CN110016471A - Recombinant ancrod enzyme and commercial scale preparation and purification method and combinations thereof - Google Patents
Recombinant ancrod enzyme and commercial scale preparation and purification method and combinations thereof Download PDFInfo
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- CN110016471A CN110016471A CN201910286716.5A CN201910286716A CN110016471A CN 110016471 A CN110016471 A CN 110016471A CN 201910286716 A CN201910286716 A CN 201910286716A CN 110016471 A CN110016471 A CN 110016471A
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- ancrod
- recombinant
- pbs
- column
- enzyme
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- A61K38/00—Medicinal preparations containing peptides
Abstract
It is related to recombinant ancrod enzyme and commercial scale preparation and purification method and combinations thereof.Specifically, which has amino acid sequence shown in specification single-stranded, which is 39~41KDa of average molecular weight, 33~52KDa of molecular weight ranges, the glycoprotein for modifying sugared content > 19%.Further relate to the preparation and purification of the recombinant ancrod enzyme and the composition of their injection form.Recombinant ancrod enzyme of the present invention is presented and the significantly superior performance of natural ancrod.The method of the present invention can obtain the recombinant ancrod enzyme of high expression quantity with industrial production specification, and yield can reach every milliliter of fermentation liquid 200IU/ml or more, and specific activity can reach 1000IU/mg.Recombinant ancrod enzyme of the present invention has more complicated and more complete level of glycosylation, and stability significantly increases, and solves the problems, such as that ancrod expression quantity is low, industrialization is difficult.Excellent pharmaceutical property is presented in gained injecta composition.
Description
Technical field
The present invention relates to a kind of recombinant ancrod enzyme, preparation method and application and their pharmaceutical compositions.More
Body, the present invention relates to using technique for gene engineering, using mammalian CHO cells culture production method preparation and reorganization albumen,
Then by purifying process, the recombinant ancrod enzyme of high expression quantity can be readily obtained with industrial production specification, yield can
Reach every milliliter of fermentation liquid 200IU/ml or more, specific activity can reach 1000IU/mg.The recombinant ancrod enzyme that the present invention obtains
With more complicated and more complete level of glycosylation, stability is significantly increased, and solves that such existing product expression amount is low, industry
Change difficult problem, the invention further relates to recombinant ancrod enzymes to treat the application in acute cerebral infarction.
Background technique
Ancrod (Ancrod) is refered in particular to from Malaysia red mouth pallas pit viper (Malayan pit viper, Calloselasma
Rhodostoma) the batroxobin isolated and purified out in snake venom is serine protein hydrolase.The blood of mammal can be hydrolyzed
Pulp fibres proteinogen is allowed to be changed into fibrin, to influence the bleeding coagulation process of animal.Snake venom thrombin-like enzyme specificity
Substrate specificity is fibrinogen, unlike fibrin ferment, the α chain of its cutting fibre proteinogen, without acting on β chain.
When it hydrolyzes the Arg in plasma fibrinogen α chain16When-GLy keys, fibrinopeptide A can be released, thus rapidly
Fibrinogen in blood is changed into fibroblast cells, since snake venom thrombin-like enzyme does not activate fibrin stabilizing factor, these lifes
At fibrin monomer be not cross-linked to form the netted structure of firm fibrin, be easy to by intracorporal fibrin hydrolase institute
Hydrolysis, therefore can be with inhibition thrombosis, having reduces blood viscosity, inhibits erythrocyte agglutination, sedimentation, the blood vessel for enhancing red blood cell
Passability and deformability reduce the effects of vascular resistence and improvement microcirculation.Keep thrombolytic effect quick, ishemic part function
Restore, to achieve the effect that treat and prevent recurrence.
The kind of snake venom thrombin-like enzyme preparation listing has: from the ancrod of the red mouth viper venom of Malaysia
(Ancrod, Viprinex, Arwin)；From the Batroxobin of Brazilian spearhead pallas pit viper (Bothrops moojeni) snake venom
(Batroxobin)；From the Defibrase (Defibrase) of agkistrodon acutus (Agkistrodon acutus) snake venom.1985
To 2000, there were the different venin-derived multicomponent preparation circulations containing snake venom thrombin-like enzyme in China, most of to be known as pallas pit viper
Ahylysantinfarctase.From 1997, homemade defibrase was instead of ahylysantinfarctase.
Natural snake venom thrombin-like enzyme is glycoprotein, and height and the specific activity of level of glycosylation are positively correlated, with activity
Stability is positively correlated.
Belong to national secondary endangered protection animal since pallas pit viper has been included in, the resource of natural snake venom is extremely limited, therefore adopts
There is realistic meaning with technique for gene engineering expression snake venom thrombin-like enzyme.Japanese scholars are most earlier than 1987
(J.Biol.Chem.262:3132-3135) by batroxobin gene in expression in escherichia coli success, Lee Chinese scholar recruits hair etc.
(Chinese patent Authorization Notice No. CN100564532C) expresses the high batroxobin protein of bioactive in saccharomycete, and yield reaches
To every milliliter of 14 Batroxobin unit (14BU/ml) of fermentation liquid, specific activity 1400BU/mg.
The amino acid sequence of natural ancrod is early in 1992 by American scholar William Burkhart etc.
(William Burkhart,et al,Amino acid sequence determination of Ancrod,the
Thrombin-like α-fibrinogenase from the venom of Akistrodon rhodostoma,
Federation of European Biochemical Societies, 1992, Vol 297 (3): 297-301) in its research
Play-by-play has been carried out in paper, is a kind of glycoprotein containing 234 amino acid.234 amino acid sequences of the albumen
It has been documented in the document of the William Burkhart, and has included global protein resource library known in the art
In http://www.uniprot.org, referring specifically to library network address http://www.uniprot.org/uniprot/
Information documented by P26324.The natural ancrod Ancrod of this 234 amino acid composition be from
Purifying obtains in Akistrodon rhodostoma snake venom, due to well-known, such as growing environment, the snake of snake
Age, the season of gathering venom etc. and snake venom raw material make a variation the limit of the influence of impurity etc. and raw material yield in related factor and snake venom
System, for carrying out this natural ancrod of industrialized production with stable scale so that it is with medicament safely, effectively, controllably
It is exceedingly difficult for being smoothly applied to clinic.
The application for a patent for invention 2018104523815 that discloses of the applicant is disclosed and is obtained so that commercial production scale is stable
The ancrod especially recombinant ancrod enzyme of high expression quantity method, however these methods still have etc. and to improve.
Therefore, this field still expects there is the method for preparing ancrod, especially obtains so that commercial production scale is stable
The method for obtaining the ancrod especially recombinant ancrod enzyme of high expression quantity.
Summary of the invention
The present invention is intended to provide a kind of recombinant ancrod enzyme, preparation method and applications, are to provide one kind especially with industry
The method of the recombinant ancrod enzyme of the stable high expression quantity of acquisition of production scale.The recombinant protein has more complicated and more complete
Level of glycosylation solves the problems, such as that the low industrialization of such existing product expression amount is difficult.Another object of the present invention also resides in offer
A kind of recombinant ancrod enzyme is treating the application in acute cerebral infarction.Recombinant ancrod enzyme of the invention has stronger external activity
With higher stability.
The first aspect of the present invention provides a kind of recombinant ancrod enzyme, which is by the end N- and C-
Terminal amino acid residue is respectively that one of 234 amino acid residues composition of valine (V) and proline (P) has following sequence
What is arranged is single-stranded:
VIGGDECNIN EHRFLVAVYE GTNWTFICGG VLIHPEWVIT AEHCARRRMN
LVFGMHRKSE KFDDEQERYP KKRYFIRCNK TRTSWDEDIM LIRLNKPVNN
SEHIAPLSLP SNPPIVGSDC RVMGWGSINR RIDVLSDEPR CANINLHNFT
MCHGLFRKMP KKGRVLCAGD LRGRRDSCNS DSGGPLICNE ELHGIVARGP
NPCAQPNKPA LYTSIYDYRD WVNNVIAGNA TCSP。
Recombinant ancrod enzyme according to a first aspect of the present invention is a kind of sugared egg that average molecular weight is 39~41KDa
White, the 5 N-Link glycosylation binding site for including is located at Asn23-Trp24-Thr25, Asn79-Lys80-Thr81, Asn99-
Asn100-Ser101, Asn148-Phe149-Thr150And Asn229-Ala230-Thr231On the asparagine residue at place, and the glycoprotein
Isoelectric point be 4.5-5.5.
It is 33~52KDa that recombinant ancrod enzyme according to a first aspect of the present invention, which is a kind of range of molecular weight distributions,
Glycoprotein.
Recombinant ancrod enzyme according to a first aspect of the present invention is a kind of sugared egg that modification sugared content is 19~49%
It is white.It is measured through careful analysis, it is known that the modification sugar being connected on glycosylation site includes galactolipin, trehalose, sialic acid
Etc. several, their combinations on amino acid chain are shown in stacked antenna shape structure.
It should be noted that being 26570Da by the single-stranded molecular weight of ancrod that 234 amino acid residues form；This
The recombinant ancrod enzyme glycoprotein that inventive method is prepared measures, mean molecule through liquid chromatography-mass spectrometry (LC-MS)
Amount is 39~41kDa, and range of molecular weight distributions is 33~52kDa (distribution span reaches 17kDa or more).In addition, ginseng of the present invention
The acquisition methods of natural ancrod glycoprotein shown in lane 3 according to the A of William Burkhart document Fig.1, using horse
Carrying out the red mouth viper venom in West Asia is raw material, obtains the i.e. natural ancrod of Natively glycosylated albumen, through being surveyed with LC-MS method of the present invention
Fixed/to calculate, average molecular weight is 37.5~38kDa, and range of molecular weight distributions is 32~41kDa (distribution span about 9kDa)；
The discrepant reason of result shown in the LC-MS measurement result and William Burkhart document is the detection method used not
Together, literature method be it is a kind of under technical conditions at that time attainable relatively accurate result.The present invention is prepared as a result,
Recombinant ancrod enzyme glycoprotein 234 amino acid residues composition the single-stranded amino acid with natural ancrod of ancrod
Residue is single-stranded identical, but (may be the amount of access sugar and the knot of access since the glycosylation modified sugar connected thereon is different
Structure/mode difference causes), recombinant protein ratio of the present invention has more complicated and more complete level of glycosylation in native protein,
The stability and biological activity significantly increased can be presented, therefore essentially present invention gained recombinant ancrod enzyme is complete
It is different from natural ancrod.The glycosylated complexity of ancrod and integrality can be on the molecular chain conformations of glycoprotein
Reflect: molecular weight is bigger, then degree of glycosylation is higher, glycosylate branch and the complexity of side chain is also higher, accordingly
Glycoprotein stability and biological activity are also higher；And the glycoprotein of more high stability makes with the work of high yield, high expression quantity
Industry scale and level prepare glycoprotein and are possibly realized.In the present invention, if not otherwise specified, the molecular weight that is related to and related to this
Parameter such as range of molecular weight distributions, average molecular weight etc., be to measure and calculate by liquid chromatography-mass spectrometry (LC-MS)
Obtained mass spectrometric determination in other words is simultaneously calculated.
