CN105524894B - The preparation method of factor XIII - Google Patents
The preparation method of factor XIII Download PDFInfo
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- CN105524894B CN105524894B CN201610105925.1A CN201610105925A CN105524894B CN 105524894 B CN105524894 B CN 105524894B CN 201610105925 A CN201610105925 A CN 201610105925A CN 105524894 B CN105524894 B CN 105524894B
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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 present invention provides the preparation method of a kind of high yield, purity is high and factor XIII with short production cycle, including the separation of blood plasma blood cell, inactivation of virus, ammonium sulfate precipitation, DEAE-FF chromatographic column, packing and step of freeze drying, auxiliary agent is wherein added when inactivation of virus, class settling, the course of dissolution of factor XIII are carried out under 0~4 DEG C of environment, different lytic agents is added, fractional solution is carried out to precipitating, improve dissolved efficiency, shorten the production cycle, bacteria breed is reduced, factor XIII yield and purity are improved.The beneficial effect of the preparation method of factor XIII provided by the invention is: extraction efficiency is high, and every liter of blood plasma can extract 10 milligrams or more of factor XIII；In factor XIII freeze-dried products obtained, factor XIII purity is 90% or more；It is easy to operate, it is with short production cycle, the preparation of factor XIII can be completed within 2 day time.
Description
Technical field
The invention belongs to medical bioengineering fields, more particularly to one kind is using human blood or mammalian as raw material
The method for preparing medical factor XIII.
Background technique
Factor XIII (also known as fibrin stabilizing factor, Fibrinoligase, transglutaminase) is with enzyme
Original (A2B2The tetramer, Mr≈ 320KD) form is present in one of blood plasma glycoprotein, and it is a kind of blood necessary to normal coagulation
Protease is starched, the effect of transaminase is played after being activated, it is anti-that the intermolecular crosslinking of soluble fibrin monomer (sFM) can be catalyzed
It answers, forms 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 cells, and there are also small from blood
It is extracted in plate, embryo, Shi Zaijing catalyzed by thrombin is clinically used, in Ca2+Under participation effect, it is transformed into active A2Knot
Structure, the further covalent cross-linking of catalysis fibre protein gel is at insoluble fibrin clot.
Factor XIII product can be extracted from human or animal's pig blood, and China is herding big country, pig breeding industry scale compared with
Greatly, pig blood resource very abundant causes the utilization rate of current China's pig blood very low since production technology is not mature enough.From pig blood
Middle extraction FXIII can not only make full use of pig blood resource, but also can reduce environmental pollution caused by due to discharge.FXIII's lacks
Weary delayed hemorrhage, galled spots bleeding, subcutaneous hematoma, tissue damage bleeding, oral mucosa and the bleeding gums of will lead to are common,
More commonly injury gained in sports or intramuscular injection cause muscle hemotoncus.In addition, FXIII is disorderly to treatment wound healing after operation
Unrest, chorionitis, ulcerative colitis, pseudomembranous colitis etc. are very effectively.FXIII be also tissue adhesive important component it
One.
Factor XIII is a kind of glycoprotein, and the extraction of general factor XIII includes six basic steps, i.e.,
The separation of blood plasma blood cell, ammonium sulfate precipitation, DEAE-FF chromatographic column, is dispensed and is lyophilized inactivation of virus, most afterwards through xeothermic
Inactivation of virus and cobalt -60 irradiate.But above-mentioned technique has the following deficiencies:
The time dissolved three times during production is too long, this is easy to cause the microbial reproductions such as bacterium in the process, from
And increase the content of the toxin such as pyrogen in product；Dissolution time is too long also to be easy to cause loss of activity, extends manufacture cycle simultaneously,
Increase cost；Product made from above-mentioned technique, purity is not high, and activity is lower.
