be not limited to the above, and in the treatment of von Willebrand disease patient the potential of viral transmission danger can be overcome to lack
Fall into, have a good application prospect.
Experimental example 2
Detect the heat resistance of human blood coagulation factor VII I products
The human blood coagulation factor VII I products prepared by preparation method that Example 1-6 is provided, contrast it in xeothermic inactivation
Front and rear human blood coagulation factor VII I potency, concrete outcome such as table 3 below.
The heat resistance testing result of the human blood coagulation factor VII I products of table 3.
As can be seen from Table 3, the loss before and after the xeothermic inactivation of human blood coagulation factor VII I potency is in 0.88%-2.5%
Between, compared with the loss 10%-25% reported on document；Using the human blood coagulation factor VII I preparation methods of the present invention xeothermic
The front and rear loss of inactivation can be ignored substantially；Being indicated above preparation prescription provided by the invention such as dialyzate has good stabilization
Property, also the heat resistance of human blood coagulation factor VII I products prepared by the explanation present invention is more preferable certainly.
It should be noted that percentage of the present invention is mass percent in addition to illustrating.
It should be noted that the numerical value in preparation method of the present invention is not limited to the concrete numerical value applied in embodiment,
In actual production, the numerical value change in smaller range does not influence result of the test, and it falls within protection scope of the present invention.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent substitution, improvement etc., should be included in the scope of the protection.
Claims (8)
1. a kind of human blood coagulation factor VII I preparation method, it is characterised in that it includes：Cryoprecipitate processing step；
The cryoprecipitate dissolving step includes：Cryoprecipitate isolated from blood plasma is dissolved with tromethamine solution, it is cold heavy to obtain
Shallow lake lysate；
Wherein, the tromethamine solution and the mass ratio of the cryoprecipitate are 3-5:1, tromethamine solution concentration is 0.015-
0.025mol/L。
2. human blood coagulation factor VII I according to claim 1 preparation method, it is characterised in that the preparation method is also wrapped
Include：Polyethylene glycol precipitation step；
The polyethylene glycol precipitation step includes：Polyglycol solution is added into the cryoprecipitate lysate, obtains the second mixing
Solution；
The final concentration control of polyethylene glycol in second mixed solution is 2-5%, the pH of the second mixed solution adjust to
6.3-6.6。
3. human blood coagulation factor VII I according to claim 2 preparation method, it is characterised in that the preparation method is also wrapped
Include：S/D inactivation steps；
The S/D inactivation steps include：The supernatant obtained by the polyethylene glycol precipitation step is taken, by the supernatant and S/
D inactivation of virus solution mixes, and obtains the 3rd mixed solution, is placed under the conditions of 24-26 DEG C, stirring inactivation 6 hours；
Wherein, the S/D inactivation of virus solution contains：Polyoxyethylene sorbitan monoleate and tributyl phosphate；
In the 3rd mixed solution：The content control of polyoxyethylene sorbitan monoleate is 1%, and the content control of tributyl phosphate is
0.3%.
4. human blood coagulation factor VII I according to claim 3 preparation method, it is characterised in that inactivate and walk in the S/D
After rapid, the preparation method also includes：Purification step；
The purification step comprises the following steps：
Equilibrium step, adsorption step, washing step and elution step；
Wherein, the equilibrium step includes：Chromatographic column is balanced with 4-5 times of column volume equilibrium liquid, the equilibrium liquid contains：Ammonia fourth three
Alcohol 16-24mmol/L, sodium chloride 80-120mmol/L, glycine 100-140mmol/L and calcium chloride 35-45 μm of ol/L, pH
6.80-7.20；
The adsorption step includes：Through described flat on the 3rd mixed solution that will handle to obtain through the S/D inactivation steps
Chromatographic column after weighing apparatus step process is adsorbed.
5. human blood coagulation factor VII I according to claim 4 preparation method, it is characterised in that the washing step bag
Include：Chromatographic column is washed with 1-2 times of column volume cleaning solution, the cleaning solution contains：Tromethamine 16-24mmol/L, sodium chloride
140-180mmol/L, glycine 100-140mmol/L, calcium chloride 35-45 μm of ol/L and polyoxyethylene sorbitan monoleate 40-80mmol/L,
pH 6.80-7.20。
6. human blood coagulation factor VII I according to claim 4 preparation method, it is characterised in that the elution step bag
Include：With 1-2 times of column volume elution chromatographic column, the eluent contains：Histidine 20-30mmol/L, calcium chloride 180-
220mmol/L, sodium chloride 140-180mmol/L and glycine 100-140mmol/L, pH 6.80-7.20.
7. human blood coagulation factor VII I according to claim 4 preparation method, it is characterised in that the purification step it
Afterwards, the preparation method also includes：Ultrafiltration step；
The ultrafiltration step includes：The liquid that flows through obtained by the purification step is concentrated, obtains concentrate, the concentrate
Dialysis 7-10 times is carried out with dialyzate；
The dialyzate contains：Rafter acid sodium 8-12mmol/L, histidine 20-30mmol/L, glycine 100-140mmol/L and
Calcium chloride 0.8-1.2mmol/L, pH 6.80-7.20.
A kind of 8. human blood coagulation factor VII I products, it is characterised in that its as people's blood coagulation any one of claim 1-7 because
Sub- VIII preparation method is prepared.
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| CN114072420A (en) * | 2019-07-04 | 2022-02-18 | 康诺贝林伦瑙有限公司 | Truncated Von Willebrand Factor (VWF) for increasing the in vitro stability of factor VIII |
| CN114787185A (en) * | 2019-12-30 | 2022-07-22 | 四川远大蜀阳药业有限责任公司 | Cryopreservation method of blood coagulation factor VIII intermediate product |
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