Further, second aspect of the present invention provides a kind of method of preparation and reorganization ancrod, and this method includes such as
Lower step:
(1) expression vector and recombinant plasmid of building coding recombinant ancrod enzyme:
For the carrier of secretion expression's recombinant protein in mammalian cells, (such as ATK-V03-aSP is carried for building
Body, the construction method of this expression vector are conventional, are also that as known to those skilled in the art, can also pass through business way
Diameter obtains；It is the ATK-V03-aSP carrier obtained by commercial sources building, at this in one embodiment of the invention
It invents in an example, is constructed by AutekBio；In one embodiment, which contains one
CMV promoter, a BGH pA tailing signal and the Neo selection markers base for screening stable expression cell in eukaryocyte
Cause；In one embodiment, which also contains the replication origin and ammonia benzyl from plasmid pMB1
Ampicillin resistance gene can be screened and be replicated in Escherichia coli), and accordingly construct ancrod recombinant plasmid (such as
ATK-V03-aSP-Ancrod, such as constructed by commercial sources)；
(2) recombinant ancrod enzyme stablizes expression in mammalian host cell:
Expression vector containing ancrod albumen is transfected into mammalian host cell, screens and stablizes expression purpose egg
White cell strain；
(3) cell culture produces recombinant ancrod enzyme:
The stable cell line screened is transferred to reactor tank and carries out large-scale culture, when cell density reaches maximum, is received
Obtain culture.
Typically, in this step, using CHO (ATK-CHO-SX) conduct of serum free medium (SFM) suspension culture
Host cell, condition of culture are 37 DEG C, 5%CO2, 120rpm (in the present invention, under this condition, when every 2-3 days secondary cultures,
Cell doubling time (PDT) about 17 hours；Highest viable cell density is up to 1 × 10 when batch cultivation7A cell/mL)；
(4) purifying of recombinant ancrod enzyme:
(41) it is pre-processed before chromatographic column on: the culture solution containing recombinant ancrod enzyme obtained by previous step is filtered
Except cell fragment, filtrate is collected, is then concentrated by ultrafiltration, molecular cut off 30KDa diaphragm plate ultrafiltration to the 1/8~1/ of original volume
10；
(42) it affinity column: is purified and (for example, in an embodiment party of the invention, is used using affinity column
Heparin Sepharose Fast Flow, GE) purifying, use NaCl, 0.05mol/L of 0.1~0.4mol/L of salinity
The elution requirement of the buffer of Tris-HCl pH7.0~7.5, elutes and collects active component；
(43) reversed-phase liquid chromatography purifies: using preparative reversed-phase liquid chromatography technology, purifies recombination peace obtained above
Crow zymoprotein, using the method for ethanol-water system gradient elution, it is miscellaneous further to remove other using the difference of polarity size
Matter；
(44) affinity column chromatography: the recombinant protein that previous step is collected is using affinity column ((for example, the present invention one
In a embodiment party, using Heparin Sepharose Fast Flow, GE) purifying, it removes ethyl alcohol and realizes the dense of recombinant protein
Contracting.
Method according to a second aspect of the present invention, wherein in step (43), using C4 preparative scale chromatography column, using following table institute
Show gradient elution:
| Time/min | A | B |
| 0~50 | 90→30 | 10→70 |
| 50~60 | 30 | 70 |
| 61.~71 | 90 | 10 |
Wherein mobile phase A is the phosphate aqueous solution of pH2.3, and Mobile phase B is ethyl alcohol, Detection wavelength 214nm, elution flow rate
For 20mL/min.In one embodiment, 1% propylene glycol is also additionally added in the mobile phase A and Mobile phase B.
Method according to a second aspect of the present invention, the wherein affinity column chromatography in step (44) and step (42) operating condition
It is identical.
Method according to a second aspect of the present invention, wherein in step (1), the gene expression of building coding recombinant ancrod enzyme
The step of carrier further include: chemical synthesis complete ancrod glycoprotein DNA encoding sequence, with T4DNA ligase by this
The DNA encoding sequence of synthesis is connected into ATK-V03-aSP carrier, obtains DNA recombinant expression plasmid ATK-V03-aSP-
Ancrod carries out the correctness of insetion sequence in verifying recombinant plasmid by DNA sequencing.
Method according to a second aspect of the present invention, wherein the mammalian cell expression vector is ATK-V03-aSP.
Method according to a second aspect of the present invention, wherein the method for transfection described in step (2) is that the electroporation of cell turns
Dyeing method (electroporation).
Method according to a second aspect of the present invention, wherein the mammalian host cell is selected from: CHO (Chinese
Hamster Ovary, Chinese hamster ovary cell), HEK293, BHK, NS0 and Sp2/0 cell.Preferably Chinese hamster ovary celI；More
It is preferred that having tamed the DHFR deficient CHO- suspension cell (DHFR-CHO) for adapting to suspension growth in serum free medium.
Method according to a second aspect of the present invention, wherein in step (3), the composition of the serum free medium (SFM) are as follows:
Glucose 0.6%, the Hepes of NaHCO3,5mM of glutamine 2mM, 3mM, 2.5 μ g/ml of insulin, transferrins 100ng/
Ml, 60 μM of butanediamine, sodium selenate 30nM, 50 μ g/ml of penicillin, 50 μ g/ml of streptomysin, DF12 add to 100ml.
The step of method according to a second aspect of the present invention, wherein cell culture produces recombinant ancrod enzyme in step (3)
Are as follows: the stable cell line for screening front is transferred to cell effect tank and carries out large-scale culture, in particular, by cell culture
The optimization of condition obtains the cell culture fluid of high expression recombinant ancrod enzyme.Cell culture processes of the invention can be realized highly dense
The glycosylation complexity for spending cell culture, quality and yield promotion and recombinant protein improves.In one embodiment, institute
The optimization for stating cell culture condition includes cooling cultivation, specifically, when cell density reaches 1 × 107The maximum of a/mL is close
When spending, temperature is down to 33 DEG C by 37 DEG C, culture is not until expression yield is further added by and (reaches most in cell density at such a temperature
1 × 10 changed greatly7When a/mL, continues culture under the conditions of 37 DEG C and be helpless to continuing growing for yield；However it has been found that
In the case of reaching this maximal density, 33 DEG C are down to by 37 DEG C of cultivation temperature, can continue to increase yield until cell density increases
About 1.8 times of level is to about 2.8 × 107A/mL or more).The activity level and recombination egg of expression albumen can be improved in this method
White cumulative production.Therefore, in one embodiment of the invention, when culture to cell density reaches maximum in step (3)
When, so that cultivation temperature is down to 33 DEG C by 37 DEG C, continues culture until cell density is not further added by.
In an other embodiment, the optimization method of the cell culture condition further include basal medium i.e.
Special additive is added in supplement in serum free medium, it is preferable that 100 μM of copper chloride, N- are additionally added in serum free medium
Acetyl group-D- epichitosamine (ManNAc) 2mM, 50 μM of potassium pyrophosphate.This method can make the glycosylation journey of recombinant ancrod enzyme
Degree dramatically increases, and the glycosylation complexities such as branch and side chain obviously increase.Therefore, in one embodiment of the invention,
100 μM of copper chloride, N- acetyl group-D- epichitosamine 2mM, burnt phosphorus have been augmented in serum free medium used in step (3)
50 μM of sour potassium.The present invention has found in methodological study test, and into serum free medium, it is one of any to augment above-mentioned three
When either any two or three do not augment all, it can not effectively increase the degree of glycosylation and complexity of recombinant ancrod enzyme
Degree has above-mentioned three only with above-mentioned amount while when increasing, could effectively increase recombinant ancrod enzyme degree of glycosylation and
Complexity, and then improve the performance of glycoprotein.
Method according to a second aspect of the present invention, the wherein method of step (3) are as follows: be transferred to the stable cell line screened
Reactor tank, with having augmented 100 μM of copper chloride, N- acetyl group-D- epichitosamine (ManNAc) 2mM, 50 μM of potassium pyrophosphate of nothing
Blood serum medium, in 37 DEG C of condition of culture, 5%CO2, cultivate under the conditions of 120rpm and be not further added by cell density, then make to train
Support temperature be down to after 33 DEG C by 37 DEG C continue culture be not further added by cell density after, harvest culture.
In the method for the present invention in step (4), in the purifying preparation step of recombinant ancrod enzyme of the present invention, the present invention is using special
Recombinant ancrod enzyme is prepared in fixed purification by chromatography, the chromatography method simple process, is easy to amplify production, mild condition,
Obtained recombinant ancrod enzyme purity reaches 98.5% or more, and stability is good, and activity is high.
Further, third aspect present invention provides recombinant ancrod enzyme of the present invention in treatment Patients With Acute Cerebral Infarction disease
In application.Such as acute cerebral infarction and improve various occlusive vascular diseases.
Further, fourth aspect present invention provides a kind of side that cell culture is carried out when producing recombinant ancrod enzyme
Method, this method comprises the following steps:
The stable cell line screened is transferred to reactor tank and carries out large-scale culture, when cell density reaches maximum, is received
Obtain culture.Typically, in this step, using CHO (ATK-CHO-SX) conduct of serum free medium (SFM) suspension culture
Host cell, condition of culture are 37 DEG C, 5%CO2, 120rpm (in the present invention, under this condition, when every 2-3 days secondary cultures,
Cell doubling time (PDT) about 17 hours；Highest viable cell density is up to 1 × 10 when batch cultivation7A cell/mL)；
Method according to a fourth aspect of the present invention, wherein the composition of the serum free medium (SFM) are as follows: glucose
0.6%, the Hepes of NaHCO3,5mM of glutamine 2mM, 3mM, 2.5 μ g/ml of insulin, transferrins 100ng/ml, fourth two
60 μM of amine, sodium selenate 30nM, 50 μ g/ml of penicillin, 50 μ g/ml of streptomysin, DF12 add to 100ml.
It is anti-to the steps include: that the stable cell line for screening front is transferred to cell for method according to a fourth aspect of the present invention
Tank is answered to carry out large-scale culture, in particular, obtaining the thin of high expression recombinant ancrod enzyme by the optimization to cell culture condition
Born of the same parents' culture solution.Cell culture processes of the invention can realize concentration cultivation, quality and yield promotion and recombinant protein
Glycosylation complexity improve.In one embodiment, the optimization of the cell culture condition includes cooling cultivation, tool
For body, when cell density reaches 1 × 107When the maximal density of a/mL, temperature is down to 33 DEG C by 37 DEG C, at such a temperature
Culture is not until expression yield is further added by and (reaches maximized 1 × 10 in cell density7When a/mL, continue under the conditions of 37 DEG C
Culture is helpless to continuing growing for yield；However it has been found that when reaching this maximal density, by 37 DEG C of cultivation temperature
33 DEG C are down to, can continue to increase yield until about 1.8 times of cell density increase of level is to about 2.8 × 107A/mL or more).
