CN101275126B - Purified high-specific-activity recombinant batroxobin - Google Patents
Purified high-specific-activity recombinant batroxobin Download PDFInfo
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- CN101275126B CN101275126B CN2007100389113A CN200710038911A CN101275126B CN 101275126 B CN101275126 B CN 101275126B CN 2007100389113 A CN2007100389113 A CN 2007100389113A CN 200710038911 A CN200710038911 A CN 200710038911A CN 101275126 B CN101275126 B CN 101275126B
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Images
Abstract
The present invention provides a recombined-batroxobin, which is characterized in that: (a) the molecular weight is 29-32Kda; (b) at least 90% batroxobin in the recombined-batroxobin has correct six pairs of disulfide bonding; Cys<7>-Cys<139>, Cys<26>-Cys<42>, Cys<74>-Cys<230>, Cys<118>-Cys<184>, Cys<150>-Cys<163> and Cys<174>-Cys<199>; (c) the 146 position and the 225 position in the SEQ ID NO:1 are suffered from glycosylation; (d) the specific activity of the batroxobin is more than 1500KU/mg.
Description
Technical field
The present invention relates to field of genetic engineering, relate in particular to the height ratio recombinant batroxobin and the application thereof alive of purifying.
Background technology
As far back as 1936 Austrian scholar Van Klobusitzky etc. just from the venom of Brazilian fer-de-lanc (Bothrops atrox) purification refine go out a kind of enzyme hemostatic agent Batroxobin, middle translated name the earliest is referred to as " batroxobin " at home.Batroxobin belongs to the serine protease quasi-molecule, and the physiological function of its molecule and bulk of molecule all have similar zymoplasm (Thrombin) part, so also be referred to as " hemocoagulase " after a while.But, given different implications in different periods to batroxobin both at home and abroad along with further investigation to it.Brazil fer-de-lanc (Bothrops atrox) has 5 subspecies, the batroxobin that extracts from the subspecies that have has hemostasia effect [1], and the batroxobin that extracts from other subspecies has the fibrinogenic effect of removal, the difference of snake kind, the protein that has caused " batroxobin " that cage traditionally claims, in fact be diverse in essence in its biological chemistry: it is main having plenty of hemostasia effect, what have then is to fall fibre to act as the master, the difference that comes from the aminoacid sequence as for not being both on this chemical nature or because the difference on the protein modifications such as glycosyl is still waiting further study tour.
Have only at present from snake venom the batroxobin of the preparation of extracting be used for the treatment of the various clinical indication (as, myocardial infarction, senile dementia, apoplexy, sudden deafness or the like) patent (US Pat.No:5595974,5869044,6106830,6399576,6416717; Eur.Pat.No:0984279,0826374,0750912,0719791) and research report, also there is not recombinant batroxobin to be used for any correlative study report of animal experiment and clinical study work.The batroxobin of biological extraction is mainly from Brazilian fer-de-lanc (Bothropsmoojeni from snake venom, Bothrops atrox) in the venom, content is very low, the acquisition difficulty of the acquisition of its raw material and the pure product of natural batroxobin is big, toward toxin and a lot of not clear impurity of contact meeting residual a small amount of snake in finished product, bring potential risks for clinical use.The theoretical molecular of the natural batroxobin after highly purified should also be 25.6kDa, but in fact this albumen secreting, expressing in the poison gland cell of poisonous snake be taken place glycosylation modified, actual molecular weight 37-43kDa, this molecular weight has on the reason of a scope or the glycosylation modified degree difference is arranged, and also has the different and sugar chain that cause of means of purification reason such as to lose and causes.Extract the natural batroxobin of preparation from snake venom, because the source of snake venom raw material is subjected to support factor affecting such as the scale, Ember Days variation of snake, its quality control is very difficult, and the specific activity of product is also very unstable.
This protein molecule biological study from snake venom is made slow progress always to batroxobin, just finishes cDNA and genomic dna examining order to batroxobin gene by the investigator of Japan up to 1987,1988.1991 Japanese rattan pool drugmaker (Fujisawa Pharmaceutical) the investigator adopt gene recombination method, in E.coli, adopt the amalgamation and expression system, formal representation with inclusion body (InclusionBody) goes out this composition, cut the method for fusion rotein open and obtain what is called the biologic activity batroxobin is arranged by preparing electrophoresis and zymoplasm again, but also applied for patent (Pat.No.:JP2124092,1990) for this reason.Adopt the albumen that disulfide linkage is rich in engineered means production always to be a technical barrier, especially producing has many serine proteinase enzyme molecules to disulfide linkage.This is because disulfide linkage pairing error rate is very high, what obtain in protokaryon almost is inclusion body entirely, though Japanese rattan pool drugmaker has reported by the denaturation renaturation to inclusion body and can obtain activated target protein, but it is lower than living, poor reproducibility does not see that up to now the said firm has the recombinant batroxobin product to come out yet.
Therefore, this area presses for and a kind ofly is not subject to seasonal restrictions, and industrial scale is the batroxobin product of the reorganization of control easily, and it has high disulfide linkage pairing accuracy, and active high.
Summary of the invention
First purpose of the present invention provides a kind of batroxobin of gene recombination.
Second purpose of wood invention provides the purposes of the batroxobin of described gene recombination.
The 3rd purpose of the present invention provides the pharmaceutical composition of the batroxobin that contains described gene recombination.
In a first aspect of the present invention, a kind of recombinant batroxobin of purifying is provided, described batroxobin has following characteristic:
(a) molecular weight is 29-32kDa;
(b) at least 90% (〉=90%) batroxobin has 6 couples of correct disulfide linkage pairing: Cys in the described recombinant batroxobin
7-Cys
139, Cys
26-Cys
42, Cys
74-Cys
230, Cys
118-Cys
184, Cys
150-Cys
163And Cys1
174-Cys
199
(c) the 146th and the 225th are glycosylation modified by the N-type in SEQ ID NO:1; With
(d) specific activity of described batroxobin is more than or equal to 1500KU/mg albumen.
In another preference, at least 95% has 6 pairs of correct disulfide linkage pairings in the described recombinant batroxobin.
In another preference, the glycosylation in the described batroxobin is at following site Asn
146-Asn
147-Thr
148And Asn
225-Lys
226-Thr
228In the Asn amino-acid residue in that N-takes place is glycosylation modified.
In another preference, the glycosylation modified molecular weight of batroxobin protein that makes of its N-type increases 4000-6000kDa on the basis of 25.6kDa.
In another preference, the specific activity 1500-3000KU/mg of described batroxobin.
In another preference, at least 99% has 6 pairs of correct disulfide linkage pairings in the described batroxobin.
In a second aspect of the present invention, provide the application of batroxobin in the preparation haemostatic medicament of the reorganization of above-mentioned purifying.
In a third aspect of the present invention, a kind of pharmaceutical composition is provided, it contains above-mentioned recombinant batroxobin and pharmaceutically acceptable carrier.
In another preference, described composition also contains gelatin hydrolysate as stablizer.
In another preference, described composition is the form of liquid or lyophilized powder.
In view of the above, the invention provides and a kind ofly be not subject to seasonal restrictions, industrial scale is the batroxobin product of the reorganization of control easily, and it has high disulfide linkage pairing accuracy, and active high.
Description of drawings
Fig. 1 has shown the C-terminal sequence of gene recombination batroxobin provided by the invention and has had glycosylation modified on the Asn225 position.
Fig. 2 shown have on the Asn146 position of gene recombination batroxobin provided by the invention glycosylation modified.
A has shown the Base Peak collection of illustrative plates of the rBAT of non-reducing Quimotrase and F1 enzymolysis among Fig. 3; B has shown the Base Peak collection of illustrative plates of the rBAT of reductive Quimotrase and F1 enzymolysis.
A has shown the Base Peak collection of illustrative plates of the rBAT of non-reducing trypsinase and F1 enzymolysis among Fig. 4; B has shown the Base Peak collection of illustrative plates of the rBAT of reductive trypsinase and F1 enzymolysis.
A has shown the Base Peak collection of illustrative plates of the rBAT of non-reducing Quimotrase and N-Glycosylase F enzymolysis among Fig. 5; B has shown the Base Peak collection of illustrative plates of the rBAT of reductive Quimotrase and N-Glycosylase F enzymolysis.