Summary of the invention
The technical problems to be solved by the present invention are: provide a kind of high yield, purity is high and blood coagulation with short production cycle because
The preparation method of sub- XIII.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention are as follows: the preparation method of factor XIII, packet
Include following steps:
Step 1 is centrifugated after anti-coagulants is added into blood, takes the anticoagulant blood plasma of supernatant；
Tributyl phosphate and Tween-80 is added as virus inactivator in step 2, Xiang Shangqing anticoagulation slurry, and is added and helps
Agent carries out S/D inactivation of virus, the blood plasma after must inactivating；
Blood plasma after inactivating obtained by step 2 is centrifuged by step 3, takes clear liquid；
Step 3 gained clear liquid is cooled to 0~4 DEG C by step 4 in advance, and after 0~4 DEG C of saturated ammonium sulfate solution is added, it is heavy to take
It forms sediment；
Step 5 washs precipitating obtained by step 4 with 0~4 DEG C of saturated ammonium sulfate solution, and the precipitating after washing is crushed
And 0~4 DEG C of a lytic agent is added and stirs, clear liquid is taken, a lytic agent is Klorvess Liquid；
Step 5 gained clear liquid is cooled to 0~4 DEG C by step 6 in advance, be added 0~4 DEG C of acetic acid volume fraction be 94%~
After 96% acetic acid solution to pH value of solution is 5.2~6.5, it is 15%~20% that ammonium sulfate solids to solution saturation degree, which is added, is taken
Precipitating；
0~4 DEG C of secondary lytic agent is added into precipitating obtained by step 6 and stirs for step 7, takes clear liquid, described secondary molten
Solution agent is the buffer containing sodium chloride, and the buffering in the buffer is to for Tris-HCl, disodium hydrogen phosphate-sodium dihydrogen phosphate
Any one of with citrate-phosphate；
Step 8, by step 7 gained clear liquid heating water bath to 53~56 DEG C, maintain 160~200 seconds after be cooled to 0~4 DEG C,
Take clear liquid；
Step 9, into step 8 gained clear liquid, addition ammonium sulfate solids to solution saturation degree is 35%~40%, takes precipitating；
0~4 DEG C of lytic agent three times is added into precipitating obtained by step 9 and stirs for step 10, takes clear liquid, it is described three times
Lytic agent is the Tris-HCl buffer containing potassium chloride；
Step 11 will be freeze-dried after step 10 gained clear liquid gradient elution to get factor XIII freeze-dried products.
The beneficial effects of the present invention are: dissolution when different lytic agents is added, while make precipitating in blood coagulation because
Sub- XIII dissolution sufficiently greatly shortens dissolution time simultaneously, reduces bacteria breed；Extraction efficiency is high, and every liter of blood plasma can extract 10 millis
Gram or more factor XIII；In factor XIII freeze-dried products obtained, factor XIII purity is 90% or more；
It is easy to operate, it is with short production cycle, the preparation of factor XIII can be completed within 2 day time；Factor XIII obtained
The redissolution time of freeze-dried products is short, and stability is good, and the holding time is long.
Detailed description of the invention
Fig. 1 is the flow chart of the preparation method of the factor XIII of Example 1 and Example 2 of the present invention.
Specific embodiment
To explain the technical content, the achieved purpose and the effect of the present invention in detail, below in conjunction with embodiment and cooperate attached
Figure is explained.
The most critical design of the present invention is: auxiliary agent is added in inactivation of virus, carried out under 0~4 DEG C of environment blood coagulation because
Class settling, the course of dissolution of sub- XIII is added different lytic agents and carries out fractional solution to precipitating, improves dissolved efficiency, contracting
Short production cycle reduces bacteria breed, improves factor XIII yield and purity.