The activity level of expression albumen and the cumulative production of recombinant protein can be improved in this method.Therefore, in an implementation of the invention
In scheme, in step (3) when culture reaches maximum to cell density, so that cultivation temperature is down to 33 DEG C by 37 DEG C, continue to cultivate
Until cell density is not further added by.
Method according to a fourth aspect of the present invention, wherein the optimization method of the cell culture condition further includes training on basis
Special additive is added in supplement in feeding base, that is, serum free medium, it is preferable that copper chloride 100 is additionally added in serum free medium
μM, N- acetyl group-D- epichitosamine (ManNAc) 2mM, 50 μM of potassium pyrophosphate.This method can make the glycosyl of recombinant ancrod enzyme
Change degree dramatically increases, and the glycosylation complexities such as branch and side chain obviously increase.Therefore, in one embodiment of the invention
In, 100 μM of copper chloride, N- acetyl group-D- epichitosamine 2mM, coke have been augmented in serum free medium used in step (3)
50 μM of potassium phosphate.The present invention has found in methodological study test, into serum free medium, augment above-mentioned three it is any it
One or any two or three when not augmenting all, it can not effectively increase the degree of glycosylation of recombinant ancrod enzyme and multiple
Miscellaneous degree has above-mentioned three only with above-mentioned amount while when increasing, and could effectively increase the degree of glycosylation of recombinant ancrod enzyme
And complexity, and then improve the performance of glycoprotein.
Method according to a fourth aspect of the present invention the steps include: that the stable cell line that will be screened is transferred to reactor tank, with increasing
100 μM of copper chloride, N- acetyl group-D- epichitosamine (ManNAc) 2mM, 50 μM of potassium pyrophosphate of serum free medium have been mended,
In 37 DEG C of condition of culture, 5%CO2, cultivate under the conditions of 120rpm and be not further added by cell density, then make cultivation temperature by 37 DEG C
Continue after being down to 33 DEG C culture be not further added by cell density after, harvest culture.
Further, fifth aspect present invention provides a kind of method purified to recombination ancrod, this method
Include the following steps:
(41) it is pre-processed before chromatographic column on: the culture solution containing recombinant ancrod enzyme obtained by previous step is filtered
Except cell fragment, filtrate is collected, is then concentrated by ultrafiltration, molecular cut off 30KDa diaphragm plate ultrafiltration to the 1/8~1/ of original volume
10；
(42) it affinity column: is purified and (for example, in an embodiment party of the invention, is used using affinity column
Heparin Sepharose Fast Flow, GE) purifying, use NaCl, 0.05mol/L of 0.1~0.4mol/L of salinity
The elution requirement of the buffer of Tris-HCl pH7.0~7.5, elutes and collects active component；
(43) reversed-phase liquid chromatography purifies: using preparative reversed-phase liquid chromatography technology, purifies recombination peace obtained above
Crow zymoprotein, using the method for ethanol-water system gradient elution, it is miscellaneous further to remove other using the difference of polarity size
Matter；
(44) affinity column chromatography: the recombinant protein that previous step is collected is using affinity column ((for example, the present invention one
In a embodiment party, using Heparin Sepharose Fast Flow, GE) purifying, it removes ethyl alcohol and realizes the dense of recombinant protein
Contracting.
Method according to a fifth aspect of the present invention, wherein in step (43), using C4 preparative scale chromatography column, using following table institute
Show gradient elution:
| Time/min | A | B |
| 0~50 | 90→30 | 10→70 |
| 50~60 | 30 | 70 |
| 61.~71 | 90 | 10 |
Wherein mobile phase A is the phosphate aqueous solution of pH2.3, and Mobile phase B is ethyl alcohol, Detection wavelength 214nm, elution flow rate
For 20mL/min.In one embodiment, 1% propylene glycol is also additionally added in the mobile phase A and Mobile phase B.
Method according to a fifth aspect of the present invention, the wherein affinity column chromatography in step (44) and step (42) operating condition
It is identical.
Further, sixth aspect present invention provides the method for purifying recombinant ancrod enzyme comprising what will be obtained
Recombinant ancrod zymoprotein after purification, carries out Heparin Sepharose 6Fast with reversed-phase liquid chromatography in the following way
The purifying of Flow gel affinity column chromatography:
Chromatographic column (column diameter 50mm, length 12cm), filler are Heparin Sepharose 6Fast Flow, PBS-
LYS buffer includes: 0.2% sodium chloride, 0.1% L-Lysine mono Hydrochloride, 15mmol/L sodium dihydrogen phosphate and adding sodium hydroxide tune
PH6.8, appropriate amount of water to full dose are saved, sample is the active main peak in step (3), it is diluted to 1000ml with PBS-LYS buffer, on
Sample, column flow rate 2mL/min.It after loading, is balanced with PBS-LYS buffer to baseline, is eluted with PBS-LYS buffer, collected
Active component.Impurity composition is eluted with PBS-LYS buffer, then rinses chromatographic column 1000ml with PBS-LYS buffer, is regenerated,
It is spare, obtain recombinant ancrod proenzyme liquid.
Method according to a sixth aspect of the present invention provides the method purified to recombination ancrod, this method
Include the following steps:
(41) it is pre-processed before chromatographic column on: the culture solution containing recombinant ancrod enzyme is filtered removal cell fragment,
Filtrate is collected, is then concentrated by ultrafiltration, molecular cut off 30KDa diaphragm plate ultrafiltration to the 1/8~1/10 of original volume；
(42) affinity column: purification is carried out using affinity column, uses 0.1~0.4mol/L's of salinity
The elution requirement of the buffer of Tris-HCl pH7.0~7.5 of NaCl, 0.05mol/L, elutes and collects active component；
(43) reversed-phase liquid chromatography purifies: using preparative reversed-phase liquid chromatography technology, purifies recombination peace obtained above
Crow zymoprotein, using the method for ethanol-water system gradient elution, it is miscellaneous further to remove other using the difference of polarity size
Matter；
(44) affinity column chromatography: the recombinant protein that previous step is collected uses affinity chromatography column purification, and removal ethyl alcohol is simultaneously
Realize the concentration of recombinant protein,
Wherein, the operation of step (44) is as follows:
Chromatographic column (column diameter 50mm, length 12cm), filler are Heparin Sepharose 6Fast Flow, PBS-
LYS buffer includes: 0.2% sodium chloride, 0.1% L-Lysine mono Hydrochloride, 15mmol/L sodium dihydrogen phosphate and adding sodium hydroxide tune
PH6.8, appropriate amount of water to full dose are saved, sample is the active main peak in step (3), it is diluted to 1000ml with PBS-LYS buffer, on
Sample, column flow rate 2mL/min.It after loading, is balanced with PBS-LYS buffer to baseline, is eluted with PBS-LYS buffer, collected
Active component.Impurity composition is eluted with PBS-LYS buffer, then rinses chromatographic column 1000ml with PBS-LYS buffer, is regenerated,
It is spare, obtain recombinant ancrod proenzyme liquid.
Method according to a sixth aspect of the present invention, in above-mentioned steps (43), using C4 preparative scale chromatography column, using following table
Shown gradient elution:
| Time/min | A | B |
| 0~50 | 90→30 | 10→70 |
| 50~60 | 30 | 70 |
| 61.~71 | 90 | 10 |
Wherein mobile phase A is the phosphate aqueous solution of pH2.3, and Mobile phase B is ethyl alcohol, Detection wavelength 214nm, elution flow rate
For 20mL/min；Such as 1% propylene glycol is also additionally added in the mobile phase A and Mobile phase B.
Further, seventh aspect present invention provides the method for preparation and reorganization ancrod injection comprising as follows
Step:
(a) purified recombinant ancrod proenzyme liquid (the optional potency for measuring the stoste) is provided；
(b) (such as being diluted to potency 20IU/mL) is diluted with PBS-LYS buffer, is removed with 0.22um filtering with microporous membrane
Bacterium, (such as with every bottle of 1ml) are dispensed into ampulla, and sealing obtains injection.
Method according to a seventh aspect of the present invention, the recombinant ancrod proenzyme liquid of the purifying are prepared into according to following method
It arrives:
(41) it is pre-processed before chromatographic column on: the culture solution containing recombinant ancrod enzyme is filtered removal cell fragment,
Filtrate is collected, is then concentrated by ultrafiltration, molecular cut off 30KDa diaphragm plate ultrafiltration to the 1/8~1/10 of original volume；
(42) affinity column: purification is carried out using affinity column, uses 0.1~0.4mol/L's of salinity
The elution requirement of the buffer of Tris-HCl pH7.0~7.5 of NaCl, 0.05mol/L, elutes and collects active component；
(43) reversed-phase liquid chromatography purifies: using preparative reversed-phase liquid chromatography technology, purifies recombination peace obtained above
Crow zymoprotein, using the method for ethanol-water system gradient elution, it is miscellaneous further to remove other using the difference of polarity size
Matter；
(44) affinity column chromatography: the recombinant protein that previous step is collected uses affinity chromatography column purification, and removal ethyl alcohol is simultaneously
Realize the concentration of recombinant protein,
Wherein, the operation of step (44) is as follows:
Chromatographic column (column diameter 50mm, length 12cm), filler are Heparin Sepharose 6Fast Flow, PBS-
LYS buffer includes: 0.2% sodium chloride, 0.1% L-Lysine mono Hydrochloride, 15mmol/L sodium dihydrogen phosphate and adding sodium hydroxide tune
PH6.8, appropriate amount of water to full dose are saved, sample is the active main peak in step (3), it is diluted to 1000ml with PBS-LYS buffer, on
Sample, column flow rate 2mL/min.It after loading, is balanced with PBS-LYS buffer to baseline, is eluted with PBS-LYS buffer, collected
Active component.Impurity composition is eluted with PBS-LYS buffer, then rinses chromatographic column 1000ml with PBS-LYS buffer, is regenerated,
It is spare, obtain recombinant ancrod proenzyme liquid.
Method according to a seventh aspect of the present invention, in above-mentioned steps (43), using C4 preparative scale chromatography column, using following table
Shown gradient elution:
| Time/min | A | B |
| 0~50 | 90→30 | 10→70 |
| 50~60 | 30 | 70 |
| 61.~71 | 90 | 10 |
Wherein mobile phase A is the phosphate aqueous solution of pH2.3, and Mobile phase B is ethyl alcohol, Detection wavelength 214nm, elution flow rate
For 20mL/min；Such as 1% propylene glycol is also additionally added in the mobile phase A and Mobile phase B.
In the above-mentioned preparation method of the invention the step of, although the specific steps of its description are in certain details or language
In description with the preparation example of following detailed description part described in step different from, however, those skilled in the art
The detailed disclosure of member's full text according to the present invention can summarize approach described above step completely.