A has shown the Base Peak collection of illustrative plates of the rBAT of non-reducing trypsinase and N-Glycosylase F enzymolysis among Fig. 6; B has shown the Base Peak collection of illustrative plates of the rBAT of reductive trypsinase and N-Glycosylase F enzymolysis.
Fig. 7 A-7D has shown the detection collection of illustrative plates of disulfide linkage C7-C139; Wherein
7A is the mass-to-charge ratio m/z collection of illustrative plates that comprises the peptide section of disulfide linkage C7-C139;
7B is the MS/MS collection of illustrative plates that comprises the peptide section of disulfide linkage C7-C139;
7C is the MS/MS collection of illustrative plates that comprises halfcystine C7;
7D is the MS/MS collection of illustrative plates that comprises halfcystine C139.
Fig. 8 A-8B has shown the detection collection of illustrative plates of disulfide linkage C26-C42; Wherein
8A is the mass-to-charge ratio m/z collection of illustrative plates that comprises the peptide section of disulfide linkage C7-C139;
8B is the MS/MS collection of illustrative plates that comprises halfcystine C26 and C42.
Fig. 9 A-9D has shown the detection collection of illustrative plates of disulfide linkage C74-C230; Wherein
9A is the mass-to-charge ratio m/z collection of illustrative plates that comprises the peptide section of disulfide linkage C74-C230;
9B is the MS/MS collection of illustrative plates that comprises the peptide section of disulfide linkage C74-C230;
9C is the MS/MS collection of illustrative plates that comprises halfcystine C74;
9D is the MS/MS collection of illustrative plates that comprises halfcystine C230.
Figure 10 A-10C has shown the detection collection of illustrative plates of disulfide linkage C118-C184; Wherein
10A is the mass-to-charge ratio m/z collection of illustrative plates that comprises the peptide section of disulfide linkage C118-C184;
10B is the MS/MS collection of illustrative plates that comprises the peptide section of disulfide linkage C118-C184;
10C is the MS/MS collection of illustrative plates that comprises halfcystine C118.
Figure 11 A-11C has shown the detection collection of illustrative plates of disulfide linkage C150-C1163; Wherein
11A is the mass-to-charge ratio m/z collection of illustrative plates that comprises the peptide section of disulfide linkage C150-C163;
11B is the MS/MS collection of illustrative plates that comprises the peptide section of disulfide linkage C150-C163;
11C is the MS/MS collection of illustrative plates that comprises halfcystine C150.
Figure 12 A-12D has shown the detection collection of illustrative plates of disulfide linkage C174-C199; Wherein
12A is the mass-to-charge ratio m/z collection of illustrative plates that comprises the peptide section of disulfide linkage C174-C199,
12B is the MS/MS collection of illustrative plates that comprises the peptide section of disulfide linkage C174-C199;
12C is the MS/MS collection of illustrative plates that comprises halfcystine C174;
12D is the MS/MS collection of illustrative plates that comprises halfcystine C199.
Figure 13 has shown fermented liquid, the non-reduced electrophoretogram of ultrafiltrated; Wherein swimming lane 1 is represented protein marker (Marker), 2 expression ultrafiltration primary fermentation supernatant liquors, 3 expression ultrafiltration effluent liquid, 4 expression ultrafiltration secondary fermentation supernatant liquors.
Figure 14 has shown wash-out collection of illustrative plates and the electrophoretogram (non-reduced SDS-PAGE) behind the cation-exchange chromatography; Wherein swimming lane 1 is represented protein marker (Marker), 2 expression ultrafiltration secondary fermentation supernatants, 3 expression cationic exchange effluent liquid, 4 expression 20mM NaAc-HAc+0.15MNaCl elutriants, 5 expression 20mM NaAc-HAc+0.50MNaCl elutriants, 6 expression 20mM NaAc-HAc+1.0MNaCl elutriants.
Figure 15 has shown wash-out collection of illustrative plates and the electrophoretogram (non-reduced SDS-PAGE) behind the anion-exchange chromatography; Wherein swimming lane 1 is represented protein marker (Marker), 2 expression cationic exchange target protein elutriants, sample effluent liquid in the 3 expression anionresins, 4 expression 20mM Tris-HCl+0.15MNaCl elutriants, 5 expression 20mM Tris-HCl+0.50MNaCl elutriants.
Figure 16 has shown wash-out collection of illustrative plates and the electrophoretogram behind the gel permeation chromatography; Wherein swimming lane 1 is represented protein marker (Marker), 2 expression ultrafiltration secondary fermentation supernatants, and 3 expression cationic exchange rBAT elutriants, 4 expression anionresin rBAT elutriants, 5-7 represents rBAT stoste.
Figure 17 has shown the aminoacid sequence (SEQ ID NO:1) of batroxobin, and wherein glycosylation site is represented with underscore.
Embodiment
The contriver by the expression and the purifying process of improvement batroxobin, thereby has made the recombinant batroxobin that a kind of height ratio is lived through extensive and deep research.It has following characteristic:
(a) molecular weight is 30-32Kda;
(b) at least 90% batroxobin has 6 couples of correct disulfide linkage pairing: Cys in the described recombinant batroxobin
7-Cys
139, Cys
26-Cys
42, Cys
74-Cys
230, Cys
118-Cys
184, Cys
150-Cys
163And Cys1
174-Cys
199
(c) in SEQ ID NO:1 the 146th and the 225th by glycosylation; With
(d) specific activity of described batroxobin is greater than 1500KU/mg albumen.
After having realized successfully that in methanol yeast (Pichia pastoris) secreting, expressing has Brazilian fer-de-lanc (Bothropsatrox) batroxobin of biologic activity, obtain purity through the multistep chromatography purification and be not less than 95% pure product of batroxobin.
As used herein, 1KU gets people's standard blood plasma of 0.1ml in standard coagulo meter (C2000-4 coagulo meter, Pulisheng Instruments Co., Ltd., Beijing) detects in the cup, 37 ℃ pre-warm 3 minutes, add batroxobin solution 0.1ml to be determined through an amount of dilution, timing if blood plasma solidified at 60 ± 20 seconds, just contains the batroxobin of a Ke Shi unit (KU) in this solution to be measured of then every 1ml.
The single chain protein that gene recombination batroxobin provided by the invention is made up of 231 amino-acid residues (SEQ ID NO:1), molecular weight is 29-32Kda, it has two N-glycosylation site: Asn
146-Asn
147-Thr
148And Asn
225-Lys
226-Thr
228
At least 90% has 6 pairs of correct disulfide linkage pairings in the gene recombination batroxobin provided by the invention, and its disulfide linkage mode of connection is: Cys
7-Cys
139, Cys
26-Cys
42, Cys
74-Cys
230, Cys
118-Cys
184, Cys
150-Cys
163And Cys
174-Cys
199Preferably 〉=95%, more preferably 〉=98% batroxobin has correct disulfide linkage pairing.
The specific activity of gene recombination batroxobin provided by the invention preferably is 1500-2000KU/mg more than or equal to 1500KU/mg albumen, more preferably is 1500-3000KU/mg.
Gene recombination batroxobin provided by the invention utilizes the codon of methanol yeast hobby, synthetic the batroxobin gene sequence, construction of expression vector has finally obtained Brazilian fer-de-lanc (Bothrops atrox) batroxobin that secreting, expressing has biologic activity in methanol yeast (Pichia pastoris).The contriver finds can guarantee in the eukaryotic cell expression system (yeast, CHO and insect cell etc.) the back modification of higher disulfide linkage pairing accuracy and expressing protein.The contriver is at methanol yeast (Pichia pastoris, Pichia methanolica) successfully given expression to the batroxobin protein that biologic activity is arranged in, output can reach every milliliter of fermented liquid 20 Ke Shi units (20KU/ml), and this output possesses the meaning of exploitation.
The fermentation supernatant can obtain the recombinant batroxobin of purifying through separation and purification.In a preference of the present invention, described purification step comprises ultrafiltration, cation-exchange chromatography, anion-exchange chromatography and gel chromatography, by the chromatography condition of optimizing, obtains disulfide linkage pairing accuracy height, high-purity product of recombinant batroxobin that specific activity is strong.