Fig. 1 is please referred to, the preparation method of factor XIII includes the following steps:
Step 1 is centrifugated after anti-coagulants is added into blood, takes the anticoagulant blood plasma of supernatant；
Tributyl phosphate and Tween-80 is added as virus inactivator in step 2, Xiang Shangqing anticoagulation slurry, and is added and helps
Agent carries out S/D inactivation of virus, the blood plasma after must inactivating；
Blood plasma after inactivating obtained by step 2 is centrifuged by step 3, takes clear liquid；
Step 3 gained clear liquid is cooled to 0~4 DEG C by step 4 in advance, and after 0~4 DEG C of saturated ammonium sulfate solution is added, it is heavy to take
It forms sediment；
Step 5 washs precipitating obtained by step 4 with 0~4 DEG C of saturated ammonium sulfate solution, and the precipitating after washing is crushed
And 0~4 DEG C of a lytic agent is added and stirs, clear liquid is taken, a lytic agent is Klorvess Liquid；
Step 5 gained clear liquid is cooled to 0~4 DEG C by step 6 in advance, be added 0~4 DEG C of acetic acid volume fraction be 94%~
After 96% acetic acid solution to pH value of solution is 5.2~6.5, it is 15%~20% that ammonium sulfate solids to solution saturation degree, which is added, is taken
Precipitating；
0~4 DEG C of secondary lytic agent is added into precipitating obtained by step 6 and stirs for step 7, takes clear liquid, described secondary molten
Solution agent is the buffer containing sodium chloride, and the buffering in the buffer is to for Tris-HCl, disodium hydrogen phosphate-sodium dihydrogen phosphate
Any one of with citrate-phosphate；
Step 8, by step 7 gained clear liquid heating water bath to 53~56 DEG C, maintain 160~200 seconds after be cooled to 0~4 DEG C,
Take clear liquid；
Step 9, into step 8 gained clear liquid, addition ammonium sulfate solids to solution saturation degree is 35%~40%, takes precipitating；
0~4 DEG C of lytic agent three times is added into precipitating obtained by step 9 and stirs for step 10, takes clear liquid, it is described three times
Lytic agent is the Tris-HCl buffer containing potassium chloride；
Step 11 will be freeze-dried after step 10 gained clear liquid gradient elution to get factor XIII freeze-dried products.
The auxiliary agent is amino acid, in glucose, sucrose, maltose, sorbierite, sodium gluconate, trisodium citrate, EDTA
One or more, the amino acid in auxiliary agent is glutamic acid, lysine, alanine, histidine, methionine, glycine, smart ammonia
The concentration of one or more of acid, L-Leu, l-Isoleucine, the solute in the auxiliary agent is 0.01~1.0mol/
L。
Auxiliary agent can improve the quality of factor XIII product, played in inactivation of virus and infall process protection blood coagulation because
The effect of the biological activity of sub- XIII.
Inventor has found in actual production practice, due to after S/D inactivation of virus, since virion is broken by extinguishing chemical
It is bad, the albumen being largely denaturalized is produced, while inactivator also has a degree of destruction to the various albumen in blood plasma, leads to egg
White matter denaturation, generates various insoluble matters, these insoluble matters, the study found that product quality, especially redissolve speed through inventor
It is had adverse effect with product purity, therefore step with centrifugal separation is added after inactivation.
The S/D inactivator, the foreign protein that are mixed in precipitating etc. are same as quality to the redissolution speed of product to be adversely affected,
Therefore using cold ammonium sulfate as detergent, to wash away the remaining S/D inactivator and foreign protein in primary sedimentation；Using
0~4 DEG C of saturated ammonium sulfate solution is because when the temperature is excessively high, detergent can dissolve factor XIII as detergent.
Wherein, during above-mentioned gradient elution, using novel DEAE Sepharose FF filler, good separating effect.
Wherein, the blood is the pig blood or human blood of aseptic collection.Wherein, potassium chloride in a lytic agent
Concentration is 0.1~0.2mol/L.
Wherein, the concentration of sodium chloride is 0.1~0.2mol/L in the secondary lytic agent, the concentration buffered pair is 0.01~
0.05mol/L, the pH value of the secondary lytic agent are 7.0~7.5.
Wherein, the pH value of the lytic agent three times is 8.0, and the concentration of potassium chloride is 0.50mM in lytic agent three times, three times
The concentration of Tris-HCL is 10mM in lytic agent.
Wherein, freeze drying protectant, the jelly is added before being freeze-dried in step 11 into the solution after gradient elution
It does protectant solute and is selected from one or more of polyhydroxy carbohydrate, trisodium citrate, sodium chloride, amino acid.
Preferably, the solute of the freeze drying protectant is polyhydroxy carbohydrate, trisodium citrate, sodium chloride and amino
Acid；In the freeze drying protectant, the concentration of polyhydroxy carbohydrate is 0.1%~5%w/v, and the concentration of sodium chloride is 0.1%
~0.9%w/v, the concentration of amino acid are 0.1%~5%w/v.The unit of w/v is g/mL in the application, such as " polyhydroxy carbon
The concentration of hydrate is that 0.1%~5%w/v " is indicated, 0.1~5 gram of polyhydroxy carbon aquation is contained in every 100 milliliters of solution
Object is closed, i.e. the concentration of polyhydroxy carbohydrate is 1~50g/L.