Any embodiment in either present invention face can be combined with the other any embodiments of the present invention,
As long as they are not in contradiction.In addition, any technical characteristic can fit in any embodiment of either side of the present invention
For the technical characteristic in the other any embodiments of the present invention, as long as they are not in contradiction.
The invention will be further described below.
All documents recited in the present invention, their full content are incorporated herein by reference, and if these are literary
When offering expressed meaning and the inconsistent present invention, it is subject to statement of the invention.In addition, the various terms that use of the present invention and
Phrase has that well known to a person skilled in the art general senses, nonetheless, the present invention remain desirable at this to these terms and
Phrase is described in more detail and explains, the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute's table of the present invention
Subject to the meaning stated.
In the present invention, term " range of molecular weight distributions " refers in mass spectrometric determination molecular weight, glycoprotein minimum point
Son amount to maximum molecular weight range, such as in Fig. 8 mass spectrogram of the present invention, the corresponding molecular weight in main peak initial position
(33980Da) to the corresponding molecular weight of main peak final position (51840Da) range.Similarly, refer to term " distribution across
Degree " refers to big difference between end value and small end value in range of molecular weight distributions.
The present invention uses Large-scale culture conditions and purification process, and the glycosylation of obtained recombinant ancrod enzyme is more complicated more
Completely, increased activity stability improves, and resists acute cerebral infarction significant in efficacy in animal body, safety evaluation is the result shows that the recombinant protein
Heart, respiratory system, the circulatory system are not influenced, there is good potential applicability in clinical practice.
Recombinant ancrod enzyme is the sugar for being expressed the gene of ancrod in Chinese hamster ovary celI using technique for gene engineering
Albumen, activated centre are serine, have batroxobin activity.Use its average molecular weight of mass spectral analysis for 39K-41kDa, carbon
Hydrate content about 19%~49%.
It is well known that may have unpredictable impurity from the product that natural snake venom extracts, to generate antigenicity, and raw
Yield and quality is uncontrollable, in comparison, recombinant protein its purity, quality controllability, antigenicity, in terms of have it is natural
The incomparable advantage of ancrod, shows wide medical prospect, but at home and abroad there is no recombinant ancrod enzyme medicines so far
Object listing.
Recombinant ancrod enzyme is obtained with gene recombination technology as better choice, and to be prepared with activity in vivo
Recombinant glycoprotein, existing prokaryotic cell (such as Escherichia coli) and yeast expression system cannot achieve industrialization, only
Recombinant protein is expressed in mammalian host cell and is just able to achieve more complete glycosylation, in fact, the glycosylation journey of albumen
The functions such as degree and albumen activity in vivo and half-life period are closely related, and recombinant protein level of glycosylation depends on cell culture condition again
And purifying process.
It is to be most suitable for industry at present using Chinese hamster ovary cell (Chinese hamster ovary celI) in mammalian cell expression system
The host cell of production.The product of expressing cho cell such as Recombinant Human Erythropoietin injection, (CHO is thin for recombinant hepatitis B vaccine
Born of the same parents), recombined human thrombopoietin injection, injection recombinant human urokinase zymogen has been applied to clinic.
There are following features with recombinant ancrod enzyme prepared by the method for the present invention:
(1) by mass spectroscopy, recombinant ancrod enzyme average molecular weight of the present invention is 39~41Kda；(2) present invention recombination
Ancrod contains 19~49% sugar；(3) the 5 N-linked glycosylation sites and sugar that recombinant ancrod enzyme of the present invention contains
Type is complicated.
Detailed description of the invention
Fig. 1: ATK-V03-aSP-Ancrod plasmid map.
Fig. 2: clone CIA8-64-33-95 batch cultivation technique (the cell growth in 10 liters of bioreactors；Vc- is living
Cell density, via- Cell viability).
Fig. 3: the clone CIA8-64-33-95 batch cultivation technique (destination protein expression) in 10 liters of bioreactors.
Fig. 4: clone CIA8-64-33-95 batch cultivation technique (the cell growth in 50L bioreactor；Vc- is living thin
Born of the same parents' density, via- Cell viability).
Fig. 5: the clone CIA8-64-33-95 batch cultivation technique (destination protein expression) in 50L bioreactor.
Fig. 6: first step Heparin Sepharose 6Fast Flow gel affinity column chromatography chromatogram.
Fig. 7: second step reversed-phase liquid chromatography purifies chromatogram.
Fig. 8: the LC-MS figure of recombinant ancrod enzyme glycoprotein.
Specific embodiment
The present invention can be further described by the following examples, however, the scope of the present invention and unlimited
In following embodiments.One of skill in the art, can be with it is understood that under the premise of without departing substantially from the spirit and scope of the present invention
Various change and modification are carried out to the present invention.The present invention carries out the material and test method arrived used in test general
And/or specific description.Although to realize the present invention many materials and operating method used in purpose be it is known in the art that
But the present invention is still described in this detail as much as possible.Following embodiment further illustrates the present invention, rather than limits this hair
It is bright.
The preparation of natural ancrod: 3 institute of lane of A of the present inventor referring to William Burkhart document Fig.1
The acquisition methods for showing natural ancrod glycoprotein use the red mouth viper venom of Malaysia for raw material, obtain Natively glycosylated
Albumen, that is, natural ancrod is used in the present invention according to test.
Embodiment 1: the expression vector of building coding ancrod recombinant protein
The present invention obtains expression vector by commercial sources (AutekBio), although the structure of this kind of expression vector
Construction method be it is known in this field, general, still details are as follows by the present invention, but these purposes being described in detail are not intended to limit this
Invention.
(1) host cell
Host cell is used as using the Chinese hamster ovary celI (ATK-CHO-SX) of serum free suspension culture, condition of culture is 37 DEG C,
5%CO2, 120rpm.When every 2-3 days secondary cultures, cell doubling time (PDT) about 17 hours.Highest is living thin when batch cultivation
Born of the same parents' density is up to 1 × 107A cell/mL.
(2) genetic engineering plasmid
ATK-V03-aSP carrier be AutekBio independently construct for secretion expression in mammalian cells recombinate
The carrier of protein.ATK-V03-aSP carrier contains a CMV promoter, a BGH pA tailing signal and in eukaryon
The Neo riddled basins for stablizing expression cell are screened in cell.ATK-V03-aSP carrier also contains from plasmid pMB1's
Replication origin and ampicillin resistance gene can be screened and be replicated in Escherichia coli.ATK-V03-aSP carrier is main
The information such as the nucleotide sequence in the source of constituent element, each control zone of function and target gene insertion site two sides are shown in Table 1 to table
3。
The main constituent element of table 1:ATK-V03-aSP carrier
| Constituent element | Source | Function |
| PUC replication origin | pMB1 | Starting plasmids replicate in prokaryotic system |
| CMV promoter | Human cytomegalovirus | Start target gene transcription |
| PolyA signal | It is artificial synthesized | Stable mRNA |
| The gene coding region Neo | It is artificial synthesized | Eukaryon screening |
| Amicillin resistance code area | pBR322 | Protokaryon screening |
The nucleotide sequence of table 2:ATK-V03-aSP carrier target gene insertion point two sides control zone
The analysis of table 3:ATK-V03-aSP carrier restriction enzyme site
| # | Name | Recognition Site | No | Position(s) |
| 1 | AgeI | A^CCGGT | 1 | 1192. |
| 2 | ApaLI | G^TGCAC | 3 | 170,4241,5487. |
| 3 | AvaI | C^YCGRG | 2 | 1308,2416. |
| 4 | BamHI | G^GATCC | 1 | 1321. |
| 5 | BglII | A^GATCT | 1 | 150. |
| 6 | EcoRI | G^AATTC | 1 | 1258. |
| 7 | EcoRV | GAT^ATC | 2 | 1275,2430. |
| 8 | HindIII | A^AGCTT | 1 | 1183. |
| 9 | KpnI | GGTAC^C | 1 | 1193. |
| 10 | MluI | A^CGCGT | 1 | 1198. |
| 11 | MunI | C^AATTG | 1 | 299. |
| 12 | NcoI | C^CATGG | 4 | 867,1208,2302,3037. |
| 13 | NdeI | CA^TATG | 1 | 741. |
| 14 | NheI | G^CTAGC | 1 | 1294. |
| 15 | NotI | GC^GGCCGC | 1 | 1314. |
| 16 | NsiI | ATGCA^T | 2 | 2145,2217. |
| 17 | PstI | CTGCA^G | 1 | 2658. |
| 18 | PvuI | CGAT^CG | 1 | 5190. |
| 19 | SacI | GAGCT^C | 1 | 1075. |
| 20 | SacII | CCGC^GG | 2 | 1162,1320. |
| 21 | SalI | G^TCGAC | 2 | 136,3553. |
| 22 | SmaI | CCC^GGG | 1 | 2418. |
| 23 | StuI | AGG^CCT | 1 | 2394. |
| 24 | XhoI | C^TCGAG | 1 | 1308. |
| 25 | XmaI | C^CCGGG | 1 | 2416. |
The DNA sequences encoding of ancrod glycoprotein is completed by chemical synthesis, is synthesized this with T4DNA ligase
DNA encoding sequence is connected into ATK-V03-aSP carrier, is obtained DNA recombinant expression plasmid ATK-V03-aSP-Ancrod, is passed through
DNA sequencing carries out the correctness of insetion sequence in verifying recombinant plasmid.Plasmid construction map is as shown in Figure 1.
Embodiment 2: recombinant ancrod enzyme stablizes expression in mammalian host cell
Ancrod glycoprotein height expresses foundation and the colony screening of high production genetically engineered cell strain
V03-aSP-Ancrod and V07-DHFR-neo plasmid is transferred to host cell jointly using the method for electric shock transfection
In ATK-CHO-S2.Cell is placed on 37 DEG C after transfection, 5%CO2It is cultivated in incubator.It, will be thin after culture 24 hours
Born of the same parents are seeded to screening and culturing medium (formula: tryptone 5.0g/L, multivalence peptone 5.0g/L, powdered beef 3.0g/L, glucose 1.0g/
L, sodium chloride 5.0g/L, disodium hydrogen phosphate 1.0g/L, glycine 10.0g/L, lithium chloride 0.5g/L, benzyl carbinol 2.5g/L, agar
15.0g/L pH value 7.3) in screened.Cell strain 27 of expression ancrod glycoprotein are obtained altogether.
According to batch cultivation as a result, choose the higher five plants of monoclonal cell strain CIAS1 of expression ancrod glycoprotein,
CIA-P4, CIA-P8, CIA-P33 and CIA-P48 carry out continuing to screen, and the inoculum density according to every hole less than 5 cells is inoculated with
Subclone screening is carried out into 96 orifice plates.The subclone of acquisition is transferred to after being cultivated 4-6 days in 24 orifice plates, carries out determination of activity.
According to determination of activity as a result, the higher clone of selection expression activity is transferred in 6 orifice plates after continuing culture 3 days, determination of activity is carried out.