Contain a large amount of foreign proteins, inorganic salt, pigment or the like in the fermentation supernatant that the present invention obtains, therefore, the present invention adopts the method desalination of ultrafiltration to remove foreign protein and exchange buffering liquid earlier, is beneficial to the ion exchange chromatography operation of back.The condition that the present invention carries out ultrafiltration is intake pressure 5-10psi, top hole pressure 2-5psi, flow velocity 100-200ml/min.Ultrafiltration level pad pH4-6, preferably pH4.5-5.5.Can this area salt commonly used in the described ultrafiltration equalizing and buffering system, such as but not limited to: acetic acid sodium salt, sodium phosphate salt, sodium citrate salt, Tris-HCl, wherein preferred sodium-acetate-acetic acid and sodium-chlor.Resulting ultrafiltrated electric conductivity value is below the 8mS/cm, and preferably electric conductivity value is below the 5mS/cm, and more preferably electric conductivity value is below the 4mS/cm.
Then, the fermentation supernatant of the present invention after to ultrafiltration carries out cation-exchange chromatography.Can use this area cation-exchange chromatography post commonly used, such as but not limited to: SP SepheroseFF, CM SepheroseFF,, preferred SP SepheroseFF.Elution buffer pH4-6, preferably pH4.5-5.5; Can be this area salt commonly used, such as but not limited to: sodium-acetate, sodium phosphate, sodium-chlor, wherein preferred sodium-acetate-acetic acid and sodium-chlor.Wherein the concentration of sodium-chlor is 0.1-1.0M, preferably is 0.15-1.0M, more preferably is 0.4-0.6M.A kind of preferred type of elution is a gradient elution.
The 3rd step was that the elutriant behind the cation-exchange chromatography post is carried out anion-exchange chromatography.Can use this area anion-exchange chromatography post commonly used, such as but not limited to: Q Sepherose, DEAE Sepherose, Source30Q, preferred Q SepheroseFF.Elution buffer pH7.5-10, preferably pH8.5-9.5; Can be this area salt commonly used, such as but not limited to: Tris-hydrochloric acid, sodium phosphate, sodium-chlor, wherein preferred Tris-HCl and sodium-chlor.Wherein the concentration of sodium-chlor is 0.05-1.0M, preferably is 0.10-0.6M, more preferably is 0.15-0.5M.A kind of preferred type of elution is a gradient elution.
At last, will carry out gel permeation chromatography through the elutriant behind the anion-exchange chromatography post.Can use this area gel permeation chromatography post commonly used, such as but not limited to: Sephacryl S, Sepharose4, Sephadex G-25, Superdex, preferred Superdex75.Elution buffer pH4-6, preferably pH4.5-5.5; Can be this area salt commonly used, such as but not limited to: sodium-acetate, sodium phosphate, sodium-chlor, wherein preferred sodium-acetate-acetic acid and sodium-chlor.Wherein the concentration of sodium-chlor is 0.05-0.5M, preferably is 0.10-0.3M, more preferably is 0.12-0.2M.
Through above-mentioned steps, obtain the gene recombination batroxobin of purifying provided by the invention.Also can use gene engineering method well known in the art to obtain containing the fermented liquid of batroxobin protein, obtain gene recombination batroxobin provided by the invention through above-mentioned purification step then.
Gene recombination batroxobin provided by the invention can be used for hemostasis.The specific effect substrate of batroxobin is a Fibrinogen, and different with zymoplasm is, the A chain of its cutting fibre proteinogen, and do not act on the B chain.When the Arg16-Gly17 position peptide bond in its hydrolysis plasma fibrinogen A chain, can discharge fibrinopeptide A, thereby apace the Fibrinogen in the blood is transformed into scleroproein, then these scleroproeins just can be gathered into loose being easy to and come wound closure by the thrombus of scleroproein enzymic hydrolysis, the effect of realization quick-acting haemostatic powder.But, using heavy dose of time marquis, batroxobin can reduce fibrinogen concentration in the blood, improves the hydrodynamic characteristic of blood viscosity and blood, thereby plays the effect (US Pat.No:3849252,1974) of fiber eliminating enzyme.
The present invention also provides a kind of pharmaceutical composition, and it contains the gene recombination batroxobin and the pharmaceutically acceptable carrier of purifying provided by the invention.
Can also contain gelatin hydrolysate in the pharmaceutical composition provided by the invention as stablizer.Pharmaceutical composition provided by the invention can be solid-state form or liquid form, wherein preferred liquid form, more preferably injection liquid.
Major advantage of the present invention is:
1, gene recombination batroxobin disulfide linkage pairing accuracy provided by the invention is high.
2, gene recombination batroxobin specific activity provided by the invention is very high.
3, the invention provides the separation purification method that obtains above-mentioned high-quality gene recombination batroxobin.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all per-cent and umber by weight.
Preparation embodiment
One, obtains to contain the fermented liquid of batroxobin protein
The fermentation of rBAT engineering bacteria in the 30L jar:
Inoculative proportion access sterilization in advance by 1:10 is equipped with in the 30L fermentor tank of 15L batch fermentation substratum, carry out the batch feeding cultivation and (pH is controlled at 4.0 with ammoniacal liquor, temperature is controlled at 30 ℃), (DO suddenly rises after carbon source exhausts, in 1 minute), stream glycerol adding (the stream rate of acceleration is kept dissolved oxygen greater than 20%).When the thalline weight in wet base is to stop feed supplement about 200 gram/L, after glycerine exhausts, mend methanol induction and express (pH is controlled to be 5.8 with ammoniacal liquor, and temperature is controlled at 20 ℃), make dissolved oxygen greater than 20% by regulating rotating speed, tank pressure, air flow quantity and feed rate, induction time is 60 hours.Fermentation ends, the centrifugal collection fermentation of 4000rpm supernatant is measured active affirmation expression output and should be not less than 20KU/ml.To ferment, go up please be frozen or directly enter purge process.
Two, separation and purification
The first step: ultrafiltration exchange buffering liquid.Select Millipore Pellicon10K ultrafiltration membrane stack for use, Millipore Masterflex peristaltic pump, ultra-fine filter intake pressure, top hole pressure are controlled to be 6psi, 3psi respectively, flow velocity is 120ml/min, ultra-filtration membrane carries out ultrafiltration to fermented supernatant fluid after successively using water for injection, damping fluid 20mM NaAc-HAc+0.15M NaCl (pH5.0) balance good.When five/for the moment of the extremely original volume of ultrafiltration, add damping fluid 20mM NaAc-HAc (pH5.0) to original volume, so repeatable operation is three times, adds the extremely original volume of damping fluid 20mM NaAc-HAc (pH5.0) at last as the cation-exchange chromatography sample solution.
Second step: cation-exchange chromatography: filler SP Sepharose FF.Level pad is 20mM NaAc-HAc (pH5.0), elution buffer is respectively 20mM NaAc-HAc+0.15M NaCl (pH5.0), 20mM NaAc-HAc+0.50M NaCl (pH5.0), 20mM NaAc-HAc+1.0M NaCl (pH5.0), collects 20mM NaAc-HAc+0.50M NaCl (pH5.0) elutriant.
The 3rd step: anion-exchange chromatography Q Sepharose FF.Cationic exchange 20mM NaAc-HAc+0.50M NaCl (pH5.0) elutriant is regulated pH value to 9.0, the dilution electricity is directed at 3.0mS/cm and goes up sample, level pad is 20mM Tris-HCl (pH9.0), elution buffer is respectively 20mM Tris-HCl+0.15M NaCl (pH9.0), 20mM Tris-HCl+0.50M NaCl (pH9.0) collects 20mM Tris-HCl+0.15M NaCl (pH9.0) elutriant.
The 4th step: gel permeation chromatography Superdex75 filler, 6.0 * 60cm prepacked column, CV1700ml.Damping fluid adopts 20mMNaAc-HAc+0.15M NaCl (pH5.0), anion-exchange chromatography 20mM Tris-HCl+0.15M NaCl (pH9.0) elutriant is gone up sample in batches, Fractional Collections rBAT main peak merges purity greater than 95% sample, be rBAT stoste I after the degerming.
Performance embodiment
The rBAT stoste I that embodiment 1 is made detects:
1. experiment significant parameter:
Detecting instrument model: LCQ DECA XP plus input mode: Microspray
Capillary temperature: 170 ℃ of chromatographic column: 0.15MM*150MM (RP-C18)
Instrument company: FINNIGAN detection mode: positive ion
2. experimental technique:
Sample rBAT ultrafiltration desalination, iodo-acid amide (IAA) modification → trypsin Trypsin) and Quimotrase (Chymotrypsin) enzymolysis → N-Glycosylase F and F1 enzymolysis → 1/2 enzymolysis product dithiothreitol (DTT) (DTT) reduction, IAA modification → mass spectroscopy → data analysis, determine rBAT proteinic C terminal sequence, glycosylation site and disulfide linkage matching method.
