CN105524894A - Preparation method of blood coagulation factor XIII - Google Patents
Preparation method of blood coagulation factor XIII Download PDFInfo
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- CN105524894A CN105524894A CN201610105925.1A CN201610105925A CN105524894A CN 105524894 A CN105524894 A CN 105524894A CN 201610105925 A CN201610105925 A CN 201610105925A CN 105524894 A CN105524894 A CN 105524894A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/104—Aminoacyltransferases (2.3.2)
- C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/02—Aminoacyltransferases (2.3.2)
- C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
Abstract
The invention provides a preparation method of a blood coagulation factor XIII. The preparation method has the advantages of high yield, high purity and short production period, and comprises the steps of blood plasma and blood cell separation, virus inactivation, ammonium sulfate graded sedimentation, DEAE-FF chromatographic column treatment, split charging and freeze drying, wherein an auxiliary agent is added during the virus inactivation; the graded sedimentation and dissolution process of the blood coagulation factor XIII is performed in an environment being 0 to 4 DEG C; different dissolving agents are added for performing graded dissolution on precipitates; the dissolution efficiency is improved; the production period is shortened; the bacteria breeding is reduced; and the yield and the purity of the blood coagulation factor XIII are improved. The preparation method of the blood coagulation factor XIII provided by the invention has the beneficial effects that the extraction efficiency is high; more than 10mg of the blood coagulation factor XIII can be extracted from per liter of blood plasma; in a prepared blood coagulation factor XIII freeze-drying product, the purity of the blood coagulation factor XIII is higher than 90 percent; the operation is simple; the production period is short; and the preparation of the blood coagulation factor XIII can be completed in two days.
Description
Technical field
The invention belongs to medical bioengineering field, particularly relate to a kind of with human blood or mammalian for the method for medical factor XIII prepared by raw material.
Background technology
Factor XIII (also known as rFXⅢ, Fibrinoligase, trans-glutaminases) is with proenzyme (A
2b
2the tetramer, M
r≈ 320KD) form is present in a kind of glycoprotein in blood plasma, it is the necessary a kind of plasma proteinase of normal coagulation, the effect of transaminase is played after being activated, can catalysis SFM (sFM) intermolecular crosslinking reaction, form the α chain polymer of γ chain homodimer and macromolecule, increase clot strength, accelerating wound healing.
The preparation of current factor XIII both domestic and external is mainly extracted from human plasma, yeast cell, extracts in addition from thrombocyte, embryo, again through catalyzed by thrombin when using clinically, at Ca
2+under participation role, be transformed into the activated A of tool
2structure, the further covalent cross-linking of catalysis fibre protein gelatin becomes insoluble fibrin clot.
Factor XIII goods can extract from human or animal's pig blood, and China is herding big country, and pig industry is larger, and pig blood resource is very abundant, because production technique is not mature enough, cause the utilization ratio of current China pig blood very low.From pig blood, extract FXIII both can make full use of pig blood resource, the environmental pollution caused because of discharge can be reduced again.The shortage of FXIII can cause that delayed hemorrhage, galled spots are hemorrhage, subcutaneous hematoma, tissue injury are hemorrhage, oral mucosa and gingival hemorrhage common, more commonly sport injury or intramuscular injection cause muscle hemotoncus.In addition, FXIII is very effective to the disorder for the treatment of wound healing after operation, scleroderma, ulcerative colitis, pseudomembranous colitis etc.One of important component of FXIII Ye Shi tissue adhesive.
Factor XIII is a kind of glycoprotein, the extraction of general factor XIII comprises six basic steps, i.e. blood plasma blood cell separation, inactivation of virus, ammonium sulfate precipitation, DEAE-FF chromatography column, carry out packing and freeze-drying, finally by xeothermic inactivation of virus and cobalt-60 irradiation.But there is following defect in above-mentioned technique:
The time that in the process of producing, three times dissolve is oversize, easily causes the microbial reproductions such as bacterium in this process, thus increases the content of the toxin such as pyrogen in product; Dissolution time is long also easily causes loss of activity, extends manufacture cycle simultaneously, increases cost; The product that above-mentioned technique is obtained, purity is not high, and activity is lower.