It is cultivated according to determination of activity as a result, choosing the higher clone of expression activity and being transferred in 50mL shaking flask.According to batch cultivation knot
Fruit chooses CIA8-64 clone.By CIA8-64 clone's further progress subclone screening, final obtain stablizes highly expressed Dan Ke
Grand cell strain CIA8-64-33-95.Activity determination is carried out using Blood coagulation instrument, considers active height by blood coagulation speed.
Embodiment 3: the cell production of recombinant ancrod zymoprotein
The present embodiment describes cell culture and scale production process.
(1) lab scale craft: CIA8-64-33-95 is in Sai Duolisi (Sartorius) BIOSTAT B 10 liters of biologies of PLUS
It is cultivated in reactor, process conditions are as follows:
Starting volume of culture: 5 liters
Basal medium: SFM
Condition of culture: 37 DEG C, 5%CO2、120rpm
Inoculum density: 0.3 × 106A/milliliter.
The composition of serum free medium (SFM) are as follows: NaHCO3,5mM of glucose 0.6%, glutamine 2mM, 3mM
Hepes, 2.5 μ g/ml of insulin, transferrins 100ng/ml, 60 μM of butanediamine, sodium selenate 30nM, 50 μ g/ml of penicillin, chain
50 μ g/ml of mycin, 100 μM of copper chloride, N- acetyl group-D- epichitosamine 2mM, 50 μM of potassium pyrophosphate, DF12 add to 100ml.
Bioreactor parameter setting is as follows:
DO (saturation of the air): 50%
PH range: 6.8~7.2
Speed of agitator: 120 revs/min
Ventilating mode: surface ventilation (air) and deep ventilation (air, oxygen and carbon dioxide).
(2) pilot process
Culture process of the CIA8-64-33-95 cell strain in 50 liters of bioreactors is as follows:
Starting volume of culture: 50 liters
Basal medium: with above
Condition of culture: 37 DEG C, 5%CO2、120rpm
Inoculum density: 0.3 × 106A/milliliter.
Bioreactor parameter setting:
DO (saturation of the air): 50%
PH range: 6.8~7.2
Speed of agitator: 120 revs/min
Ventilating mode: surface ventilation (air) and deep ventilation (air, oxygen and carbon dioxide).
In above-mentioned (1) lab scale craft and (2) pilot process, copper chloride, N- acetyl are added in serum free medium
Base-D- epichitosamine and potassium pyrophosphate three, when culture was to the 6th~7 day under the conditions of 37 DEG C, 1.0~1.2 can be reached ×
107The level of a cell/mL；Then so that temperature is down to 33 DEG C by 37 DEG C, continue to cultivate (about 3~4 days) to cell density no longer
After increase, culture is harvested, cell density maximum can reach 2.8~3.0 × 10 at this time7The level of a cell/mL.
Complementary testing: referring to embodiment 3 its " (1) lab scale craft " and " (2) pilot process ", different is only to cultivate extremely
After 7th day reaches highest viable cell density, bioreactor did not adjusted continuation in 37 DEG C of cultures to 12 days, found at the 6th~7 day
Cell density reaches about 1.0~1.2 × 107The horizontal of a cell/mL can not be further added by density later, or even at the 8th~9 day
Cell density has the tendency that reduction.In the test for not changing (reduction) cultivation temperature herein, the cell growth of lab scale craft and mesh
Protein expression situation it is as shown in Figures 2 and 3, the growth of the cell of pilot process and destination protein expression such as Fig. 4 and Fig. 5 institute
Show.
(3) quantitative analysis method of ancrod glycoprotein
Since ancrod glycoprotein causes blood plasma agglutination, the quantitative analysis method of ancrod glycoprotein is solidifying using blood plasma
Collection method.
Instrument: the raw C2000-1 coagulo meter of Puli, single-time measurement cup and the dedicated steel ball of coagulo meter.
Step: human plasma,frozen is placed in and melts in 37 ± 0.5 DEG C of waters bath with thermostatic control and gently shakes up.By coagulo meter and survey
Determine cup and is preheated to 37 ± 0.5 DEG C.200 μ l blood plasma and steel ball is added into measurement cup.Measurement cup is added simultaneously in 100 μ l of sample to be tested
Start timing.Agglutination time time for making steel ball stop swinging.Sample to be tested should be dense according to the content of ancrod glycoprotein
Degree is diluted in right amount.
This quantitative analysis method, can during monitoring process the yield of recombinant ancrod enzyme, yield, the blood coagulation of material it is living
Property etc..Such as have been found that the method for the present invention readily can obtain high expression quantity with industrial production specification in the above-described embodiments
Recombinant ancrod enzyme, yield can reach every milliliter of fermentation liquid 200IU/ml or more, specific activity can reach 1000IU/mg with
On.
Embodiment 4: the purifying of recombinant ancrod enzyme
(1) concentration of recombinant ancrod enzyme fermentation liquid
Culture solution containing recombinant ancrod enzyme obtained by previous step is filtered removal cell fragment, collects filtrate；
Harvest 50L fermentation liquid every time, the sample volume for needing to purify is big, inside there is a large amount of small-molecule substances, is unfavorable for purifying.First with retention
Molecular weight is the ultrafiltration membrane packet ultrafiltration fermentation liquid of 30K, and the blood coagulation activity of Preliminary fermentation liquid is 10-20S, is concentrated into film upper liquid volume
5-6L, the blood coagulation activity of liquid is greater than 200S under film, discards.Film upper liquid carries out the purifying of next step ultrafiltration membrane until reduction in bulk is original
The 1/8~1/10 of volume.
Liquid concentration technique flow example of fermenting is as follows: fermentation liquid 50L (blood coagulation activity 10-20S) --- the ultrafiltration of > 30K
Film --- > film upper liquid 10L, liquid discards under film, and the blood coagulation activity of liquid is greater than 200S under film --- and > film upper liquid carries out pure in next step
Change.
(2) Heparin Sepharose 6Fast Flow gel affinity column chromatography
Chromatographic column (column diameter 50mm, length 12cm), filler are Heparin Sepharose 6Fast Flow, and balance is slow
Fliud flushing is 0.05mol/L Tris-HCl pH7.0-7.5 buffer, and sample is the film upper liquid 1000ml in (1) step, with balance
Buffer is diluted to 2000ml, loading, column flow rate 24mL/min.After loading, with equilibration buffer to baseline, use
The elution of 0.25mol/L NaCl, 0.05mol/L Tris-HCl pH7.0-7.5 buffer, collects active component.Use 1.0mol/L
NaCl, 0.05mol/L Tris-HCl pH7.0-7.5 buffer elute impurity composition, then with Equilibration buffer wash chromatographic column
1000ml, regeneration are spare.The color of purification process first step Heparin Sepharose 6Fast Flow gel affinity column chromatography
Spectrogram such as Fig. 6.
(3) reversed-phase liquid chromatography purifies
Using preparative reversed-phase liquid chromatography technology, recombinant ancrod zymoprotein obtained above is purified, it is big using polarity
Small difference further removes other impurities using the method for ethanol-water system gradient elution.Sample usesSystem
Standby type high performance liquid chromatograph carries out separation preparation, chromatographic column be Dubbe C4 chromatographic column (10 μm, 20 × 250mm of Φ,)。
With the ethyl alcohol in 500mL pure water cleaning system flow path and chromatographic column is balanced first, flow velocity 10mL/min, then by step (2)
The active component of middle collection, by liquid chromatogram infusion pump loading, loading flow velocity is 10mL/min, after completion of the sample, uses 100mL
Pure water detergent line and chromatographic column.
Gradient elution is carried out according to table 4, wherein mobile phase A is phosphate aqueous solution (pH2.3), and Mobile phase B is ethyl alcohol, and is flowed
1% propylene glycol, Detection wavelength 214nm, elution flow rate 20mL/min are additionally added in dynamic phase A, B.
Table 4: elution requirement table
| Time/min | A | B |
| 0~50 | 90→30 | 10→70 |
| 50~60 | 30 | 70 |
| 61.~71 | 90 | 10 |
Active main peak is collected, is diluted with water 10 times.Carry out lower step purifying.
Purification process second step reversed-phase liquid chromatography purifying chromatogram is shown in Fig. 7.As shown in fig. 7, in 2000~2200ml model
Target glycoprotein can be eluted out in enclosing.The present inventor has found in the test of supplement, if do not added in mobile phase A, B
Volume required for adding propylene glycol to be then eluted out target glycoprotein up to 2500~2900 ranges, not only elution time extend but also
Mobile phase volume used is bigger, and protein concentration is lower in gained eluent.
(4) Heparin Sepharose 6Fast Flow gel affinity column chromatography
Chromatographic column (column diameter 50mm, length 12cm), filler are Heparin Sepharose 6Fast Flow, and balance is slow
Fliud flushing is 0.05mol/L Tris-HCl pH7.0-7.5 buffer, and sample is the active main peak in step (3), uses equalizing and buffering
Liquid is diluted to 1000ml, loading, column flow rate 2mL/min.After loading, with equilibration buffer to baseline, 0.25mol/L is used
The elution of NaCl, 0.05mol/L Tris-HCl pH7.0-7.5 buffer, collects active component.With 1.0mol/L NaCl,
0.05mol/L Tris-HCl pH7.0-7.5 buffer elutes impurity composition, then with Equilibration buffer wash chromatographic column
1000ml, regeneration are spare.
The purification process of the present embodiment 4 can efficiently separate out recombinant protein, inverted chromatography, and glycoprotein purity is equal
Greater than 99%.
Embodiment 5: recombinant ancrod enzymic structure analysis
(1) end N- and the amino acid residue measurement of the end C- 15:
Protein sequence is identified using LTQ-MS, the end N- 1-15 have been carried out to 4 gained recombinant ancrod enzyme of embodiment
The sequencing of amino acid residue, sequence are as follows: VIGGDECNINEHRFL is consistent with known array；C-terminal 217-234 amino acids
Sequence D YRDWVNNVIAGNATCSP is consistent with known array.Through complete determined amino acid sequence, the results showed that, recombination peace gram
Lip river enzyme sequence is identical as the amino acid sequence of the contained ancrod of http://www.uniprot.org.
(2) disulfide bond:
After measured, the primary structure of recombinant ancrod enzyme is single-stranded containing 234 amino acid residues, there is 6 pairs of disulfide bond,
It is located at C7-141, C28-44, C78-232, C120-C188, C152-167, C178-203, it is consistent with known array.
(3) glycosylation structure:
Glycosylation modified peptide fragment, high-precision LC-MS Mass Spectrometer Method and spectra count are obtained by protease hydrolyzed protein
The qualitative and quantitative analysis of glycopeptide level is carried out according to analysis software Pepfinder TM.
The result shows that recombinant ancrod enzyme of the present invention contains 5 N-Link glycosylation sites, it is located at: Asn23-
Trp24-Thr25, Asn79-Lys80-Thr81, Asn99-Asn100-Ser101, Asn148-Phe149-Thr150And Asn229-Ala230-
Thr231Place, it is identical as the contained known glycosylation site of ancrod of http://www.uniprot.org.