Wherein, the polyhydroxy carbohydrate is selected from one of sucrose, trehalose, glucose, mannitol, sorbierite
Or it is several；Amino acid in freeze drying protectant is selected from glutamic acid, lysine, alanine, histidine, methionine, glycine, smart ammonia
One or more of acid, L-Leu, l-Isoleucine.
Wherein, also successively include: after step 11
Step 12 is dispensed the factor XIII freeze-dried products in step 11, vacuum degree measurement and after rolling lid,
98~100 DEG C of water-baths carry out xeothermic virus to the non-lipid-coated virus in factor XIII product under conditions of keeping the temperature 30 minutes
Inactivation；
Factor XIII product after inactivation xeothermic in step 12 is carried out the irradiation of cobalt -60 by step 13, is obtained medical solidifying
Blood factor XIII sterile product.
Former blood plasma employed in the embodiment of the present invention is both from the same day through the blood plasma of aseptic collection, addition
The sterile apyrogeneity anti-coagulants 10000ml of the trisodium citrate of 0.1mol/L, sterile blood collection total amount is about 90000ml, by real
It applies the requirement in example 1,2 and 3 and tests progress in three times.
Embodiment 1
Fig. 1 is please referred to, all operations of technique carry out in sterile ten thousand grades of purifying areas, wherein packing and freeze-drying operation exist
Local laminar flow purifying area carries out, and the operation carried out meets clean area operation and requires.Production specification is 1.5ml.
(1) the pig blood 10000ml of aseptic collection is taken；
(2) it is centrifugated, takes the anticoagulant blood plasma 4000ml of supernatant；
(3) 0.3%w/v tributyl phosphate is added to the anticoagulant blood plasma of step (2) resulting supernatant and 1%w/v Tween-80 is made
For virus inactivator, the glucose of 0.2mol/L is added as auxiliary agent, is virus inactivated 6 hours at 25 DEG C；
By the blood plasma centrifuge separation after inactivation, sediment is discarded, clear liquid is taken；
(4) primary sedimentation: will be cooled in advance 0~4 DEG C through clear liquid obtained by step (3), 1000ml saturated ammonium sulfate solution is added,
The temperature of the saturated ammonium sulfate solution is 0~4 DEG C；
(5) the factor XIII primary sedimentation object for separating and once being settled in collection step (4), with 0~4 DEG C,
Ammonium sulfate washs the factor XIII primary sedimentation object once settled in step (4), discards cleaning solution；
(6) primary dissolution: shredding the factor XIII primary sedimentation object after washing in step (5) and is added primary molten
Agent is solved, under the conditions of 4 DEG C and is stirred, step (5) are collected to obtained factor XIII primary sedimentation object and are dissolved, are removed insoluble
Object collects dissolution clear liquid 500ml；
(7) insoluble matter in collection step (6), is added an isometric lytic agent, and 25 DEG C of water-bath 1h are removed insoluble
Object collects dissolution clear liquid 300ml；
(8) secondary settlement: after the dissolution clear liquid 800ml that merging step (6) (7) obtains is cooled to 0~4 DEG C in advance, to supernatant
It is middle that cold acetic acid is added, pH is adjusted, solid ammonium sulfate is added, the factor XIII in clear liquid is dissolved in sedimentation, the cold acetic acid
Temperature is 0~4 DEG C；
(9) the factor XIII secondary precipitate that secondary dissolution: separating and secondary settlement obtains in collection step (8),
It is added secondary lytic agent 400ml, under the conditions of 4 DEG C and stirs, factor XIII secondary precipitate is dissolved；
(10) the secondary dissolution clear liquid heating water bath for obtaining step (9) is maintained 3 minutes, is put into rapidly to 53~56 DEG C
4 DEG C are cooled in ice bath, precipitation and separation collects supernatant；
(11) it settles three times: in the supernatant obtained to step (10), solid ammonium sulfate is added, settle solidifying in supernatant
Blood factor XIII；
(12) it dissolves: separating three times and the factor XIII settled three times in collection step (11) precipitates three times
Object, is added lytic agent three times, under the conditions of 4 DEG C and stirs, by factor XIII, sediment dissolves three times；
(13) DEAE column on the clear liquid of dissolution three times for obtaining step (12), gradient elution collect active part；
(14) freeze drying protectant is added in the active eluent obtained to step (13), is lyophilized, obtains coagulation factor
XIII freeze-dried products.