Summary of the invention
Technical problem to be solved by this invention is: the preparation method providing the factor XIII that a kind of yield is high, purity is high and with short production cycle.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: the preparation method of factor XIII, comprises the steps:
Step 1, in blood, add centrifugation after antithrombotics, get supernatant anticoagulate plasma;
Step 2, in supernatant anticoagulate plasma, add tributyl phosphate and tween-80 as virus inactivating agent, and add auxiliary agent, carry out S/D inactivation of virus, obtain the blood plasma after deactivation;
Step 3, by centrifugal for the blood plasma after the deactivation of step 2 gained, get clear liquid;
Step 4, step 3 gained clear liquid is chilled to 0 ~ 4 DEG C in advance, after adding the saturated ammonium sulphate solution of 0 ~ 4 DEG C, gets precipitation;
Step 5, by the step 4 gained precipitation saturated ammonium sulphate solution washing of 0 ~ 4 DEG C, the precipitation fragmentation after washing is added a solvating agent of 0 ~ 4 DEG C and stirs, and get clear liquid, a described solvating agent is Klorvess Liquid;
Step 6, step 5 gained clear liquid is chilled to 0 ~ 4 DEG C in advance, the acetic acid volume fraction adding 0 ~ 4 DEG C be 94% ~ 96% acetic acid solution to pH value of solution be after 5.2 ~ 6.5, adding ammonium sulfate solids to solution saturation ratio is 15% ~ 20%, gets precipitation;
Step 7, add the secondary solvating agent of 0 ~ 4 DEG C and stir in step 6 gained precipitation, get clear liquid, described secondary solvating agent is the damping fluid containing sodium-chlor, and the buffering in described damping fluid is to being any one in Tris-HCl, Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC and citrate-phosphate;
Step 8, by step 7 gained clear liquid heating in water bath to 53 ~ 56 DEG C, maintain and be cooled to 0 ~ 4 DEG C after 160 ~ 200 seconds, get clear liquid;
Step 9, in step 8 gained clear liquid, add ammonium sulfate solids to solution saturation ratio be 35% ~ 40%, gets precipitation;
Step 10, add three solvating agents of 0 ~ 4 DEG C and stir in step 9 gained precipitation, get clear liquid, described three solvating agents are the Tris-HCl damping fluid containing Repone K;
Step 11, by step 10 gained clear liquid gradient elution postlyophilization, obtain factor XIII freeze-dried products.
Beneficial effect of the present invention is: add different solvating agents when dissolving, and makes the factor XIII in precipitating dissolve simultaneously and greatly shortens dissolution time fully simultaneously, reduce bacteria breed; Extraction efficiency is high, and often liter of blood plasma can extract the factor XIII of more than 10 milligrams; In obtained factor XIII freeze-dried products, factor XIII purity is more than 90%; Simple to operate, with short production cycle, the preparation of factor XIII can be completed within 2 day time; The redissolution time of obtained factor XIII freeze-dried products is short, and good stability, the shelf time is long.
Accompanying drawing explanation
Fig. 1 is the schema of the preparation method of the factor XIII of the embodiment of the present invention 1 and embodiment 2.
Embodiment
By describing technology contents of the present invention in detail, realized object and effect, accompanying drawing is coordinated to be explained below in conjunction with embodiment.
The design of most critical of the present invention is: add auxiliary agent when inactivation of virus, class settling, the dissolution process of factor XIII is carried out under 0 ~ 4 DEG C of environment, add different solvating agents and fractional solution is carried out to precipitation, improve dissolved efficiency, shorten the production cycle, reduce bacteria breed, improve factor XIII yield and purity.