The recombinant ancrod enzyme being prepared in each test of the present invention, after measured, isoelectric point is in 4.5-5.5 range
It is interior.
The measurement of glycoprotein molecule amount: using liquid chromatography-mass spectrometry to recombinant ancrod enzyme made from the method for the present invention
Glycoprotein and natural ancrod glycoprotein carry out the measurement of molecular weight, as the result is shown the recombination peace of present invention gained each batch
The average molecular weight of Crow enzyme glycoprotein is within the scope of 39~41kDa, and range of molecular weight distributions is within the scope of 33~52kDa
(distribution span reaches 17kDa or more)；Such as batch of the present invention be 20140509 recombinant ancrod enzyme glycoprotein sample it is flat
Average molecular weight is 40544Da, and range of molecular weight distributions is 33980~51841Da (distribution span reaches 17.9kDa), the sample
LC-MS figure is shown in Fig. 8；And the average molecular weight of natural ancrod glycoprotein is 37.7kDa, range of molecular weight distributions is 32.1~
40.8kDa (distribution span is 8.7kDa), distribution span is much smaller than recombinant glycoprotein of the present invention (natural ancrod glycoprotein
LC-MS figure do not show).
The complementary testing that process conditions influence degree of glycosylation: different referring to the method for implementing Examples 1 to 4 above
Be only to change serum free medium composition used in the lab scale craft and pilot process of embodiment 3, that is, cancel wherein chlorine
Change copper, N- acetyl group-D- epichitosamine, potassium pyrophosphate three or both any or one of any, (nothing under the conditions of various
By being small formula or pilot scale), after measured, average molecular weight is in 38.3~39.1kDa range for glycoprotein obtained by such condition
Interior, range of molecular weight distributions (distribution span is no more than 11kDa) within the scope of 34~45kDa shows the sugar of these glycoprotein
Base level is relatively low.In addition, being measured lower obtained by this complementary testing according to the measuring method of specific activity documented by embodiment 6
The specific activity of glycosylated glycoprotein, as a result in the range of the lower specific activity of 883~976IU/mg.In addition, according to embodiment 6
Documented study on the stability method measures the stability of lower glycosylated glycoprotein obtained by this complementary testing, as a result relatively
Activity is in 74~88% ranges.This complementary testing prompt, when carrying out cell culture, using being added to copper chloride, N- second
Acyl group-D- epichitosamine, potassium pyrophosphate three serum free medium for obtain the superior glycoprotein of performance be beneficial
's.
Embodiment 6: recombinant ancrod enzyme active determination in vitro
Sample: recombinant ancrod enzyme obtained by recombinant ancrod enzyme, various complementary testings obtained by embodiment 4 each batch, natural
Ancrod etc..
Activity determination method:
Principle: since the recombinant ancrod enzyme fibrinogen in blood that can degrade is fibrin, making blood clotting, can be with
The activity of recombinant ancrod enzyme is determined by the setting time of measurement blood plasma or fibrinogen.
Instrument and material:
Coagulo meter, trishydroxymethylaminomethane, sodium hydroxide, calcium chloride, concentrated hydrochloric acid are domestic analytical reagents；Water is double
Distilled water.Bovine fibrinogen (ox blood) (88mg soluble protein /), lot number: 140607-201137；Ox blood albumin be into
Mouth packing reagent.
Test solution is prepared
2mol/L Tris- hydrochloric acid liquid stock solution: taking trishydroxymethylaminomethane 12.1g, water 80ml added to make to dissolve,
After 1mol/L hydrochloric acid solution tune pH value to 7.4, add water to 100ml, shake up to obtain the final product.
20mmol/L Tris- hydrochloric acid liquid: it takes above-mentioned stock solution 0.5mL, adds water to be settled to 50mL, mix to obtain the final product.
Phosphate dilution (pH6.0): disodium hydrogen phosphate 0.17g, sodium chloride 0.85g is taken to be dissolved in 80mL water, 1mol/L
It after sodium hydroxide tune pH to 6.0, adds water to 100mL, shakes up to obtain the final product.
0.5% ox blood albumin -20mNTris- hydrochloric acid liquid: ox blood albumin 0.05g is taken, 20mNTris- hydrochloric acid is added
Dilution dissolution is settled to 10mL, shakes up to obtain the final product.
0.5% ox blood albumin-phosphate dilution (pH6.0): ox blood albumin 0.05g is taken, 20mNTris- phosphoric acid is added
The dissolution of salt dilution is settled to 10mL, shakes up to obtain the final product.
Bovine fibrinogen working solution: National Institute for Food and Drugs Control's bovine fibrinogen standard reagent 1 is taken, is added
Dilution 11mL is prepared into the stock solution that concentration is 8mg/mL, then the working solution for being 4mg/mL with diluted.
Measuring method:
The preparation of reference substance solution: recombinant ancrod enzyme working reference substance 1 is taken, appropriate dilution is added, is made into 10IU/
Then the reference substance mother liquor of mL takes 800 μ L, 600 μ L, 400 μ L, 200 μ L standard solution respectively, dilution is added to be settled to 1mL, quasi-
It is really diluted to the solution that concentration is 6.0,4.0,2.0,1.0IU/ml, faces the used time and sets in the heat preservation hole of coagulo meter, kept the temperature at 37 DEG C.
The preparation of test solution: precision measurement this product is appropriate, adds dilution to be made molten containing about 2~6IU in every 1ml
Liquid.
The preparation of standard curve: bovine fibrinogen working solution 0.2mL, 3 be respectively placed in the test sample hole of coagulo meter are taken
In a measurement cup, 3 minutes are kept the temperature at 37 DEG C, it is accurate respectively to measure warmed-up each 0.1mL of reference substance solution, it is rapidly added fibre
In each cup of fibrillarin original solution, instrument automatically records setting time.Every kind of concentration is measured in parallel 3 times, calculates average value and mark
Quasi- deviation (when standard deviation is greater than 3.0 seconds, need to go again measurement).It is flat with the logarithm of reference substance solution concentration and its setting time
The Logarithmic calculation regression equation of mean value.
Measuring method: precision measures this product 0.1ml, sets after preheating in 37 DEG C of water-baths of coagulo meter, by the preparation of standard curve
Lower method measures setting time, calculates the average value and standard deviation (system of the standard deviation with standard curve of 3 measurement results
It is standby to require), by the potency of regression equation calculation recombinant ancrod enzyme to get.
Sample measurement: the four batch weight group ancrod stostes for taking 4 method of the embodiment of the present invention to obtain measure, as a result in accordance with the law
As shown in table 5, potency is all larger than 270IU/mL or more.
Table 5: recombinant ancrod proenzyme liquid titration result
| Lot number | Potency/(IU/mL) |
| 20140509 | 362.11 |
| 20140521 | 271.29 |
| 20140610 | 503.15 |
| 20140524 | 649.40 |
The measurement of specific activity: the recombinant ancrod enzyme that different batches of the embodiment of the present invention are prepared measures its albumen and contains
Then amount is measured its activity, the specific activity of glycoprotein is characterized with IU/mg, as the result is shown recombinant ancrod produced by the present invention
The specific activity of enzyme glycoprotein is up to the range of the high specific acitivity of 1135~1462IU/mg；Regrettably, day is similarly measured
The specific activity of right ancrod glycoprotein is only 865IU/mg.The difference of this specific activity may be due to being repaired outside amino acid chain
Caused by the difference for adoring sugar, and prompt the activity of the more more complicated more complete then glycoprotein of modification sugar higher.
Study on the stability: the recombinant ancrod enzyme (5 four batches, table) and day that different batches of the embodiment of the present invention are prepared
Right ancrod makes it dissolve in the buffer of pH6.5, protective agent is not added, and places 6 months under 2~6 DEG C of refrigerated conditions,
The results show that external activity of recombinant ancrod enzyme under the conditions of this study on the stability has no decline substantially, for 0 month
Relative activity in June is 93~96%；Surprisingly the relative activity of natural ancrod is only 61.5%, far below recombination
Glycoprotein.Term " relative activity " refers to that certain a sample is active divided by the active multiplied by 100% of measurement in 0 month what is measured June
Gained percentage.The difference of this stability may be due to quilt outside recombinant ancrod enzyme and natural ancrod modification sugar not
Caused by together, and prompt the stability of the more more complicated more complete then glycoprotein of modification sugar higher.
Embodiment 7: recombinant ancrod enzyme damages the dissolution of dog arteria cerebri media thrombotic clot and cerebral ischaemia
It influences
Purpose: the protective effect that observation recombinant ancrod enzyme damages the dissolution of arterial thrombus in dog and cerebral ischaemia with
And blood coagulation and fibrinolysis activity after dog thrombus.
Method: dog arteria cerebri media injects thrombotic clot and causes persistence local cerebral ischemia model.Test is divided into 5 groups, mould
Type control group, recombinant ancrod enzyme high dose group (0.68IU/kg), middle dose group (0.34IU/kg), low dose group (0.17IU/
) and Batroxobin group (0.34BU/kg) kg.Test medicine is drawn by weight, is injected in 100ml physiological saline, 10min is quiet after surgery
Arteries and veins administered by infusion, administration time are controlled in 35~45min.Control group intravenous drip same amount of normal saline.
As a result: the high, medium and low dosage of recombinant ancrod enzyme has dissolution well to make dog arteria cerebri media thrombotic clot
With cerebral infarct size reduces 89.9%, 82.5% and 59.2% than model group control group respectively.Recombinant ancrod enzyme is low dose of
Group and Batroxobin group, reducing, the effect of cerebral infarction area is close.24 hours right internal carotid artery restoration of blood flow rates are respectively after medicine
42.7%, 36.2%, 23.6%.
Recombinant ancrod enzyme substantially reduces neuromuscular function behavior symptom, the scoring of neurobehavioral index is reduced, to stalk
Dead dog arteria cerebri media thrombotic clot causes local cerebral ischemia damage to have significant protective effect.
After the high, medium and low dosage 6h of intravenous drip recombinant ancrod enzyme, arteria cerebri media thrombosis dog plasma factor
Time (PT), thrombin time (TT), Activated partial thromboplastin time (APTT) have significant extension.Recombinant ancrod enzyme height,
In, low dose group and Batroxobin group extend dog plasma FIB and be greater than incidence of the 60s and FIB content lower than 0.5g/L and be
100%, 83.3%, 100%, 66.7%.After high dose group medicine for 24 hours, dog plasma thrombin time (TT) significantly extends, dog plasma
It is 83.3% that FIB, which extends the incidence greater than 60s and FIB content lower than 0.5g/L,.