Using the protein concentration C1 (being shown in Table 1) in Bradford method detection eluent；
(15) it according to the testing result in (14), uses PEG to be concentrated into the protein concentration in solution as 5mg/ml, is added and protects
Agent is protected, at being grouped as in preparation are as follows:
Factor XIII 5mg/ml
Glucose 2.5mg/ml
Glycine 2mg/ml
(16) it dispenses, every packing 1.5ml, is lyophilized after the completion of packing, freeze-drying carries out rolling lid immediately after offeing for sale, finally
Cobalt -60 is carried out to dried frozen aquatic products to irradiate, and factor XIII product B1 (being shown in Table 2) can be obtained.
(17) the factor XIII freeze-dried products in step (16) are dispensed, vacuum degree measurement and after rolling lid,
98~100 DEG C of water-baths carry out xeothermic virus to the non-lipid-coated virus in factor XIII product under conditions of keeping the temperature 30 minutes
Inactivation；
(18) the factor XIII product after step (17) xeothermic inactivation is carried out cobalt -60 to irradiate, obtains medical blood coagulation
Factor XIII sterile product.
Embodiment 2
Fig. 1 is please referred to, all operations of technique carry out in sterile ten thousand grades of purifying areas, wherein packing and freeze-drying operation exist
Local laminar flow purifying area carries out, and the operation carried out meets clean area operation and requires.Production specification is 1.5ml.
(1) the pig blood 24000ml of aseptic collection is taken；
(2) it is centrifugated, takes the anticoagulant blood plasma 12000ml of supernatant；
(3) 0.3%w/v tributyl phosphate is added to the anticoagulant blood plasma of step (2) resulting supernatant and 1%w/v Tween-80 is made
For virus inactivator, the glucose of 0.2mol/L is added as auxiliary agent, is virus inactivated 6 hours at 25 DEG C；
By the blood plasma centrifuge separation after inactivation, sediment is discarded, clear liquid is taken；
(4) primary sedimentation: will be cooled in advance 0~4 DEG C through clear liquid obtained by step (3), 3000ml saturated ammonium sulfate solution is added,
The temperature of the saturated ammonium sulfate solution is 0~4 DEG C；
(5) the factor XIII primary sedimentation object for separating and once being settled in collection step (4), with 0~4 DEG C,
Ammonium sulfate washs the factor XIII primary sedimentation object once settled in step (4), discards cleaning solution；
(6) primary dissolution: shredding the factor XIII primary sedimentation object after washing in step (5) and is added primary molten
Agent is solved, under the conditions of 4 DEG C and is stirred, step (5) are collected to obtained factor XIII primary sedimentation object and are dissolved, are removed insoluble
Object collects dissolution clear liquid 1500ml；
(7) insoluble matter in collection step (6), is added an isometric lytic agent, and 25 DEG C of water-bath 1h are removed insoluble
Object collects dissolution clear liquid 900ml；
(8) secondary settlement: after the dissolution clear liquid 2400ml that merging step (6) (7) obtains is cooled to 0~4 DEG C in advance, to supernatant
It is middle that cold acetic acid is added, pH is adjusted, solid ammonium sulfate is added, the factor XIII in clear liquid is dissolved in sedimentation, the cold acetic acid
Temperature is 0~4 DEG C；
(9) the factor XIII secondary precipitate that secondary dissolution: separating and secondary settlement obtains in collection step (8),
It is added secondary lytic agent 1200ml, under the conditions of 4 DEG C and stirs, factor XIII secondary precipitate is dissolved；
(10) the secondary dissolution clear liquid heating water bath for obtaining step (9) is maintained 3 minutes, is put into rapidly to 53~56 DEG C
4 DEG C are cooled in ice bath, precipitation and separation collects supernatant；
(11) it settles three times: in the supernatant obtained to step (10), solid ammonium sulfate is added, settle solidifying in supernatant
Blood factor XIII；
(12) it dissolves: separating three times and the factor XIII settled three times in collection step (11) precipitates three times
Object, is added lytic agent three times, under the conditions of 4 DEG C and stirs, by factor XIII, sediment dissolves three times；
(13) DEAE column on the clear liquid of dissolution three times for obtaining step (12), gradient elution collect active part；
(14) freeze drying protectant is added in the active eluent obtained to step (13), is lyophilized, obtains coagulation factor
XIII freeze-dried products.