Please refer to Fig. 1, the preparation method of factor XIII, comprises the steps:
Step 1, in blood, add centrifugation after antithrombotics, get supernatant anticoagulate plasma;
Step 2, in supernatant anticoagulate plasma, add tributyl phosphate and tween-80 as virus inactivating agent, and add auxiliary agent, carry out S/D inactivation of virus, obtain the blood plasma after deactivation;
Step 3, by centrifugal for the blood plasma after the deactivation of step 2 gained, get clear liquid;
Step 4, step 3 gained clear liquid is chilled to 0 ~ 4 DEG C in advance, after adding the saturated ammonium sulphate solution of 0 ~ 4 DEG C, gets precipitation;
Step 5, by the step 4 gained precipitation saturated ammonium sulphate solution washing of 0 ~ 4 DEG C, the precipitation fragmentation after washing is added a solvating agent of 0 ~ 4 DEG C and stirs, and get clear liquid, a described solvating agent is Klorvess Liquid;
Step 6, step 5 gained clear liquid is chilled to 0 ~ 4 DEG C in advance, the acetic acid volume fraction adding 0 ~ 4 DEG C be 94% ~ 96% acetic acid solution to pH value of solution be after 5.2 ~ 6.5, adding ammonium sulfate solids to solution saturation ratio is 15% ~ 20%, gets precipitation;
Step 7, add the secondary solvating agent of 0 ~ 4 DEG C and stir in step 6 gained precipitation, get clear liquid, described secondary solvating agent is the damping fluid containing sodium-chlor, and the buffering in described damping fluid is to being any one in Tris-HCl, Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC and citrate-phosphate;
Step 8, by step 7 gained clear liquid heating in water bath to 53 ~ 56 DEG C, maintain and be cooled to 0 ~ 4 DEG C after 160 ~ 200 seconds, get clear liquid;
Step 9, in step 8 gained clear liquid, add ammonium sulfate solids to solution saturation ratio be 35% ~ 40%, gets precipitation;
Step 10, add three solvating agents of 0 ~ 4 DEG C and stir in step 9 gained precipitation, get clear liquid, described three solvating agents are the Tris-HCl damping fluid containing Repone K;
Step 11, by step 10 gained clear liquid gradient elution postlyophilization, obtain factor XIII freeze-dried products.
Described auxiliary agent is one or more in amino acid, glucose, sucrose, maltose, sorbyl alcohol, sodium gluconate, trisodium citrate, EDTA, amino acid in auxiliary agent is one or more in L-glutamic acid, Methionin, L-Ala, Histidine, methionine(Met), glycine, arginine, L-Leu, ILE, and the concentration of the solute in described auxiliary agent is 0.01 ~ 1.0mol/L.
Auxiliary agent can improve the quality of factor XIII goods, plays the effect of the biologic activity of protection factor XIII in inactivation of virus and settling process.
Contriver finds in actual production practice, due to after S/D inactivation of virus, because virosome is destroyed by fire-fighting medium, create the albumen of a large amount of sex change, simultaneously inactivator also has destruction to a certain degree to the various albumen in blood plasma, cause protein denaturation, produce various insolubles, these insolubless study discovery through contriver, to quality product, especially redissolution speed and product purity have disadvantageous effect, therefore after deactivation, add step with centrifugal separation.
The S/D inactivator, foreign protein etc. be mixed in precipitation has disadvantageous effect to the redissolution speed of product is same with quality, therefore adopts cold ammoniumsulphate soln as washing composition, to wash away S/D inactivator and the foreign protein of the remnants in primary sedimentation; The saturated ammonium sulphate solution of 0 ~ 4 DEG C is adopted to be because when temperature is too high, washing composition can dissolve factor XIII as washing composition.
Wherein, in above-mentioned gradient elution process, adopt novel DEAESepharoseFF filler, good separating effect.
Wherein, described blood is pig blood or the human blood of aseptic collection.Wherein, in a described solvating agent, the concentration of Repone K is 0.1 ~ 0.2mol/L.
Wherein, in described secondary solvating agent, the concentration of sodium-chlor is 0.1 ~ 0.2mol/L, and cushioning right concentration is 0.01 ~ 0.05mol/L, and the pH value of described secondary solvating agent is 7.0 ~ 7.5.
Wherein, the pH value of described three solvating agents is the concentration of Repone K in 8.0, three solvating agents is 0.50mM, and in three solvating agents, the concentration of Tris-HCL is 10mM.
Wherein, carry out adding lyophilized vaccine in the solution after lyophilize forward direction gradient elution in step 11, the solute of described lyophilized vaccine is selected from one or more in poly-hydroxy carbohydrate, trisodium citrate, sodium-chlor, amino acid.
Preferably, the solute of described lyophilized vaccine is poly-hydroxy carbohydrate, trisodium citrate, sodium-chlor and amino acid; In described lyophilized vaccine, the concentration of poly-hydroxy carbohydrate is 0.1% ~ 5%w/v, and the concentration of sodium-chlor is 0.1% ~ 0.9%w/v, and amino acid whose concentration is 0.1% ~ 5%w/v.In the application, the unit of w/v is g/mL, and as " concentration of poly-hydroxy carbohydrate is 0.1% ~ 5%w/v " represents, containing the poly-hydroxy carbohydrate of 0.1 ~ 5 gram in every 100 ml solns, namely the concentration of poly-hydroxy carbohydrate is 1 ~ 50g/L.