3. experimental result and analysis:
3.1C terminal sequence analysis
The principle that PROTEIN C-terminal sequence mass spectroscopy is identified: with proteolytic enzyme testing protein is carried out enzymolysis earlier, obtain containing the peptide section of C terminal sequence, the similarity (Xcorr) of second order ms that obtains by comparative experiments and the theoretical second order ms that obtains then, thus the C-terminal sequence is verified.
Handle if N-Glycosylase of no use (PNGase F) carries out desugar to rBAT albumen, then can not confirm the proteinic C-terminal sequence of rBAT.Yet after rBAT proteolysis product being carried out the desugar processing with N-Glycosylase (PNGase F), experimental data and the theoretical second order ms that obtains meet finely, and its C-terminal sequence can be verified as IQSIIAGDKTATCP.Notice that PNGase F also changes Asn into Asp when removing the sugar chain that is connected on the Asn, simultaneously, Cys is also modified, and becomes to be acetylizad halfcystine.Experimental result as shown in Figure 1.Observe a series of b and y ion in the experiment, as b2, b3, b5-b12, y1-y4, y6, y8-y12.B and y are modal fragmentions in the experiment, because the fracture of the peptide chain of peptide section produces.If post-rift electric charge then is the b ion at the N of peptide section end, if electric charge then is the y ion at the C of peptide section end.As the y1-8 ion among this figure, it is GDKTATCP
+, y represents that this ion is a y series ion, the charge number of 1 this ion band of expression is 1,8 position of explanation peptide bond rupture then.
3.2 determining of glycosylation site
Glycosyl turns to an appraisal principle: with proteolytic enzyme testing protein is carried out enzymolysis earlier, obtain the peptide section of different sizes.Then, the sample behind the part enzymolysis excises the sugar chain that is connected on the Asn with PNGase F, and changes Asn into Asp.Then the sample of handling and not handling through desugar through desugar being carried out HPLC-MS-MS respectively analyzes.Peptide section fraction of coverage by comparative experiments obtains can identify whether glycosylation modified and glycosylation modified site is arranged.
Two N-glycosylation sites are found in experiment, and one is Asn
146, another is Asn
225Asn
225Fig. 1 is seen in the evaluation of glycosylation site.Asn
146Fig. 2 is seen in the evaluation of glycosylation site.
Attention: the N-Glycosylase with the N-sugar chain excision after, change Asn into Asp simultaneously, simultaneously, cys is also modified, and becomes to be acetylizad halfcystine.The b and the y ion of the overwhelming majority all are detected, as b1-b8, and y1-8, this has fully confirmed the sequence of this peptide section.
3.3 disulfide linkage matching method
Mass spectroscopy is identified the principle of disulfide linkage: at first use iodo-acid amide (IAA) that the free halfcystine of testing protein is closed, under non-reduced state testing protein is carried out enzymolysis with proteolytic enzyme then.Part enzymolysis product directly carries out HPLC-MS-MS to be analyzed, and another part is then used dithiothreitol (DTT) (DTT) reduction, with iodo-acid amide halfcystine is modified then, prevents to form new disulfide linkage.At last, by the difference of more detected peptide section, draw the new peptide section that produces by the DTT reduction, thereby infer the mode of connection of disulfide linkage.At last, under non-reduced state the second order ms figure of enzymolysis product,, thereby confirm the mode of connection of disulfide linkage by the molecular weight and the second order ms figure of contrast parent ion.
Because in rBAT albumen, exist glycosylation modified, and from top data, find, the existence of halfcystine is arranged in glycosylation modified site, this can have influence on detecting of disulfide linkage matching method, therefore, behind protease hydrolyzed, we have added the step of removing sugar chain with PNGase F, are the condition of providing convenience that detects of disulfide linkage matching method like this.We have designed the non-reduced and rBAT-Quimotrase of rBAT-Quimotrase-N-Glycosylase F--N-Glycosylase F-reduction, rBAT-trypsinase-N-Glycosylase F-is non-reduced and rBAT-trypsinase-N-Glycosylase F-reduction, rBAT-Quimotrase-F1-is non-reduced and rBAT-Quimotrase-F1-reduction, rBAT-trypsinase-F1-is non-reduced and rBAT-trypsinase-F1-reduces four groups of control experiments.From these four groups of data, we have analyzed 75 kinds of possible peptide sections that comprise disulfide linkage, totally 47 kinds of disulfide linkage matching methods.Use strict screening criteria, six pairs of disulfide linkage are detected: C7-C139, C26-C42, C74-C230, C118-C184, C150-C163 and C174-C199; Wei Liang halfcystine peptide section C7, the C118 and the C150 that do not form disulfide linkage also is detected in addition.Concrete outcome is seen experimental patterns 3-6 and Fig. 8-12.
To the peptide section by disulfide-bonded, because it comprises two sections peptide chains, the mode of its fracture is many.It may be some splitting of chain wherein, and the another one peptide chain by disulfide-bonded then remains unchanged.Also might be that two peptide chains rupture respectively.At this, be example with the peptide section that is connected with Cys139 by Cys7, the decision method of disulfide linkage mode of connection is described.In the non-reduced and rBAT-Quimotrase of rBAT-Quimotrase-N-Glycosylase F--N-Glycosylase F-reductive experiment, we detect VIGGDECDINEHPF and these two peptide sections of GAITTSEDTYPDVPHCANIN (Fig. 7 C and Fig. 7 D), and in rBAT-Quimotrase-non-reduced experiment of N-Glycosylase F-, not being detected of this two peptide sections.In non-reduced sample, we detect specific charge is 916.1Da, the peak of 1221.3Da and 1831.4Da (as Fig. 7 A), by the analysis of deconvoluting, find that these three peaks all are 3660.3 peptide section corresponding to molecular weight, they are respectively the tetravalence of this peptide section, trivalent and divalent ion.And the molecular weight of the peptide section that is connected by disulfide linkage by VIGGDECDINEHPF and GAITTSEDTYPDVPHCANIN just is 3660.9Da..Thus, can infer that these two sections peptides are by disulfide-bonded.Further the second order ms data acknowledgement this deduction.Fig. 7 B is by the VIGGDECDINEHPF of disulfide linkage connection and the second order ms of GAITTSEDTYPDVPHCANIN.In the experiment, detect a series of C
139-y2 ion (Cys
139-y2-5 is to Cys
139-y2-18).This has fully confirmed the existence of its disulfide linkage.With Cys
139-y2-10 is an example, and its detected specific charge is 1311.33Da.C
139Represent that this ion is by containing Cys
139The fracture of the peptide chain of residue produces, above the another one peptide chain then still is connected by disulfide linkage; Y2 illustrates that this ion is the ion of y series, and has 2 electric charges; Position (Y-P) key of 10 expression peptides bond rupture.
The result shows that the rBAT disulfide linkage pairing accuracy among the prepared rBAT stoste I is 〉=95%.
3.4 the rBAT albumen stoste I that obtains behind the chromatography purification by fermentation:
Purity: 98%
Concentration: 1.02mg/ml
Specific activity: 2000KU/mg
Purifying process research embodiment
1. hydrophobic chromatography Study on Conditions
Because minimal medium (pH value about 6.0) is adopted in fermentation, so the fermentation supernatant contains higher salt, electricity is led higher, thus want to adopt hydrophobic chromatography as the purifying the first step, thick pure rBAT.
1.1pH6.0 the time hydrophobic chromatography Study on Conditions
Get fermentation supernatant 100ml respectively, press following operation:
| Fermentation supernatant volume (ml) | 3M ammonium sulfate volume (ml) | Water for injection volume (ml) | Cumulative volume (ml) | Ammonium sulfate final concentration (M) | |
| The |
100 | 150 | 50 | 300 | 1.5 |
| The |
100 | 100 | 100 | 300 | 1.0 |
| The |
100 | 50 | 150 | 300 | 0.5 |
Above-mentioned A, B, C group, regulate pH value to 6.0 after, 12000rpm, room temperature, centrifugal 10 minutes collect supernatant, measure activity respectively.