Using the protein concentration C2 (being shown in Table 1) in Bradford method detection eluent；
(15) it according to the testing result in (14), uses PEG to be concentrated into the protein concentration in solution as 5mg/ml, is added and protects
Agent is protected, at being grouped as in preparation are as follows:
Factor XIII 5mg/ml
Glucose 2mg/ml
Glycine 1mg/ml
(16) it dispenses, every packing 1.5ml, is lyophilized after the completion of packing, freeze-drying carries out rolling lid immediately after offeing for sale, finally
Cobalt -60 is carried out to dried frozen aquatic products to irradiate, and factor XIII product B2 (being shown in Table 2) can be obtained.
(17) the factor XIII freeze-dried products in step (16) are dispensed, vacuum degree measurement and after rolling lid,
98~100 DEG C of water-baths carry out xeothermic virus to the non-lipid-coated virus in factor XIII product under conditions of keeping the temperature 30 minutes
Inactivation；
(18) the factor XIII product after step (17) xeothermic inactivation is carried out cobalt -60 to irradiate, obtains medical blood coagulation
Factor XIII sterile product.
All operations of 3 technique of embodiment carry out in ten thousand grades of purifying areas, wherein packing and freeze-drying operation are in part hundred
Grade purifying area carries out, and the operation carried out meets clean area operation and requires.Production specification is 1.5ml.
(1) the pig blood 50000ml of aseptic collection is taken；
(2) it is centrifugated, takes the anticoagulant blood plasma 20000ml of supernatant；
(3) 0.3%w/v tributyl phosphate is added to the anticoagulant blood plasma of step (2) resulting supernatant and 1%w/v Tween-80 is made
For virus inactivator, it is virus inactivated 6 hours at 25 DEG C；
By the blood plasma centrifuge separation after inactivation, sediment is discarded, clear liquid is taken；
(4) primary sedimentation: will be cooled in advance 0~4 DEG C through clear liquid obtained by step (3), 5000ml saturated ammonium sulfate solution is added,
The temperature of the saturated ammonium sulfate solution is 0~4 DEG C；
(5) the factor XIII primary sedimentation object for separating and once being settled in collection step (4), with 0~4 DEG C,
Ammonium sulfate washs the factor XIII primary sedimentation object once settled in step (4), discards cleaning solution；
(6) primary dissolution: shredding the factor XIII primary sedimentation object after washing in step (5) and is added primary molten
Agent is solved, under the conditions of 4 DEG C and is stirred, step (5) are collected to obtained factor XIII primary sedimentation object and are dissolved, are removed insoluble
Object collects dissolution clear liquid 2400ml；
(7) insoluble matter in collection step (6), is added an isometric lytic agent, and 25 DEG C of water-bath 1h are removed insoluble
Object collects dissolution clear liquid 1400ml；
(8) secondary settlement: after the dissolution clear liquid 3800ml that merging step (6) (7) obtains is cooled to 0~4 DEG C in advance, to supernatant
It is middle that cold acetic acid is added, pH is adjusted, solid ammonium sulfate is added, the factor XIII in clear liquid is dissolved in sedimentation, the cold acetic acid
Temperature is 0~4 DEG C；
(9) the factor XIII secondary precipitate that secondary dissolution: separating and secondary settlement obtains in collection step (8),
It is added secondary lytic agent 2000ml, under the conditions of 4 DEG C and stirs, factor XIII secondary precipitate is dissolved；
(10) the secondary dissolution clear liquid heating water bath for obtaining step (9) is maintained 3 minutes, is put into rapidly to 53~56 DEG C
4 DEG C are cooled in ice bath, precipitation and separation collects supernatant；
(11) it settles three times: in the supernatant obtained to step (10), solid ammonium sulfate is added, settle solidifying in supernatant
Blood factor XIII；
(12) it dissolves: separating three times and the factor XIII settled three times in collection step (11) precipitates three times
Object, is added lytic agent three times, under the conditions of 4 DEG C and stirs, by factor XIII, sediment dissolves three times；
(13) DEAE column on the clear liquid of dissolution three times for obtaining step (12), gradient elution collect active part；
(14) freeze drying protectant is added in the active eluent obtained to step (13), is lyophilized, obtains coagulation factor
XIII freeze-dried products.