Wherein, described poly-hydroxy carbohydrate is selected from one or more in sucrose, trehalose, glucose, N.F,USP MANNITOL, sorbyl alcohol; Amino acid in lyophilized vaccine is selected from one or more in L-glutamic acid, Methionin, L-Ala, Histidine, methionine(Met), glycine, arginine, L-Leu, ILE.
Wherein, also comprise successively after step 11:
Step 12, the factor XIII freeze-dried products in step 11 carried out packing, vacuum degree measurement and after rolling lid, under 98 ~ 100 DEG C of water bath heat preservations condition of 30 minutes, xeothermic inactivation of virus is carried out to the non-lipid-coated virus in factor XIII goods;
Step 13, the factor XIII goods after deactivation xeothermic in step 12 are carried out cobalt-60 irradiation, obtain medical factor XIII sterile product.
The former blood plasma adopted in the embodiment of the present invention all comes from the same day through the blood plasma of aseptic collection, add the trisodium citrate aseptic pyrogen-free antithrombotics 10000ml of 0.1mol/L, aseptic blood collection total amount is about 90000ml, divides three experiments to carry out by the service requirements in embodiment 1,2 and 3.
Embodiment 1
Please refer to Fig. 1, technique all operations all carries out in aseptic ten thousand grades of purifying areas, and wherein packing and freeze-drying operation are all carried out in local laminar flow purifying area, and the operation carried out all meets clean area operational requirement.Production specification is 1.5ml.
(1) the pig blood 10000ml of aseptic collection is got;
(2) centrifugation, gets supernatant anticoagulate plasma 4000ml;
(3) 0.3%w/v tributyl phosphate and 1%w/v tween-80 are added as virus inactivating agent to the supernatant anticoagulate plasma of step (2) gained, add the glucose of 0.2mol/L as auxiliary agent, carry out inactivation of virus at 25 DEG C 6 hours;
By the blood plasma centrifugation after deactivation, discard throw out, get clear liquid;
(4) sedimentations: 0 ~ 4 DEG C will be chilled in advance through step (3) gained clear liquid, and add 1000ml saturated ammonium sulphate solution, the temperature of described saturated ammonium sulphate solution is 0 ~ 4 DEG C;
(5) be separated and collect the factor XIII primary sedimentation thing that a sedimentation in step (4) obtains, with 0 ~ 4 DEG C, ammoniumsulphate soln washs the factor XIII primary sedimentation thing that a sedimentation in step (4) obtains, and discards washings;
(6) once dissolve: the factor XIII primary sedimentation thing after washing in step (5) is shredded and adds a solvating agent, stir under 4 DEG C of conditions, step (5) is collected the factor XIII primary sedimentation thing obtained to dissolve, remove insolubles, collect and dissolve clear liquid 500ml;
(7) collect the insolubles in step (6), add an isopyknic solvating agent, 25 DEG C of water-bath 1h, remove insolubles, collect and dissolve clear liquid 300ml;
(8) secondary settlement: after the dissolving clear liquid 800ml that combining step (6) (7) obtain is chilled to 0 ~ 4 DEG C in advance, cold acetic acid is added in supernatant liquor, adjust pH, add solid ammonium sulfate again, the factor XIII in clear liquid is dissolved in sedimentation, and the temperature of described cold acetic acid is 0 ~ 4 DEG C;
(9) secondary dissolves: be separated and collect the factor XIII secondary precipitate that the middle secondary settlement of step (8) obtains, adding secondary solvating agent 400ml, stirs, dissolved by factor XIII secondary precipitate under 4 DEG C of conditions;
(10) secondary step (9) obtained dissolves clear liquid heating in water bath to 53 ~ 56 DEG C, maintains 3 minutes, puts into rapidly ice bath and be cooled to 4 DEG C, precipitation separation, collects supernatant liquor;
(11) three sedimentations: in the supernatant liquor that step (10) obtains, add solid ammonium sulfate, the factor XIII in sedimentation supernatant liquor;
To dissolve for (12) three times: be separated and collect factor XIII three throw outs that three sedimentations in step (11) obtain, adding three solvating agents, stir, by factor XIII three precipitate dissolves under 4 DEG C of conditions;
(13) dissolve DEAE post on clear liquid, gradient elution three times that step (12) are obtained, collect and have active part;
(14) add lyophilized vaccine, freeze-drying in the activated elutriant obtained to step (13), obtain factor XIII freeze-dried products.