Select Phenyl Sepharose FastFlow1ml prepacked column for use, through damping fluid 1:20mM PB, 1.5M (NH
4)
2SO
4, after the pH6.0 balance, last A group (going up sample volume 100ml, as follows) is collected effluent liquid, measures active; After the regeneration of Phenyl Sepharose FastFlow1ml prepacked column, through damping fluid 2:20mMPB, 1.0M (NH
4)
2SO
4, after the pH6.0 balance, last B group is collected effluent liquid, measures active; After the regeneration of Phenyl Sepharose FastFlow1ml prepacked column, through damping fluid 3:20mM PB, 0.5M (NH
4)
2SO
4After the pH6.0 balance, last C group is collected effluent liquid, measures active.Result such as following table:
| The fermentation supernatant | A | B | C | |
| Sample solution activity (KU/ml) | 10 | 3 | 3 | 3 |
| Effluent liquid activity (KU/ml) | 1 | 1 | 2 |
By last table data as seen, rBAT combines badly with Phenyl Sepharose Fast Flow with this understanding, has quite a few to flow out, so this kind condition is unsuitable for the purifying of rBAT.
1.2pH5.0 the time hydrophobic chromatography Study on Conditions
Get fermentation supernatant 100ml respectively, press following operation:
| Fermentation supernatant volume (ml) | 3M ammonium sulfate volume (ml) | Water for injection volume (ml) | Cumulative volume (ml) | Ammonium sulfate final concentration (M) | |
| The |
100 | 150 | 50 | 300 | 1.5 |
| The |
100 | 100 | 100 | 300 | 1.0 |
| The |
100 | 50 | 150 | 300 | 0.5 |
Above-mentioned D, E, F group, regulate pH value to 5.0 after, 12000rpm, room temperature, centrifugal 10 minutes collect supernatant, measure activity respectively.
Select Phenyl Sepharose Fast Flow 1ml prepacked column for use, through damping fluid 4:20mM NaAc-HAc, 1.5M (NH
4)
2SO
4, after the pH5.0 balance, last D group (going up sample volume 100ml, as follows) is collected effluent liquid, measures active; After the regeneration of PhenylSepharose FastFlow1ml prepacked column, through damping fluid 5:20mM NaAc-HAc, 1.0M NH
4)
2SO
4, after the pH5.0 balance, last E group is collected effluent liquid, measures active; After the regeneration of Phenyl Sepharose FastFlow1ml prepacked column, through damping fluid 6:20mM NaAc-HAc, 0.5M (NH
4)
2SO
4, after the pH5.0 balance, last F group is collected effluent liquid, measures active.Result such as following table:
| The fermentation supernatant | D | E | F | |
| Sample solution activity (KU/ml) | 10 | 3 | 3 | 3 |
| Effluent liquid activity (KU/ml) | 1 | 1 | 2 |
After pH reduced, the proteic hydrophobicity of rBAT increased to some extent, compares during with pH6.0, and the activity of effluent liquid does not have much variations, still has the outflow of considerable part, did further research so should change dewatering filling.
1.3pH5.0 the time, the research of Butyl and Octyl filler
Get fermentation supernatant 100ml respectively, press following operation:
| Fermentation supernatant volume (ml) | 3M ammonium sulfate volume (ml) | Water for injection volume (ml) | Cumulative volume (ml) | Ammonium sulfate final concentration (M) | |
| The |
100 | 150 | 50 | 300 | 1.5 |
| The |
100 | 150 | 50 | 300 | 1.5 |
Above-mentioned G, H group, regulate pH value to 5.0 after, 12000rpm, room temperature, centrifugal 10 minutes collect supernatant, measure activity respectively.
Select Butyl Sepharose FastFlow1ml prepacked column for use, through damping fluid 7:20mM NaAc-HAc, 1.5M (NH
4)
2SO
4, after the pH5.0 balance, last G group (going up sample volume 100ml, as follows) is collected effluent liquid, measures active; Select Octyl Sepharose FastFlow 1ml prepacked column for use, through damping fluid 8:20mM NaAc-HAc, 1.5M (NH
4)
2SO
4, after the pH5.0 balance, last H group is collected effluent liquid, measures active.Result such as following table:
| The fermentation supernatant | G | H | |
| Sample solution activity (KU/ml) | 10 | 3 | 3 |
| Effluent liquid activity (KU/ml) | 1 | 1 |
With this understanding, rBAT does not improve with combining of filler, and the ratio of rBAT is still very high in the effluent liquid.
In sum, because the bonding properties of rBAT and dewatering filling is bad, even reduced the pH value of entire operation system, the effect that improves is little, and above operation is all at room temperature carried out, if the temperature when increasing operation again may be influential to the proteic activity of rBAT to improve hydrophobicity, so this points out hydrophobic chromatography and is not suitable for the proteic purifying of rBAT.
2. cation-exchange chromatography Study on Conditions
Owing to contain a large amount of foreign proteins, inorganic salt, pigment or the like in the fermentation supernatant, intend adopting the method desalination of ultrafiltration to remove foreign protein and exchange buffering liquid, be beneficial to the ion exchange chromatography operation of back.But owing to do not know which kind of damping fluid rBAT albumen adopts be fit to ion exchange chromatography, change which kind of damping fluid when promptly not knowing ultrafiltration, so should at first study the absorption behavior of the ion exchange chromatography of rBAT albumen under different condition,, study the condition of ultrafiltration again to determine damping fluid.
2.1 it is preferred that best pH of sample solution and electricity are led
According to the iso-electric point (theoretical iso-electric point is 7.39) of rBAT, fermented supernatant fluid, regulating the pH value respectively with the HAc of 1M is 4.5,5.0,5.5 and 6.0.Under each pH value, respectively the fermentation supernatant is diluted to electricity with water for injection and leads as shown in the table.Select 1ml SP Sepharose Fast Flow prepacked column for use, applied sample amount is a 50ml fermentation supernatant (before the dilution), and flow velocity is 1.0ml/min, and the collection effluent liquid is also measured active, carries out the sample solution condition optimization, and the result is as follows:
PH=4.5, level pad 20mM NaAc-HAc, pH4.5
| The electric conductivity value of sample solution (mS/cm) | 4.0 | 8.0 | 12.0 | 16.0 |
| The fermented |
12 | 6 | 4 | 3 |
| Clotting time | Blood coagulation in 100 seconds | Blood coagulation in 35 seconds | Blood coagulation in 22 seconds | Blood coagulation in 16 seconds |
| Clotting time | Not blood coagulation in 10 fens | Blood coagulation in 305 seconds | Blood coagulation in 125 seconds | Blood coagulation in 75 seconds |
PH=5.0, level pad 20mM NaAc-HAc, pH5.0
| The electric conductivity value of sample solution (mS/cm) | 4.0 | 8.0 | 12.0 | 16.0 |
| The fermented |
12 | 6 | 4 | 3 |
| Clotting time | Blood coagulation in 98 seconds | Blood coagulation in 35 seconds | Blood coagulation in 23 seconds | Blood coagulation in 15 seconds |
| Clotting time | Not blood coagulation in 10 fens | Blood coagulation in 280 seconds | Blood coagulation in 108 seconds | Blood coagulation in 64 seconds |
PH=5.5, level pad 20mM NaAc-HAc, pH5.5
| The electric conductivity value of sample solution (mS/cm) | 4.0 | 8.0 | 12.0 | 16.0 |
| The fermented |
12 | 6 | 4 | 3 |
| Clotting time | Blood coagulation in 98 seconds | Blood coagulation in 36 seconds | Blood coagulation in 25 seconds | Blood coagulation in 15 seconds |
| Clotting time | Blood coagulation in 450 seconds | Blood coagulation in 265 seconds | Blood coagulation in 85 seconds | Blood coagulation in 56 seconds |
PH=6.0, level pad 20mM NaAc-HAc, pH6.0
| The electric conductivity value of sample solution (mS/cm) | 4.0 | 8.0 | 12.0 | 16.0 |
| The fermented |
12 | 6 | 4 | 3 |
| Clotting time | Blood coagulation in 102 seconds | Blood coagulation in 36 seconds | Blood coagulation in 25 seconds | Blood coagulation in 16 seconds |
| Clotting time | Blood coagulation in 380 seconds | Blood coagulation in 240 seconds | Blood coagulation in 63 seconds | Blood coagulation in 48 seconds |
By each data in the above table as can be known, under the condition of pH=4.5 and 5.0, when the electricity of diluent is led to 4.0mS/cm, rBAT does not flow out basically, considers the influence of pH value to the rBAT protein-active, so select for use the fermentation supernatant is regulated pH value to 5.0, the dilution electricity is directed at 4.0mS/cm, level pad is 20mM NaAc-HAc, and the condition of pH5.0 is studied the wash-out behavior of rBAT albumen on cation-exchange chromatography.So the damping fluid that ultrafiltration is changed is 20mM NaAc-HAc, pH5.0, and answer ultrafiltration to electricity to lead below the 4.0mS/cm.