Using the protein concentration C3 (table 1) in Bradford method detection eluent；
(15) it according to the testing result in (14), uses PEG to be concentrated into the protein concentration in solution as 5mg/ml, is added and protects
Agent is protected, at being grouped as in preparation are as follows:
Factor XIII 5mg/ml
Glucose 2mg/ml
Glycine 2mg/ml
(16) it dispenses, every packing 1.5ml, is lyophilized after the completion of packing, freeze-drying carries out rolling lid immediately after offeing for sale, finally
Cobalt -60 is carried out to dried frozen aquatic products to irradiate, and factor XIII product B3 (table 2) can be obtained.
(17) the factor XIII freeze-dried products in step (16) are dispensed, vacuum degree measurement and after rolling lid,
98~100 DEG C of water-baths carry out xeothermic virus to the non-lipid-coated virus in factor XIII product under conditions of keeping the temperature 30 minutes
Inactivation；
(18) by step (17) heat inactivation after factor XIII product carry out cobalt -60 irradiate, obtain medical blood coagulation because
Sub- XIII sterile product.
Embodiment 4 takes standard people's blood of human blood coagulation XIII shortage fibrinogen or blood plasma, different dilutions respectively
Slurry or test sample (1:2~1:256), human thrombin/CaCl2Solution (uses 0.05mol/L CaCl2Redissolve freeze dried human zymoplasm extremely
50IU/ml) each 0.1ml is mixed, that is, forms grumeleuse.37 DEG C: heat preservation 60 minutes adds 1% chloroacetic acid of lml, shakes on the mixer
It shakes, bottom grumeleuse is made to float, shake 1 time within every 10 minutes, observe insoluble grumeleuse.The standard that grumeleuse does not dissolve also is recorded after 30 minutes
The greatest dilution of blood plasma and test sample.Human blood coagulation XIII potency in sample is calculated as follows, 1.0U/ml should be not less than.
Grumeleuse or suspended matter greatest dilution/standard plasma observable can be observed in human blood coagulation XIII potency (U/ml)=test sample
To grumeleuse or suspended matter greatest dilution.For standard plasma FXIII activity based on 1U/ml, the FX III that sample to be tested can be obtained is living
Property value, the results are shown in Table 1.
Embodiment 5 is according to the 5th method in 2015 editions four general rules " protein determination " of the Pharmacopoeia of the People's Republic of China
" Coomassie Brilliant Blue " detects the protein concentration of factor XIII, and the results are shown in Table 1.Referring to " the People's Republic of China (PRC)
Pharmacopeia " 2015 editions three page 274~275 of related " human fibrinogen " standard, to " pyrogen " in product, " tricresyl phosphate
The detections such as butyl ester " and " Tween-80 " are detected, and the results are shown in Table 2.
Table 1
Table 2
Remarks: medical factor XIII freeze-dried products are using 1ml distilled water as double solvents.
In conclusion the beneficial effect of the preparation method of factor XIII provided by the invention is: adding in dissolution
Enter different lytic agents, while making the factor XIII dissolution in precipitating abundant while greatly shortening dissolution time, reduces
Bacteria breed；Extraction efficiency is high, and every liter of blood plasma can extract 10 milligrams or more of factor XIII；Factor XIII obtained
In freeze-dried products, factor XIII purity is 90% or more；It is easy to operate, it is with short production cycle, it can be completed within 2 day time
The preparation of factor XIII；The redissolution time of factor XIII freeze-dried products obtained is short, and stability is good, the holding time
It is long.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalents made by bright specification and accompanying drawing content are applied directly or indirectly in relevant technical field, similarly include
In scope of patent protection of the invention.