Adopt the protein concentration C1 (see table 1) in Bradford method detection elutriant;
(15) according to the detected result in (14), the protein concentration adopting PEG to be concentrated in solution is 5mg/ml, and add protective material, in preparation, composition consists of:
Factor XIII 5mg/ml
Glucose 2.5mg/ml
Glycine 2mg/ml
(16) packing, often props up packing 1.5ml, carries out freeze-drying after packing completes, and freeze-drying is carried out after offeing for sale rolling lid immediately, finally carries out cobalt-60 irradiation to dried frozen aquatic products, can obtain factor XIII goods B1 (see table 2).
(17) the factor XIII freeze-dried products in step (16) is carried out packing, vacuum degree measurement and after rolling lid, under 98 ~ 100 DEG C of water bath heat preservations condition of 30 minutes, xeothermic inactivation of virus is carried out to the non-lipid-coated virus in factor XIII goods;
(18) the factor XIII goods after step (17) xeothermic deactivation are carried out cobalt-60 irradiation, obtain medical factor XIII sterile product.
Embodiment 2
Please refer to Fig. 1, technique all operations all carries out in aseptic ten thousand grades of purifying areas, and wherein packing and freeze-drying operation are all carried out in local laminar flow purifying area, and the operation carried out all meets clean area operational requirement.Production specification is 1.5ml.
(1) the pig blood 24000ml of aseptic collection is got;
(2) centrifugation, gets supernatant anticoagulate plasma 12000ml;
(3) 0.3%w/v tributyl phosphate and 1%w/v tween-80 are added as virus inactivating agent to the supernatant anticoagulate plasma of step (2) gained, add the glucose of 0.2mol/L as auxiliary agent, carry out inactivation of virus at 25 DEG C 6 hours;
By the blood plasma centrifugation after deactivation, discard throw out, get clear liquid;
(4) sedimentations: 0 ~ 4 DEG C will be chilled in advance through step (3) gained clear liquid, and add 3000ml saturated ammonium sulphate solution, the temperature of described saturated ammonium sulphate solution is 0 ~ 4 DEG C;
(5) be separated and collect the factor XIII primary sedimentation thing that a sedimentation in step (4) obtains, with 0 ~ 4 DEG C, ammoniumsulphate soln washs the factor XIII primary sedimentation thing that a sedimentation in step (4) obtains, and discards washings;
(6) once dissolve: the factor XIII primary sedimentation thing after washing in step (5) is shredded and adds a solvating agent, stir under 4 DEG C of conditions, step (5) is collected the factor XIII primary sedimentation thing obtained to dissolve, remove insolubles, collect and dissolve clear liquid 1500ml;
(7) collect the insolubles in step (6), add an isopyknic solvating agent, 25 DEG C of water-bath 1h, remove insolubles, collect and dissolve clear liquid 900ml;
(8) secondary settlement: after the dissolving clear liquid 2400ml that combining step (6) (7) obtain is chilled to 0 ~ 4 DEG C in advance, cold acetic acid is added in supernatant liquor, adjust pH, add solid ammonium sulfate again, the factor XIII in clear liquid is dissolved in sedimentation, and the temperature of described cold acetic acid is 0 ~ 4 DEG C;
(9) secondary dissolves: be separated and collect the factor XIII secondary precipitate that the middle secondary settlement of step (8) obtains, adding secondary solvating agent 1200ml, stirs, dissolved by factor XIII secondary precipitate under 4 DEG C of conditions;
(10) secondary step (9) obtained dissolves clear liquid heating in water bath to 53 ~ 56 DEG C, maintains 3 minutes, puts into rapidly ice bath and be cooled to 4 DEG C, precipitation separation, collects supernatant liquor;
(11) three sedimentations: in the supernatant liquor that step (10) obtains, add solid ammonium sulfate, the factor XIII in sedimentation supernatant liquor;
To dissolve for (12) three times: be separated and collect factor XIII three throw outs that three sedimentations in step (11) obtain, adding three solvating agents, stir, by factor XIII three precipitate dissolves under 4 DEG C of conditions;
(13) dissolve DEAE post on clear liquid, gradient elution three times that step (12) are obtained, collect and have active part;
(14) add lyophilized vaccine, freeze-drying in the activated elutriant obtained to step (13), obtain factor XIII freeze-dried products.