2.2 ultrafiltration Study on Conditions
Adopt Millipore Pellicon10K ultra-fine filter, Millipiore Masterflex peristaltic pump, operating parameters: intake pressure 10psi, top hole pressure 5psi, flow velocity 200ml/min.Successively with water for injection, damping fluid 20mM NaAc-HAc, pH5.0 is good with the ultra-fine filter balance, gets the 5L fermented liquid then and begins ultrafiltration, when remaining the 1L fermented liquid, add damping fluid 20mM NaAc-HAc, pH5.0 to 5L circulates 3 times so repeatedly, uses damping fluid 20mM NaAc-HAc at last, pH5.0 complements to 5L (pH5.0 after measured, electricity is led 3.5mS/cm), get fermented liquid, ultrafiltrated, effluent liquid mensuration activity respectively, the result is as follows:
| Fermented liquid | Ultrafiltrated | Effluent liquid | |
| Volume (L) | 5 | 5 | 12 |
| Active (KU/ml) | 10 | 8 | Blood coagulation in 150 seconds |
Ultrafiltration is lost in about 20% under this condition, doubt for the nonspecific absorption of membrane stack or working pressure excessive due to, so do when testing for the second time, earlier intake pressure, top hole pressure are adjusted into 6psi, 3psi respectively with injection water, flow velocity is adjusted into 120ml/min, with damping fluid 20mM NaAc-HAc, 0.15M NaCl, pH5.0 balance ultrafiltration system.Get the 5L fermented liquid and begin ultrafiltration, when remaining the 1L fermented liquid, add damping fluid 20mM NaAc-HAc, pH5.0 to 5L circulates 3 times so repeatedly, uses damping fluid 20mMNaAc-HAc at last, pH5.0 complements to 5L (pH5.0 after measured, electricity is led 3.5mS/cm), get fermented liquid, ultrafiltrated, effluent liquid mensuration activity respectively, the result is as follows:
| Fermented liquid | Ultrafiltrated | Effluent liquid | |
| Volume (L) | 5 | 5 | 12 |
| Active (KU/ml) | 10 | 9 | Blood coagulation in 10 minutes |
The result shows that by ultrafiltration exchange buffering liquid, the active yield of rBAT is on 90%.Fermentation supernatant after the ultrafiltration is very beneficial for next step cation-exchange chromatography.
2.3 the research of cation-exchange chromatography elution requirement
Select 1ml SP Sepharose F F prepacked column for use, level pad A:20mM NaAc-HAc, pH5.0, elution buffer is B:20mM NaAc-Hac, 1.0M NaCl, pH5.0, with sample on the flow velocity of 1.0ml/min, last sample finishes the back with level pad A balance, carries out gradient elution 50ml ultrafiltration secondary fermentation supernatant liquor, condition is 0-100%B, 20CV, flow velocity 1.0ml/min found that and contain three elution peaks altogether in the elution process of 0-100%B, go out the peak position and lay respectively at 18%B, 45%B and 60%B place, sampling is surveyed and is lived respectively; With 2M NaCl regeneration pillar, collection regeneration peak is also surveyed and is lived at last.All results are as follows:
| 18% peak leading portion | Stage casing, 18% peak | 18% peak back |
45% peak leading portion | Stage casing, 45% peak | |
| Active (KU/ml) | Not blood coagulation in 10 |
1 | 10 | 40 | 300 |
| 45% peak back |
60% peak leading portion | Stage casing, 60 |
60% peak back segment | The 2M peak of regenerating | |
| Active (KU/ml) | 50 | 8 | 125 seconds | Not blood coagulation in 10 fens | Not blood coagulation in 10 fens |
By last table data as can be known, rBAT albumen mainly concentrates in 45%B (0.45MNaCl) elution peak, but a spot of rBAT albumen is also arranged at the posterior segment of 18%B (0.18MNaCl) elution peak, the middle leading portion of 60%B (0.60MNaCl) elution peak, so should suitably reduce 18%B, 45%B suitably raises, and makes rBAT albumen mainly concentrate in the elution peak.Therefore repeat above process of the test, just adopt 15%B, 50%B and 100%B stepwise elution when wash-out, collect each elution peak respectively, measure activity, the result is as follows:
| 15% peak leading portion | Stage casing, 15 |
15% peak back |
50% peak leading portion | Stage casing, 50% peak | |
| Active (KU/ml) | Not blood coagulation in 10 fens | Not blood coagulation in 10 fens | Blood coagulation in 135 |
50 | 320 |
| 50% peak back |
100% peak leading portion | Stage casing, 100% peak | 100% peak back segment | The 2M peak of regenerating | |
| Active (KU/ml) | 65 | Blood coagulation in 114 seconds | Not blood coagulation in 10 fens | Not blood coagulation in 10 fens | Not blood coagulation in 10 fens |
By the data of last table as can be known, the rBAT albumen overwhelming majority concentrates in 50%B (0.50M NaCl) elution peak, the back segment of 15%B (0.15MNaCl) elution peak and 100%B (1.0M NaCl) are though the leading portion of elution peak has a spot of rBAT albumen, but with respect to fermented liquid, activity is very low, so at last elution requirement is defined as 20mM NaAc-HAc, 0.15M NaCl, pH5.0,20mMNaAc-HAc, 0.50MNaCI, pH5.0 and 20mM NaAc-HAc, 1.0M NaCl, pH5.0, collect 20mM NaAc-HAc, 0.50MNaCl the pH5.0 elution peak is the rBAT protein peak.
2.4 the analysis and research of cation-exchange chromatography process
Selecting specification for use is the chromatography column of 1.6x15cm, interior dress 20ml SP Sephrose FF filler, and last sample flow velocity is 10ml/min, sample on the 1000ml ultrafiltration secondary fermentation supernatant liquor.Level pad is 20mM NaAc-HAc, pH5.0, elution buffer is respectively 20mMNaAc-HAc, 0.15M NaCl, pH5.0,20mM NaAc-HAc, 0.50M NaCl, pH5.0 and 20mM NaAc-HAc, 1.0MNaCl, pH5.0 collects each elution peak (1.0M elution peak pigment is heavier) respectively, and wash-out collection of illustrative plates and electrophoretogram (non-reduced SDS-PAGE) are as Figure 14
Each is collected liquid take a sample respectively, measure activity, the result is as follows:
| Fermented liquid | 0.15M elution peak | 0.50M elution peak | 1.0M elution peak | |
| Active (KU/ml) | 10 | Not blood coagulation in 10 fens | 230 | Not blood coagulation in 10 fens |
The result shows, 20mM NaAc-HAc, and 0.50MNaCl, the pH5.0 elution peak is the rBAT protein peak.