Claims (6)
1. the preparation method of factor XIII, which comprises the steps of:
Step 1 is centrifugated after anti-coagulants is added into blood, takes the anticoagulant blood plasma of supernatant；
Tributyl phosphate and Tween-80 is added as virus inactivator in step 2, Xiang Shangqing anticoagulation slurry, and auxiliary agent is added, into
Row S/D inactivation of virus, the blood plasma after must inactivating；
Blood plasma after inactivation is centrifuged by step 3, takes clear liquid；
Step 3 gained clear liquid is cooled to 0~4 DEG C by step 4 in advance, after 0~4 DEG C of saturated ammonium sulfate solution is added, takes precipitating；
Step 5 washs precipitating obtained by step 4 with 0~4 DEG C of saturated ammonium sulfate solution, and the precipitating after washing is crushed and is added
Enter 0~4 DEG C of a lytic agent and stir, take clear liquid, a lytic agent is Klorvess Liquid；
Step 5 gained clear liquid is cooled to 0~4 DEG C by step 6 in advance, and the acetic acid volume fraction for being added 0~4 DEG C is 94%~96%
After acetic acid solution to pH value of solution is 5.2~6.5, it is 15%~20% that ammonium sulfate solids to solution saturation degree, which is added, takes precipitating；
0~4 DEG C of secondary lytic agent is added into precipitating obtained by step 6 and stirs for step 7, takes clear liquid, the secondary lytic agent
For the buffer containing sodium chloride, buffering in the secondary lytic agent is to for Tris-HCl, disodium hydrogen phosphate-sodium dihydrogen phosphate
Any one of with citrate-phosphate；
Step 8, by step 7 gained clear liquid heating water bath to 53~56 DEG C, be cooled to 0~4 DEG C after maintaining 160~200 seconds, take clear
Liquid；
Step 9, into step 8 gained clear liquid, addition ammonium sulfate solids to solution saturation degree is 35%~40%, takes precipitating；
0~4 DEG C of lytic agent three times is added into precipitating obtained by step 9 and stirs for step 10, takes clear liquid, described to dissolve three times
Agent is the Tris-HCl buffer containing potassium chloride；
Step 11 will be freeze-dried after step 10 gained clear liquid gradient elution to get factor XIII freeze-dried products；
The concentration of potassium chloride is 0.1~0.2mol/L in lytic agent；The concentration of sodium chloride in the secondary lytic agent
For 0.1~0.2mol/L, the concentration buffered pair is 0.01~0.05mol/L, and the pH value of the secondary lytic agent is 7.0~7.5；
The glucose that auxiliary agent in step 2 is 0.2mol/L.
2. the preparation method of factor XIII according to claim 1, it is characterised in that: the blood is aseptic collection
Pig blood or human blood.
3. the preparation method of factor XIII according to claim 1, it is characterised in that: it is dry to carry out freezing in step 11
Freeze drying protectant is added in solution after dry forward direction gradient elution, the solute of the freeze drying protectant is selected from polyhydroxy carbon hydrate
One or more of object, trisodium citrate, sodium chloride, amino acid.
4. the preparation method of factor XIII according to claim 3, it is characterised in that: the freeze drying protectant it is molten
Matter includes polyhydroxy carbohydrate, trisodium citrate, sodium chloride and amino acid；In the freeze drying protectant, polyhydroxy carbon water
The concentration of compound is 0.1%~5%w/v, and the concentration of sodium chloride is 0.1%~0.9%w/v, and the concentration of amino acid is 0.1%
~5%w/v.
5. the preparation method of factor XIII according to claim 3, it is characterised in that: the polyhydroxy carbon hydrate
Object is selected from one or more of sucrose, trehalose, glucose, mannitol, sorbierite；
The amino acid be selected from glutamic acid, lysine, alanine, histidine, methionine, glycine, arginine, L-Leu,
One or more of l-Isoleucine.
6. the preparation method of factor XIII according to claim 1, it is characterised in that: after step 11 also successively
Include:
Step 12 is dispensed the factor XIII freeze-dried products in step 11, vacuum degree measurement and after rolling lid, 98~
100 DEG C of water-baths carry out xeothermic virus to the non-lipid-coated virus in factor XIII product under conditions of keeping the temperature 30 minutes and go out
It is living；
Step 13, by after inactivation xeothermic in step 12 factor XIII product carry out cobalt -60 irradiate, obtain medical blood coagulation because
Sub- XIII sterile product.
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| CN107167613B (en) * | 2017-06-22 | 2018-10-02 | 深圳清华大学研究院 | Albumen spotting buffer for plasma gold chip |
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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 |