Adopt the protein concentration C2 (see table 1) in Bradford method detection elutriant;
(15) according to the detected result in (14), the protein concentration adopting PEG to be concentrated in solution is 5mg/ml, and add protective material, in preparation, composition consists of:
Factor XIII 5mg/ml
Glucose 2mg/ml
Glycine 1mg/ml
(16) packing, often props up packing 1.5ml, carries out freeze-drying after packing completes, and freeze-drying is carried out after offeing for sale rolling lid immediately, finally carries out cobalt-60 irradiation to dried frozen aquatic products, can obtain factor XIII goods B2 (see table 2).
(17) the factor XIII freeze-dried products in step (16) is carried out packing, vacuum degree measurement and after rolling lid, under 98 ~ 100 DEG C of water bath heat preservations condition of 30 minutes, xeothermic inactivation of virus is carried out to the non-lipid-coated virus in factor XIII goods;
(18) the factor XIII goods after step (17) xeothermic deactivation are carried out cobalt-60 irradiation, obtain medical factor XIII sterile product.
Embodiment 3 technique all operations all carries out in ten thousand grades of purifying areas, and wherein packing and freeze-drying operation are all carried out in local laminar flow purifying area, and the operation carried out all meets clean area operational requirement.Production specification is 1.5ml.
(1) the pig blood 50000ml of aseptic collection is got;
(2) centrifugation, gets supernatant anticoagulate plasma 20000ml;
(3) 0.3%w/v tributyl phosphate and 1%w/v tween-80 are added as virus inactivating agent to the supernatant anticoagulate plasma of step (2) gained, carry out inactivation of virus at 25 DEG C 6 hours;
By the blood plasma centrifugation after deactivation, discard throw out, get clear liquid;
(4) sedimentations: 0 ~ 4 DEG C will be chilled in advance through step (3) gained clear liquid, and add 5000ml saturated ammonium sulphate solution, the temperature of described saturated ammonium sulphate solution is 0 ~ 4 DEG C;
(5) be separated and collect the factor XIII primary sedimentation thing that a sedimentation in step (4) obtains, with 0 ~ 4 DEG C, ammoniumsulphate soln washs the factor XIII primary sedimentation thing that a sedimentation in step (4) obtains, and discards washings;
(6) once dissolve: the factor XIII primary sedimentation thing after washing in step (5) is shredded and adds a solvating agent, stir under 4 DEG C of conditions, step (5) is collected the factor XIII primary sedimentation thing obtained to dissolve, remove insolubles, collect and dissolve clear liquid 2400ml;
(7) collect the insolubles in step (6), add an isopyknic solvating agent, 25 DEG C of water-bath 1h, remove insolubles, collect and dissolve clear liquid 1400ml;
(8) secondary settlement: after the dissolving clear liquid 3800ml that combining step (6) (7) obtain is chilled to 0 ~ 4 DEG C in advance, cold acetic acid is added in supernatant liquor, adjust pH, add solid ammonium sulfate again, the factor XIII in clear liquid is dissolved in sedimentation, and the temperature of described cold acetic acid is 0 ~ 4 DEG C;
(9) secondary dissolves: be separated and collect the factor XIII secondary precipitate that the middle secondary settlement of step (8) obtains, adding secondary solvating agent 2000ml, stirs, dissolved by factor XIII secondary precipitate under 4 DEG C of conditions;
(10) secondary step (9) obtained dissolves clear liquid heating in water bath to 53 ~ 56 DEG C, maintains 3 minutes, puts into rapidly ice bath and be cooled to 4 DEG C, precipitation separation, collects supernatant liquor;
(11) three sedimentations: in the supernatant liquor that step (10) obtains, add solid ammonium sulfate, the factor XIII in sedimentation supernatant liquor;
To dissolve for (12) three times: be separated and collect factor XIII three throw outs that three sedimentations in step (11) obtain, adding three solvating agents, stir, by factor XIII three precipitate dissolves under 4 DEG C of conditions;
(13) dissolve DEAE post on clear liquid, gradient elution three times that step (12) are obtained, collect and have active part;
(14) add lyophilized vaccine, freeze-drying in the activated elutriant obtained to step (13), obtain factor XIII freeze-dried products.