3. anion-exchange chromatography Study on Conditions
3.1 it is preferred that best pH of sample solution and electricity are led
According to the iso-electric point (theoretical iso-electric point is 7.39) of rBAT, last step 20mM NaAc-HAc, 0.50MNaCl, the pH5.0 elution peak, regulating the pH value respectively with the NaoH of 1M is 8.0,8.5 and 9.0.Under each pH value, be diluted to electricity respectively with water for injection and lead as shown in the table.Select 1ml Q Sepharose Fast Flow prepacked column for use, applied sample amount is that 10ml cationic exchange rBAT collects liquid (before the dilution), and flow velocity is 1.0ml/min, and the collection effluent liquid is also measured active, carries out the sample solution condition optimization, and the result is as follows:
PH=8.0, level pad 20mM Tris-HCl, pH8.0
| The electric conductivity value of sample solution (mS/cm) | 3.0 | 6.0 | 9.0 | 12.0 |
| |
15 | 8 | 5 | 4 |
| Sample solution activity (KU/ml) | 15 | 28 | 44 | 55 |
| Effluent liquid activity (KU/ml) | 1 | 5 | 16 | 27 |
PH=8.5, level pad 20mM Tris-HCl, pH8.5
| The electric conductivity value of sample solution (mS/cm) | 3.0 | 6.0 | 9.0 | 12.0 |
| |
15 | 8 | 5 | 4 |
| Sample solution activity (KU/ml) | 15 | 28 | 43 | 54 |
| Effluent liquid activity (KU/ml) | Blood coagulation in 275 |
3 | 14 | 25 |
PH=9.0, level pad 20mM Tris-HCl, pH9.0
| The electric conductivity value of sample solution (mS/cm) | 3.0 | 6.0 | 9.0 | 12.0 |
| |
15 | 8 | 5 | 4 |
| Sample solution activity (KU/ml) | 14 | 27 | 41 | 52 |
| Effluent liquid activity (KU/ml) | Not blood coagulation in 10 |
1 | 10 | 21 |
By each data in the above table as can be known, under the pH9.0 condition, when the electricity of diluent is led to 3.0mS/cm, rBAT does not flow out basically, regulate pH value to 9.0 so select for use a cationic exchange rBAT to collect liquid, the dilution electricity is directed at 3.0mS/cm, and level pad is 20mM Tris-HCl, pH9.0 studies the elution requirement of rBAT albumen on anion-exchange chromatography.
3.2 the research of anion-exchange chromatography elution requirement
Select 1ml Q Sepharose F F prepacked column for use, level pad A is: 20mM Tris-HCl, pH9.0, elution buffer B is: 20mM Tris-HCl, 1.0M NaCl, pH9.0 collects liquid to 10ml cationic exchange rBAT and regulates pH value to 9.0, the dilution electricity is directed at 3.0mS/cm, sample on the flow velocity of 1.0ml/min.At first carry out gradient elution, condition is 0-100%B, 20CV, flow velocity 1.0ml/min found that and contain two elution peaks altogether in the elution process of 0-100%B, goes out the peak position and lays respectively at 10%B and 50%B place, but the hangover of 10%B peak is more serious, the survey work of collecting respectively and take a sample; At last with the 2MNaCL pillar of regenerating, collect the regeneration peak and survey and live.The result is as follows:
| 0.10M elution peak | 0.10M wash-out tail of the peak | 0.50M elution peak | The 2M peak of regenerating | |
| Active (KU/ml) | 800 | 50 | 10 | Not blood coagulation in 10 fens |
Wash the tailing problem that drags the peak in order to overcome 10%B, elution requirement changes into from 0-40%B, 5CV, after rise to 50%B, flow velocity 1.0ml/min, the hangover of first peak has clear improvement, the peak point is positioned at the 15%B place, collects respectively and the sampling and measuring activity.The result is as follows:
| 0.15M elution peak | 0.15M wash-out tail of the peak | 0.50M elution peak | The 2M peak of regenerating | |
| Active (KU/ml) | 1100 | 30 | 2 | Not blood coagulation in 10 fens |
So at last elution requirement is defined as 20mM Tris-HCl, 0.15M NaCl, pH9.0,20mM Tris-HCl, 0.50MNaCl, pH9.0 collects 20mMTris-HCl, 0.15MNaCl, pH9.0 eluted protein peak is the rBAT protein peak.
3.3 the analysis and research of anion-exchange chromatography process
Selecting specification for use is the chromatography column of 1.6x15cm, interior dress 20ml Q Sephrose F F filler, and last sample flow velocity is 10ml/min, 200ml cationic exchange rBAT is collected liquid regulate pH value to 9.0, the dilution electricity is directed at 3.0mS/cm and goes up sample.Level pad is 20mMTris-HCl, and pH9.0, elution buffer are respectively 20mM Tris-HCl, 0.15M NaCl, pH9.0 and 20mM Tris-HCl, 0.50MNaCl, pH9.0 collects each elution peak respectively, and wash-out collection of illustrative plates and electrophoretogram (non-reduced SDS-PAGE) are as Figure 15
Each is collected liquid take a sample respectively, measure active.The result is as follows:
| Sample solution | 0.15M elution peak | 0.50M elution peak | |
| Active (KU/ml) | 15 | 1060 | 3 |
This shows: 20mM Tris-HCl, 0.15M NaCl, the pH9.0 elution peak is the rBAT protein peak.
4. the condition of gel permeation chromatography
Select the Superdex75 of Pharmacia company filler for use, 6.0 * 60cm prepacked column, CV1700ml, level pad is 20mMNaAc-HAc, 0.15M NaCl, pH5.0 is with anion-exchange chromatography 20mM Tris-HCl, 0.15M NaCl, the pH9.0 elutriant is gone up sample in batches, and each applied sample amount is no more than 85ml (column volume 5%), Fractional Collections elution peak, purity is merged greater than 95% sample, be rBAT stoste after the degerming.
Wash-out collection of illustrative plates and electrophoretogram such as Figure 16
5. the research of purifying process repeatability
According to above technical process, with continuous three the checking purifying process of a batch fermentation supernatant that embodiment 1 method obtains, the result is as follows:
*Average total yield is 33.6%.
6. the pilot scale of purifying process
6.1 ultrafiltration exchange buffering liquid: select Millipore Pellicon10K ultrafiltration membrane stack for use, Millipore Masterflex peristaltic pump, ultra-fine filter intake pressure, top hole pressure are controlled to be 6psi, 3psi respectively, flow velocity is 120ml/min, ultra-filtration membrane is successively used water for injection, damping fluid 20mM NaAc-HAc, 0.15M NaCl after the pH5.0 balance is good, carries out ultrafiltration to fermented supernatant fluid.When five/for the moment of the extremely original volume of ultrafiltration, add damping fluid 20mM NaAc-HAc, pH5.0 is to original volume, and so repeatable operation is three times, adds damping fluid 20mM NaAc-HAc at last, and the extremely original volume of pH5.0 is as the cation-exchange chromatography sample solution.
6.2 cation-exchange chromatography: with SP Sepharose F F cation-exchange chromatography chromatography column on the ultrafiltration secondary fermentation supernatant liquor, the post specification is Index70/500mm, packed height 7.5cm, column volume is 300ml, level pad is 20mM NaAc-HAc, pH5.0, elution buffer are respectively 20mM NaAc-HAc, 0.15M NaCl, pH5.0,20mM NaAc-HAc, 0.50M NaCl, pH5.0,20mM NaAc-HAc, 1.0M NaCl, pH4.0, collect 20mM NaAc-HAc, 0.50M NaCl, pH5.0 elution peak.
6.3 anion-exchange chromatography: with cation-exchange chromatography 20mMNaAc-HAc, 0.50MNaCl, the pH5.0 elutriant is regulated pH value to 9.0, and the dilution electricity is directed at below the 3.0mS/cm last Q Sepharose F F anion-exchange chromatography, specification is 36/30, packed height 6cm, column volume are 60ml, level pad 20mM Tris-HCl, pH9.0, elution buffer is respectively 20mM Tris-HCl, 0.15M NaCl, pH9.0, with 20mM Tris-HCl, 0.5M NaCl, pH9.0 collects 20mM Tris-HCl, 0.15MNaCl, the pH9.0 elution peak.
6.4 gel permeation chromatography: with anion-exchange chromatography 20mM Tris-HCl, 0.15M NaCl, pH9.0 elutriant branch is pulled on molecular sieve Superdex75, and specification is the 60/600mm prepacked column, column volume is 1700ml, each applied sample amount is 5% (85ml) that is no more than column volume, and damping fluid is 20mMNaAc-HAc, 0.15MNaCl, pH5.0, Fractional Collections merges purity greater than 95% sample, be rBAT stoste after the degerming.
6.5 continuous three batches of pilot product purification result see the following form
Lot number: 20030601
Lot number: 20030602
Lot number: 20030603
Embodiment 4-10
Pharmaceutical composition
The gene recombination batroxobin (rBAT stoste I) of the high purifying that embodiment 1 is made mixes with the aqueous solution for injection of each auxiliary material component in containing following table, obtains the recombinant batroxobin liquid preparation.
On behalf of this assembly side, √ selected this auxiliary material.
Embodiment 11-17
Stability test embodiment
Prescription referring to embodiment 4-10 is formed.
37 ℃ of stability experiment results
The recombinant batroxobin preparation specification of embodiment 4-10 is 1KU/ml, that is to say at each check point, the solution that 100ul is contained a certain prescription of recombinant batroxobin mixes with people's standard blood plasma of handling through anti-freezing of 100ul, under 37 ℃, can solidify in 60 ± 20 seconds.Off-limits all defective.