After the high, medium and low dosage of venoclysis recombinant ancrod enzyme, dog wound has a large amount of blood to ooze out, 6h after administration
Wound blood ooze out incidence be 100%, 66.7%, 50%, after administration for 24 hours wound blood exudation incidence be 66.7%,
16.7%, 0.Between 20ml~100ml, wound oozing of blood amount is related with dosage for wound oozing of blood amount valuation, high dose group wound
Mouth oozing of blood amount is more, and the duration is also grown.Batroxobin group is 50% 6h blood oozes out incidence after medicine.
Embodiment 8:SD rat intravenous injection gives recombinant ancrod enzyme single-dose toxicity test
This test gives recombinant ancrod enzyme by SD rat single intravenous injection, observes anxious caused by recombinant ancrod enzyme
Property toxic reaction situation.
This test sets 3 groups, and every group of 10 animals, half male and half female, single intravenous injection gives Vehicle controls product (0 μ g/ respectively
Kg, 0.9% sodium chloride injection), 160 and 320 μ g/kg recombinant ancrod enzymes, administration capacity is 5mL/kg, the observation period 14 days.
Following index is observed or detected during test: animal dead and dying situation, clinical signs, weight.Observation period foot couple
Animal carries out gross anatomy inspection.Because of gross anatomy no abnormality seen, therefore histopathological examination is not carried out.
320 μ g/kg groups and 160 μ g/kg groups have 1 animal dead in the same day is administered respectively, see that activity is reduced, no before dead
It can stand, double hind limb paralysis, above-mentioned animal dead is related to test sample.
Activity reduction, Bu Nengzhan are seen to animal in after administration 5 minutes from about 1 minute after 320 and 160 μ g/kg group self administration of medication
Vertical, double hind limb paralysis or it is stiff, be short of breath, righting reflex loss, and be in certain dosage correlation.320 and 160 μ g/kg
About 10 minutes and 30 minutes observation Shi Junjian urines are in peony after the jenny administration of group survival.In addition, 320 μ g/kg groups
Still see movable reduction when observation in 30 minutes after 3 bucks administration of survival, wherein 2 males see also perpendicular hair, and continue to
2 hours after administration.Only 1 male is shown in movable reduction when observation in 30 minutes after the administration of 160 μ g/kg groups.320 and 160 μ g/kg groups are deposited
Female animals living are showed no exception to observation end of term clinical observation from 4 and 2 hours after self administration of medication respectively.0 μ g/kg during test
Group female animals are showed no exception.
During test, test sample group the weight of animals and pathological examination are showed no exception relevant to test sample.
In conclusion SD rat single intravenous injection gives 160 and 320 μ g/kg recombinant ancrods under this experimental condition
Enzyme.Dosage >=160 μ g/kg recombinant ancrod enzymes may be actuated object appearance activity reduce, cannot stand, double hind limb paralysis or it is stiff,
It is short of breath, righting reflex loss, urine are in peony, can also occur perpendicular hair to animal under 320 μ g/kg dosage, above-mentioned to change
It can restore in 2~4 hours after becoming administration.Therefore, under this experimental condition, SD rat single intravenous injection gives recombinant ancrod
The maximum tolerated dose (Maximal Tolerance Dose, MTD) of enzyme is less than 160 μ g/kg.
Embodiment 9:SD rat intravenous injection gives recombinant ancrod enzyme surrounding convalescence surrounding repeated dose toxicity test
The purpose of this test is to give recombinant ancrod enzyme by the continuous surrounding intravenous injection of SD rat, is administered once every other day,
Convalescence surrounding observes toxic reaction caused by recombinant ancrod enzyme and studies its Drug Pharmacokinetics feature, provides recombination peace gram
The target organ of the toxic reaction of Lip river enzyme observes the reversible case of damage after convalescence four weeks, provides reference for clinical test.
This test use SD rat 144, every group 30, half male and half female；Separately set TK group, 6/group, half male and half female.Vein
Recombinant ancrod enzyme, 20,40,80 μ g/kg of dosage are given in injection.It is administered once every other day, administration capacity is 5mL/kg, and the phase is administered
It is 29 days (D1~D29), while is arranged convalescence 28 days (R1~R28).
Observed or detected as follows during test: dead and dying situation observation, clinical signs observation, weighing body weight,
Food ration detection, eye examination, Drug Pharmacokinetics analysis, urinalysis, hematology and coagulation indexes inspection, Serum bichemisbry refer to
Mark inspection, gross anatomy, organ weights and histopathological examination.
Drug Pharmacokinetics are as the result is shown: female and male rat blood plasma recombinant ancrod under first same dose after the last administration
Enzyme exposed amount has no difference, and exposed amount is linearly related, and after successive administration 29 days, the exposed amount of recombinant ancrod enzyme has no storage
Product.
Test result is shown: during experiment, the discovery 1 in about 5 minutes or so after D3 administration of 80 μ g/kg dosage group bucks
Example is dead, and activity reduction is mainly seen before dead, cannot stand and urinate discoloration (dark red), gross anatomy and histopathological examination are not
See and test sample relevant abnormalities.
Urine discoloration (pale red) is daily shown in administration of the 40 μ g/kg group jennies during administration in first week.80 μ g/kg groups are female
Tom sees movable reduction in about 2~5 minutes after urine discoloration (dark red) is daily shown in the administration during administration and is administered, in addition, male
Property animal administration phase administration occur day it is transient cannot stand, the pollution of soft stool and crissum.The above animal is in administration phase non-administration
Day and convalescence clinical observation are showed no obvious abnormalities.
80 μ g/kg group bucks, which are administered, saw that food ration declines for the phase first week, weight no abnormality seen.
Be administered the end of term, compared with 0mg/kg group, 80 μ g/kg group jennies be averaged RBC be remarkably decreased and female animals put down
Equal %RETIC conspicuousness increases.It is more than convalescence abnormal to see recovery.
The end of term is administered, 80 μ g/kg group female animals urines are brown to arrive peony, blood urine and Urine proteins, and buck is also
See urine muddiness.Restore the end of term and sees recovery.
Histopathological examination is as it can be seen that the administration end of term, 80 μ g/kg group buck spleen extramedullary hematopoiesis.Restoring the end of term can
See recovery.
During experiment, all dosage group the weight of animals, eye examination, serum biochemistry are showed no obvious abnormalities.
In conclusion recombinant ancrod enzyme is given in the continuous surrounding intravenous injection of SD rat, and dosage is under this experimental condition
0 (0.9% sodium chloride injection), 20,40,80 μ g/kg, once every two days, convalescence surrounding.When dosage is 40 μ g/kg, it is seen that
There is transient urine discoloration (pale red) in jenny；When dosage is 80 μ g/kg, female animals urine discoloration aggravates (dark red), lives
It is dynamic reduce, %RETIC is increased, blood urine and Urine proteins, jenny sees also RBC decline, buck see also it is transient cannot
It stands and spleen extramedullary hematopoiesis is shown in food ration decline, soft stool and crissum pollution, histopathological examination.Convalescence is all of above different
Chang Junjian restores.The exposed amount of each dosage group blood plasma recombinant ancrod enzyme has no gender differences, and the linear correlation of exposed amount,
After successive administration 29 days, accumulation is had no.Female animals do not observe toxic reaction dosage (NOAEL, No observed
Adverse effect level) it is respectively 20 and 40 μ g/kg.
Embodiment 10: the injecta composition of recombinant ancrod enzyme
Recombinant ancrod enzyme of the present invention, clinically wishes the direct injection in the form of injection, and such dosage form has
Help the acute treatment as acute cerebral infarction medication.However due to comprising Tris in the recombinant ancrod enzyme product being obtained above,
It should not be used as people's direct injection, therefore the composition especially liquid composition for preparing adaptation human body direct injection is to compel to be essential
It wants.
1, the preparation of stoste
According to of the invention " embodiment 4: the purifying of recombinant ancrod enzyme ", but by step "(4)Heparin Sepharose
Equilibration buffer and elution buffer in 6Fast Flow gel affinity column chromatography " are changed to PBS-LYS buffer, the PBS-
LYS buffer includes: 0.2% sodium chloride, 0.1% L-Lysine mono Hydrochloride, 15mmol/L sodium dihydrogen phosphate and adding sodium hydroxide tune
Save pH6.8, appropriate amount of water to full dose.
Specific step " (4) Heparin Sepharose 6Fast Flow gel affinity column chromatography " is as follows: chromatographic column
(column diameter 50mm, length 12cm), filler are that Heparin Sepharose 6Fast Flow, PBS-LYS buffer include:
0.2% sodium chloride, 0.1% L-Lysine mono Hydrochloride, 15mmol/L sodium dihydrogen phosphate and adding sodium hydroxide adjusting pH6.8, appropriate amount of water
To full dose, sample is the active main peak in step (3), is diluted to 1000ml, loading with PBS-LYS buffer, column flow rate is
2mL/min.It after loading, is balanced with PBS-LYS buffer to baseline, is eluted with PBS-LYS buffer, collect active component.With
PBS-LYS buffer elutes impurity composition, then rinses chromatographic column 1000ml with PBS-LYS buffer, and regeneration is spare.So far, it obtains
To the recombinant ancrod proenzyme liquid of the present embodiment 10, wherein only including 0.25% sodium chloride, 0.1% lysine hydrochloride, 15mM phosphorus
Acid dihydride sodium and recombinant ancrod zymoprotein.
2, the recombinant ancrod enzyme of the present embodiment 10 is detected
The purification process of the present embodiment 10 can efficiently separate out recombinant protein, inverted chromatography, gained recombination peace gram
Lip river enzyme glycoprotein purity is 99.8%.
Referring to " embodiment 5: recombinant ancrod enzymic structure analysis ", the knot of 10 gained recombinant protein of the present embodiment as the result is shown
Fruit and result shown in embodiment are completely the same, such as isoelectric point is 4.83.The mean molecule of recombinant ancrod enzyme glycoprotein sample
Amount is 40650Da, and range of molecular weight distributions is 34200~51750Da (distribution span reaches 17.55kDa), the LC-MS of the sample
Scheme essentially identical with Fig. 8.
Referring to " embodiment 6: recombinant ancrod enzyme active determination in vitro ", egg is recombinated obtained by the present embodiment 10 as the result is shown
White: potency 635IU/mL, specific activity 1532IU/mg, June was relative to 0 month after placing 6 months under 2~6 DEG C of refrigerated conditions of stoste
Relative activity be 99.8%, PBS of the stoste described in the present embodiment comprising lysine etc. is diluted to after 20IU/mL at 2~6 DEG C
After being placed 6 months under refrigerated condition June relative to 0 month relative activity be 99.4%, show the present embodiment stoste and dilution
It is likewise supplied with excellent stability.