Adopt the protein concentration C3 (table 1) in Bradford method detection elutriant;
(15) according to the detected result in (14), the protein concentration adopting PEG to be concentrated in solution is 5mg/ml, and add protective material, in preparation, composition consists of:
Factor XIII 5mg/ml
Glucose 2mg/ml
Glycine 2mg/ml
(16) packing, often props up packing 1.5ml, carries out freeze-drying after packing completes, and freeze-drying is carried out after offeing for sale rolling lid immediately, finally carries out cobalt-60 irradiation to dried frozen aquatic products, can obtain factor XIII goods B3 (table 2).
(17) the factor XIII freeze-dried products in step (16) is carried out packing, vacuum degree measurement and after rolling lid, under 98 ~ 100 DEG C of water bath heat preservations condition of 30 minutes, xeothermic inactivation of virus is carried out to the non-lipid-coated virus in factor XIII goods;
(18) the factor XIII goods after step (17) hot deactivation are carried out cobalt-60 irradiation, obtain medical factor XIII sterile product.
Embodiment 4 is got human blood coagulation XIII respectively and is lacked Fibrinogen or blood plasma, different dilution standard human plasma or trial-product (1:2 ~ 1:256), human thrombin/CaCl
2solution (uses 0.05mol/LCaCl
2redissolve freeze dried human zymoplasm to 50IU/ml) each 0.1ml mixing, namely form grumeleuse.37 DEG C: be incubated 60 minutes, add lml1% Monochloro Acetic Acid, on the mixer jolting, bottom grumeleuse is floated, and jolting 1 time in every 10 minutes, observes insoluble grumeleuse.The greatest dilution that grumeleuse also standard plasma and the trial-product do not dissolved is recorded after 30 minutes.Be calculated as follows human blood coagulation XIII in sample to tire, should 1.0U/ml be not less than.Human blood coagulation XIII (U/ml)=trial-product of tiring can be observed grumeleuse or suspended substance greatest dilution/standard plasma can be observed grumeleuse or suspended substance greatest dilution.Standard plasma FXIII is active in 1U/ml, and can obtain FX III activity value of testing sample, its result is as shown in table 1.
Embodiment 5 detects the protein concentration of factor XIII according to the 5th method " Coomassie Brilliant Blue " in the Pharmacopoeia of the People's Republic of China 2015 editions four general rules " protein determination ", and its result is as shown in table 1.With reference to relevant " human fibrinogen " standard of the Pharmacopoeia of the People's Republic of China 2015 editions three 274th ~ 275 pages, detect detections such as " pyrogens ", " tributyl phosphate " and " tween-80 " in goods, its result is as shown in table 2.
Table 1
Table 2
Remarks: medical factor XIII freeze-dried products adopts 1ml distilled water as double solvents.
In sum, the beneficial effect of the preparation method of factor XIII provided by the invention is: add different solvating agents when dissolving, and makes the factor XIII in precipitating dissolve simultaneously and greatly shortens dissolution time fully simultaneously, reduce bacteria breed; Extraction efficiency is high, and often liter of blood plasma can extract the factor XIII of more than 10 milligrams; In obtained factor XIII freeze-dried products, factor XIII purity is more than 90%; Simple to operate, with short production cycle, the preparation of factor XIII can be completed within 2 day time; The redissolution time of obtained factor XIII freeze-dried products is short, and good stability, the shelf time is long.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every equivalents utilizing specification sheets of the present invention and accompanying drawing content to do, or be directly or indirectly used in relevant technical field, be all in like manner included in scope of patent protection of the present invention.