When carrying out the preparation stability test, it is that 1KU/ml carries out that stoste is diluted to active concentration.
The recombinant batroxobin preparation of embodiment 4-10 leaves under 37 ℃, in the clotting time (second) of each check point.See the following form:
The 196s maximum range of representing to exceed coagulo meter still do not have blood coagulation to take place.
The result shows, uses gelatin hydrolysate as protective material, can make recombinant batroxobin demonstrate advantages of higher stability, can stablize more than 100 days under 37 ℃.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (8)
1. the recombinant batroxobin of a purifying is characterized in that, the recombinant batroxobin of described purifying has following characteristic:
(a) molecular weight is 29-32kDa;
(b) at least 90% batroxobin has 6 couples of correct disulfide linkage pairing: Cys in the recombinant batroxobin of described purifying
7-Cys
139, Cys
26-Cys
42, Cys
74-Cys
230, Cys
118-Cys
184, Cys
150-Cys
163And Cys
174-Cys
199
(c) the 146th and the 225th are glycosylation modified by the N-type in the SEQ of accompanying drawing 17 ID NO:1; With
(d) specific activity 385.5,405.1 or the 426.7KU/mg albumen of the recombinant batroxobin of described purifying;
The recombinant batroxobin of described purifying prepares by following steps:
(1) ultrafiltration exchange buffering liquid: select Millipore Pellicon 10K ultrafiltration membrane stack for use, Millipore Masterflex peristaltic pump, ultra-fine filter intake pressure, top hole pressure are controlled to be 6psi, 3psi respectively, flow velocity is 120ml/min, ultra-filtration membrane is successively used water for injection, damping fluid 20mM NaAc-HAc, 0.15M NaCl after the pH5.0 balance is good, carries out ultrafiltration to fermented supernatant fluid; When five/for the moment of the extremely original volume of ultrafiltration, add damping fluid 20mM NaAc-HAc, pH5.0 is to original volume, and so repeatable operation is three times, adds damping fluid 20mM NaAc-HAc at last, and the extremely original volume of pH5.0 is as the cation-exchange chromatography sample solution;
(2) cation-exchange chromatography: with SP Sepharose F F cation-exchange chromatography chromatography column on the ultrafiltration secondary fermentation supernatant liquor, the post specification is Index70/500mm, packed height 7.5cm, column volume is 300ml, level pad is 20mMNaAc-HAc, pH5.0, elution buffer are respectively 20mM NaAc-HAc, 0.15M NaCl, pH5.0,20mM NaAc-HAc, 0.50MNaCl, pH5.0,20mM NaAc-HAc, 1.0M NaCl, pH4.0, collect 20mM NaAc-HAc, 0.50M NaCl, pH5.0 elution peak;
(3) anion-exchange chromatography: with cation-exchange chromatography 20mM NaAc-HAc, 0.50M NaCl, the pH5.0 elutriant is regulated pH value to 9.0, the dilution electricity is directed at below the 3.0mS/cm, last Q Sepharose F F anion-exchange chromatography, and specification is 36/30, packed height 6cm, column volume is 60ml, level pad 20mM Tris-HCl, pH9.0, elution buffer is respectively 20mMTris-HCl, 0.15M NaCl, pH9.0 and 20mM Tris-HCl, 0.5M NaCl, pH9.0, collect 20mM Tris-HCl, 0.15MNaCl, pH9.0 elution peak;
(4) gel permeation chromatography: with anion-exchange chromatography 20mM Tris-HCl, 0.15M NaCl, pH9.0 elutriant branch is pulled on molecular sieve Superdex 75, and specification is the 60/600mm prepacked column, column volume is 1700ml, each applied sample amount is that to be no more than 5% of column volume be 85ml, and damping fluid is 20mM NaAc-HAc, 0.15M NaCl, pH5.0, Fractional Collections merges purity greater than 95% sample, be the recombinant batroxobin of purifying after the degerming.
2. the recombinant batroxobin of purifying according to claim 1 is characterized in that, at least 95% batroxobin has 6 pairs of correct disulfide linkage pairings in the described recombinant batroxobin.
3. as the recombinant batroxobin of purifying as described in the claim 2, it is characterized in that the glycosylation modified molecular weight of batroxobin protein that makes of its N-type increases 4000-6000kDa on the basis of 25.6kDa.
One kind as claim 1-3 arbitrary as described in the preparation method of recombinant batroxobin of purifying, it is characterized in that described method comprises step:
(1) ultrafiltration exchange buffering liquid: select Millipore Pellicon 10K ultrafiltration membrane stack for use, Millipore Masterflex peristaltic pump, ultra-fine filter intake pressure, top hole pressure are controlled to be 6psi, 3psi respectively, flow velocity is 120ml/min, ultra-filtration membrane is successively used water for injection, damping fluid 20mM NaAc-HAc, 0.15M NaCl after the pH5.0 balance is good, carries out ultrafiltration to fermented supernatant fluid; When five/for the moment of the extremely original volume of ultrafiltration, add damping fluid 20mM NaAc-HAc, pH5.0 is to original volume, and so repeatable operation is three times, adds damping fluid 20mM NaAc-HAc at last, and the extremely original volume of pH5.0 is as the cation-exchange chromatography sample solution;
(2) cation-exchange chromatography: with SP Sepharose F F cation-exchange chromatography chromatography column on the ultrafiltration secondary fermentation supernatant liquor, the post specification is Index70/500mm, packed height 7.5cm, column volume is 300ml, level pad is 20mMNaAc-HAc, pH5.0, elution buffer are respectively 20mM NaAc-HAc, 0.15M NaCl, pH5.0,20mM NaAc-HAc, 0.50MNaCl, pH5.0,20mM NaAc-HAc, 1.0M NaCl, pH4.0, collect 20mM NaAc-HAc, 0.50M NaCl, pH5.0 elution peak;
(3) anion-exchange chromatography: with cation-exchange chromatography 20mM NaAc-HAc, 0.50M NaCl, the pH5.0 elutriant is regulated pH value to 9.0, the dilution electricity is directed at below the 3.0mS/cm, last Q Sepharose F F anion-exchange chromatography, and specification is 36/30, packed height 6cm, column volume is 60ml, level pad 20mM Tris-HCl, pH9.0, elution buffer is respectively 20mMTris-HCl, 0.15M NaCl, pH9.0 and 20mM Tris-HCl, 0.5M NaCl, pH9.0, collect 20mM Tris-HCl, 0.15MNaCl, pH9.0 elution peak;
(4) gel permeation chromatography: with anion-exchange chromatography 20mM Tris-HCl, 0.15M NaCl, pH9.0 elutriant branch is pulled on molecular sieve Superdex 75, and specification is the 60/600mm prepacked column, column volume is 1700ml, each applied sample amount is that to be no more than 5% of column volume be 85ml, and damping fluid is 20mM NaAc-HAc, 0.15M NaCl, pH5.0, Fractional Collections merges purity greater than 95% sample, be the recombinant batroxobin of purifying after the degerming.
5. the application of the recombinant batroxobin of a purifying as claimed in claim 1 in the preparation haemostatic medicament.
6. a pharmaceutical composition is characterized in that, it contains the recombinant batroxobin and the pharmaceutically acceptable carrier of the described purifying of claim 1.
7. pharmaceutical composition as claimed in claim 6 is characterized in that, described pharmaceutical composition also contains gelatin hydrolysate as stablizer.
8. pharmaceutical composition as claimed in claim 6 is characterized in that, described pharmaceutical composition is the form of liquid or lyophilized powder.
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| CN106153746B (en) * | 2015-03-31 | 2020-11-06 | 三生国健药业（上海）股份有限公司 | IgG2 type monoclonal antibody disulfide bond pairing analysis method |
| CN106153745B (en) * | 2015-03-31 | 2020-11-10 | 三生国健药业（上海）股份有限公司 | Antibody protein disulfide bond pairing analysis method |
| CN105606831A (en) * | 2016-01-29 | 2016-05-25 | 河南中医学院 | Method for detecting quality of thrombus clear freeze-dried powder needle based on blood agglutination activity |
| CN116334052B (en) * | 2023-04-14 | 2023-12-29 | 上海腾瑞制药股份有限公司 | Fermentation medium and fermentation method of batroxobin |
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