Referring to " embodiment 7: recombinant ancrod enzyme damages the dissolution of dog arteria cerebri media thrombotic clot and cerebral ischaemia
The influence of wound " test 10 gained recombinant ancrod enzyme of the present embodiment, the results show that the height of 10 recombinant ancrod enzyme of embodiment, in,
Low dosage has good dissolution to dog arteria cerebri media thrombotic clot, and cerebral infarct size is respectively than model group control group
Reduce 94.3%, 88.5% and 67.3%, after medicine 24 hours right internal carotid artery restoration of blood flow rates be respectively 56.6%,
43.7%, 29.3%.After the 10 high, medium and low dosage of recombinant ancrod enzyme of venoclysis embodiment, dog wound has a large amount of blood
Exudation, 6h wound blood exudation incidence is 96.3%, 61.4%, 46 after administration.3%, the hair of wound blood exudation for 24 hours after administration
Raw rate is 43.2%, 8.3%, 0.
This reality is tested referring to " embodiment 8:SD rat intravenous injection gives recombinant ancrod enzyme single-dose toxicity test "
10 gained recombinant ancrod enzyme of example is applied, every result and 8 result of embodiment are essentially identical.
Referring to " embodiment 9:SD rat intravenous injection gives recombinant ancrod enzyme surrounding convalescence surrounding repeated dose toxicity
Test " test 10 gained recombinant ancrod enzyme of the present embodiment, every result and 9 result of embodiment are essentially identical.
3, the preparation of injection
(1) recombinant ancrod proenzyme liquid obtained by the present embodiment 10 is taken, is diluted to potency with PBS-LYS buffer
20IU/mL is dispensed into ampulla with 0.22um filtering with microporous membrane degerming with every bottle of 1ml, and sealing obtains injection 101.
(2) recombinant ancrod proenzyme liquid obtained by Example 4, with 1.0mol/L NaCl, 0.05mol/L Tris-
HCl pH7.0-7.5 buffer is diluted to potency 20IU/mL, with 0.22um filtering with microporous membrane degerming, is dispensed into every bottle of 1ml
In ampulla, sealing obtains injection 102.
(3) referring to 10 method of the present embodiment but do not add lysine, recombinant ancrod obtained in PBS-LYS buffer
This raw material is diluted to potency 20IU/mL with the PBS buffer solution for not adding lysine by proenzyme liquid, with 0.22um miillpore filter mistake
Bacterium is filtered out, is dispensed into ampulla with every bottle of 1ml, seals, obtains injection 103.
(4) referring to 10 method of the present embodiment but do not add sodium chloride, recombinant ancrod obtained in PBS-LYS buffer
This raw material is diluted to potency 20IU/mL with the PBS-LYS buffer for not adding sodium chloride by proenzyme liquid, is filtered with 0.22um micropore
Film filtration sterilization is dispensed into ampulla with every bottle of 1ml, and sealing obtains injection 104.
(5) lysine is changed to equivalent L- R-gene referring in 10 method of the present embodiment but PBS-LYS buffer
(being basic amino acid as lysine), recombinant ancrod proenzyme liquid obtained, by this raw material PBS- arginine buffer
Liquid is diluted to potency 20IU/mL, with 0.22um filtering with microporous membrane degerming, is dispensed into ampulla with every bottle of 1ml, and sealing obtains
Injection 105.
Above-mentioned 101~injection of injection 105 is placed 18 months under 2~6 DEG C of refrigerated conditions, is measured 0 month and 18 months
When each injection activity, and calculate 18 months for 0 month relative activities.As a result: 101 relative activity of injection
94.2%, 102 relative activity 76.3% of injection, 103 relative activity 68.7% of injection, 104 relative activity of injection
74.4%, 105 relative activity 77.5% of injection.As it can be seen that carrying out final protein purification simultaneously by using PBS-LYS buffer
And using the buffer as the carrier of injection formulation, excellent stability is presented in gained injection.
101~injection of injection 105 obtained is all achromatism and clarity solution；They are placed at room temperature
A month, observe whether each injection appearance changes, as a result: injection 101 is still maintained in achromatism and clarity solution, injection
102~injection 104 has opalescence and generation flocculation, injection 105 have white depositions.As it can be seen that injection 101 has obviously
Superior physical stability and biological stability.
The present invention relates to a kind of recombinant ancrod enzyme, preparation method and application and their pharmaceutical compositions.More
Body, the present invention relates to using technique for gene engineering, using mammalian CHO cells culture production method preparation and reorganization albumen,
Then by purifying process, the recombinant ancrod enzyme of high expression quantity can be readily obtained with industrial production specification, yield can
Reach every milliliter of fermentation liquid 200IU/ml or more, specific activity can reach 1000IU/mg.The recombinant ancrod enzyme that the present invention obtains
With more complicated and more complete level of glycosylation, stability is significantly increased, and solves that such existing product expression amount is low, industry
Change difficult problem, the invention further relates to recombinant ancrod enzymes to treat the application in acute cerebral infarction.
Spirit of the invention is elaborated above by present pre-ferred embodiments.Those skilled in the art's reason
Solution, all any modification, equivalent variations and modification to the above embodiments according to the technical essence of the invention, all falls within this hair
In bright protection scope.
Claims (10)
1. the method for purifying recombinant ancrod enzyme comprising pure in the recombinant ancrod zymoprotein reversed-phase liquid chromatography that will be obtained
After change, the purifying of Heparin Sepharose6 Fast Flow gel affinity column chromatography is carried out in the following way:
Chromatographic column, filler are Heparin Sepharose6 Fast Flow, and sample is the active main peak in step (3), are used
PBS-LYS buffer is diluted to 1000ml, loading, column flow rate 2mL/min；After loading, balanced with PBS-LYS buffer to base
Line is eluted with PBS-LYS buffer, collects active component；Impurity composition is eluted with PBS-LYS buffer, then slow with PBS-LYS
Fliud flushing rinses chromatographic column 1000ml, and regeneration is spare, obtains recombinant ancrod proenzyme liquid.
2. the method according to claim 1, the PBS-LYS buffer includes: 0.2% sodium chloride, 0.1% hydrochloric acid L- rely ammonia
Acid, 15mmol/L sodium dihydrogen phosphate and adding sodium hydroxide adjust pH6.8, appropriate amount of water to full dose.
3. the method according to claim 1, the column diameter 50mm of the chromatographic column, length 12cm.
4. the method that pair recombination ancrod is purified, this method comprises the following steps:
(41) it is pre-processed before chromatographic column on: the culture solution containing recombinant ancrod enzyme is filtered removal cell fragment, collected
Filtrate is then concentrated by ultrafiltration, molecular cut off 30KDa diaphragm plate ultrafiltration to the 1/8~1/10 of original volume；
(42) affinity column: using affinity column carry out purification, using the NaCl of 0.1~0.4mol/L of salinity,
The elution requirement of the buffer of Tris-HCl pH7.0~7.5 of 0.05mol/L, elutes and collects active component；
(43) reversed-phase liquid chromatography purifies: using preparative reversed-phase liquid chromatography technology, purifies recombinant ancrod obtained above
Zymoprotein further removes other impurities using the method for ethanol-water system gradient elution using the difference of polarity size；
(44) affinity column chromatography: the recombinant protein that previous step is collected uses affinity chromatography column purification, removes ethyl alcohol and realizes
The concentration of recombinant protein,
Wherein, the operation of step (44) is as follows:
Chromatographic column, filler are Heparin Sepharose6 Fast Flow, and sample is the active main peak in step (3), are used
PBS-LYS buffer is diluted to 1000ml, loading, column flow rate 2mL/min；After loading, balanced with PBS-LYS buffer to base
Line is eluted with PBS-LYS buffer, collects active component；Impurity composition is eluted with PBS-LYS buffer, then slow with PBS-LYS
Fliud flushing rinses chromatographic column 1000ml, and regeneration is spare, obtains recombinant ancrod proenzyme liquid.
5. method according to claim 4, the PBS-LYS buffer includes: 0.2% sodium chloride, 0.1% hydrochloric acid L- rely ammonia
Acid, 15mmol/L sodium dihydrogen phosphate and adding sodium hydroxide adjust pH6.8, appropriate amount of water to full dose.
6. method according to claim 4, the column diameter 50mm of the chromatographic column, length 12cm.
7. method according to claim 4, in above-mentioned steps (43), using C4 preparative scale chromatography column, using ladder shown in following table
Degree elution:
Wherein mobile phase A is the phosphate aqueous solution of pH2.3, and Mobile phase B is ethyl alcohol, Detection wavelength 214nm, and elution flow rate is
20mL/min；Such as 1% propylene glycol is also additionally added in the mobile phase A and Mobile phase B.
8. the method for preparation and reorganization ancrod injection comprising following steps:
(a) purified recombinant ancrod proenzyme liquid (the optional potency for measuring the stoste) is provided；
(b) (such as being diluted to potency 20IU/mL) is diluted with PBS-LYS buffer, with 0.22um filtering with microporous membrane degerming,
(such as with every bottle of 1ml) is dispensed into ampulla, and sealing obtains injection.
9. the recombinant ancrod proenzyme liquid of method according to claim 8, the purifying is prepared according to following method:
(41) it is pre-processed before chromatographic column on: the culture solution containing recombinant ancrod enzyme is filtered removal cell fragment, collected
Filtrate is then concentrated by ultrafiltration, molecular cut off 30KDa diaphragm plate ultrafiltration to the 1/8~1/10 of original volume；
(42) affinity column: using affinity column carry out purification, using the NaCl of 0.1~0.4mol/L of salinity,
The elution requirement of the buffer of Tris-HCl pH7.0~7.5 of 0.05mol/L, elutes and collects active component；
(43) reversed-phase liquid chromatography purifies: using preparative reversed-phase liquid chromatography technology, purifies recombinant ancrod obtained above
Zymoprotein further removes other impurities using the method for ethanol-water system gradient elution using the difference of polarity size；
(44) affinity column chromatography: the recombinant protein that previous step is collected uses affinity chromatography column purification, removes ethyl alcohol and realizes
The concentration of recombinant protein,
Wherein, the operation of step (44) is as follows:
Chromatographic column (column diameter 50mm, length 12cm), filler are Heparin Sepharose6 Fast Flow, and PBS-LYS is slow
Fliud flushing includes: 0.2% sodium chloride, 0.1% L-Lysine mono Hydrochloride, 15mmol/L sodium dihydrogen phosphate and adding sodium hydroxide adjusting
PH6.8, appropriate amount of water to full dose, sample are the active main peak in step (3), are diluted to 1000ml with PBS-LYS buffer, on
Sample, column flow rate 2mL/min；It after loading, is balanced with PBS-LYS buffer to baseline, is eluted with PBS-LYS buffer, collected
Active component；Impurity composition is eluted with PBS-LYS buffer, then rinses chromatographic column 1000ml with PBS-LYS buffer, is regenerated,
It is spare, obtain recombinant ancrod proenzyme liquid.
10. method according to claim 9, in above-mentioned steps (43), using C4 preparative scale chromatography column, using ladder shown in following table
Degree elution:
Wherein mobile phase A is the phosphate aqueous solution of pH2.3, and Mobile phase B is ethyl alcohol, Detection wavelength 214nm, and elution flow rate is
20mL/min；Such as 1% propylene glycol is also additionally added in the mobile phase A and Mobile phase B.
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