Claims (9)
1. the preparation method of factor XIII, is characterized in that, comprises the steps:
Step 1, in blood, add centrifugation after antithrombotics, get supernatant anticoagulate plasma;
Step 2, in supernatant anticoagulate plasma, add tributyl phosphate and tween-80 as virus inactivating agent, and add auxiliary agent, carry out S/D inactivation of virus, obtain the blood plasma after deactivation;
Step 3, by centrifugal for the blood plasma after deactivation, get clear liquid;
Step 4, step 3 gained clear liquid is chilled to 0 ~ 4 DEG C in advance, after adding the saturated ammonium sulphate solution of 0 ~ 4 DEG C, gets precipitation;
Step 5, by the step 4 gained precipitation saturated ammonium sulphate solution washing of 0 ~ 4 DEG C, the precipitation fragmentation after washing is added a solvating agent of 0 ~ 4 DEG C and stirs, and get clear liquid, a described solvating agent is Klorvess Liquid;
Step 6, step 5 gained clear liquid is chilled to 0 ~ 4 DEG C in advance, the acetic acid volume fraction adding 0 ~ 4 DEG C be 94% ~ 96% acetic acid solution to pH value of solution be after 5.2 ~ 6.5, adding ammonium sulfate solids to solution saturation ratio is 15% ~ 20%, gets precipitation;
Step 7, add the secondary solvating agent of 0 ~ 4 DEG C and stir in step 6 gained precipitation, get clear liquid, described secondary solvating agent is the damping fluid containing sodium-chlor, and the buffering in described secondary solvating agent is to being any one in Tris-HCl, Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC and citrate-phosphate;
Step 8, by step 7 gained clear liquid heating in water bath to 53 ~ 56 DEG C, maintain and be cooled to 0 ~ 4 DEG C after 160 ~ 200 seconds, get clear liquid;
Step 9, in step 8 gained clear liquid, add ammonium sulfate solids to solution saturation ratio be 35% ~ 40%, gets precipitation;
Step 10, add three solvating agents of 0 ~ 4 DEG C and stir in step 9 gained precipitation, get clear liquid, described three solvating agents are the Tris-HCl damping fluid containing Repone K;
Step 11, by step 10 gained clear liquid gradient elution postlyophilization, obtain factor XIII freeze-dried products.
2. the preparation method of factor XIII according to claim 1, is characterized in that: described blood is pig blood or the human blood of aseptic collection.
3. the preparation method of factor XIII according to claim 1, it is characterized in that: described auxiliary agent is one or more in amino acid, glucose, sucrose, maltose, sorbyl alcohol, sodium gluconate, trisodium citrate, EDTA, described amino acid is one or more in L-glutamic acid, Methionin, L-Ala, Histidine, methionine(Met), glycine, arginine, L-Leu, ILE.
4. the preparation method of factor XIII according to claim 1, is characterized in that: in a described solvating agent, the concentration of Repone K is 0.1 ~ 0.2mol/L.
5. the preparation method of factor XIII according to claim 1, it is characterized in that: in described secondary solvating agent, the concentration of sodium-chlor is 0.1 ~ 0.2mol/L, cushioning right concentration is 0.01 ~ 0.05mol/L, and the pH value of described secondary solvating agent is 7.0 ~ 7.5.
6. the preparation method of factor XIII according to claim 1; it is characterized in that: carry out in step 11 adding lyophilized vaccine in the solution after lyophilize forward direction gradient elution, the solute of described lyophilized vaccine is selected from one or more in poly-hydroxy carbohydrate, trisodium citrate, sodium-chlor, amino acid.
7. the preparation method of factor XIII according to claim 6, is characterized in that: the solute of described lyophilized vaccine comprises poly-hydroxy carbohydrate, trisodium citrate, sodium-chlor and amino acid; In described lyophilized vaccine, the concentration of poly-hydroxy carbohydrate is 0.1% ~ 5%w/v, and the concentration of sodium-chlor is 0.1% ~ 0.9%w/v, and amino acid whose concentration is 0.1% ~ 5%w/v.
8. the preparation method of factor XIII according to claim 6, is characterized in that: described poly-hydroxy carbohydrate is selected from one or more in sucrose, trehalose, glucose, N.F,USP MANNITOL, sorbyl alcohol;
Described amino acid is selected from one or more in L-glutamic acid, Methionin, L-Ala, Histidine, methionine(Met), glycine, arginine, L-Leu, ILE.
9. the preparation method of factor XIII according to claim 1, is characterized in that: also comprise successively after step 11:
Step 12, the factor XIII freeze-dried products in step 11 carried out packing, vacuum degree measurement and after rolling lid, under 98 ~ 100 DEG C of water bath heat preservations condition of 30 minutes, xeothermic inactivation of virus is carried out to the non-lipid-coated virus in factor XIII goods;
Step 13, the factor XIII goods after deactivation xeothermic in step 12 are carried out cobalt-60 irradiation, obtain medical factor XIII sterile product.
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Denomination of invention: Preparation of coagulation factor XIII Effective date of registration: 20200820 Granted publication date: 20190326 Pledgee: Jingdong sub branch of Fujian Sanming Rural Commercial Bank Co., Ltd Pledgor: FUJIAN HUAGENE BIOSCIENCES Co.,Ltd. Registration number: Y2020980005205 |
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