CN104946659A - Preparation method of recombinant human growth hormone and preparation method of PEGylation modification substance - Google Patents
Preparation method of recombinant human growth hormone and preparation method of PEGylation modification substance Download PDFInfo
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Abstract
The invention relates to a gene recombinant human growth hormone modification substance for treating microsoma caused by children's endogenous recombinant human growth hormone deficiency or insufficiency and for surgeries for injuries and serious burns and subsequent treatment, particularly a preparation method of a polyethyleneglycol (PEG) modification substance of the growth hormone. The invention aims to provide a preparation method of a recombinant human growth hormone, which is higher in expression level and convenient for operation, can enhance the purity and activity of the effective drug and can enhance the purity and activity of the product on the premise of not increasing the cost, thereby enhancing the activity of the drug. The invention also aims to provide a preparation method of the recombinant human growth hormone PEGylation modification substance. According to the method, the PEGylation is utilized to modify the drug, so that the PEG and human growth hormone are connected through covalence, thereby prolonging the half life of the drug and lowering the toxic or side effect of the drug protein.
Description
Technical field
The present invention relates to a kind of children's for the treatment of endogenous recombinant human somatropin lack or the short stature that causes of deficiency and for damaging, the operation of serious burn and recombinant somatropin's modifier of successive treatment, particularly relate to the preparation method of the carbowax modifier of tethelin.
Background technology
Human growth hormone (human grow th ho rmone, hGH) is the important hormone of human pituitary's frontal lobe eosinophil secretion, has and promotes growth in humans to grow and maintain the biological function of tissue organ function.Recombinant human somatropin (rhGH) is mainly used in treating the short stature of children clinically, is also widely used in damage, the operation of serious burn and successive treatment simultaneously.Human growth hormone (human growth hormone, HGH), be also called somatotropin (somatotorpin), the wetting ability strand sphaeroprotein be made up of 191 amino acid, its relative molecular mass is at about 22kD, iso-electric point is 4.9, there are two pairs of disulfide linkage in molecule.
Genentech Inc (US) first has cloned rhGH, and it is described in patent EP-B22242.2006, comprise Nutropin (Genentech), Humatrope (Eli Lilly), Genotropin (Pfizer), Norditropin (Novo Nordisk), Saizen (Merck Serono) and Omnitrope (Sandoz) at the tethelin of US and European (and their manufacturer) obtainable synthesis.
Recombinant human somatropin (rhGH) is the medicine of the various diseases such as lipodystrophy being widely used in treating cretinism, serious burn, AIDS patient clinically, the transformation period in body short (0.5 hour).Although rhGH clinical application is extensive, because its transformation period is in vivo short, need inject every day, patient can be very painful and can produce antibody, is badly in need of developing rhGH long lasting drug formulations.Addressing this problem method ideal is at present carry out molecular modification with polyoxyethylene glycol (polyethyleneglyco, l PEG) to it.
Polyoxyethylene glycol (polyethylene glycol, PEG) is a kind of safe, non-activity, nontoxic polymkeric substance, is usually used in molecular modification.Comprise by the benefit of polyethylene glycol modified protein in treatment and reduce kidney and cell clearance, prolong half-life; Strengthen proteoclastic protection; Reduce toxicity.Make the object of biological activity protein Pegylation be improve its pharmacokinetics and pharmacodynamics performance, and retain the inherent biologic activity of natural protein.For improving pharmacological activity and clinical efficacy, need be optimized the pharmacokinetics of polyethylene glycol protein and pharmacodynamics, and the structure of PEG polymer chain, length, molecular weight and modifying method etc. are all factors that impact is optimized.
Polyethylene glycol modified protein technology has been talked and has been widely used in biomedicine field, and the polyethylene glycol modified protein that up to the present FDA batch is accurate has polyoxyethylene glycol adenosine deaminase, polyoxyethylene glycol N enzyme, Peg-IFN alpha-2b etc.
Traditional utilizes escherichia coli expression recombinant human somatropin, has the technological approaches that two kinds different, and a kind of is the recombinant human somatropin producing secretor type, and one is then produce born of the same parents' inner mold.Through practical proof, produce the recombinant human somatropin of secretor type, although step is simple, foreign protein interference is less, and the environment of oxidized form is beneficial to protein folding, and its expression amount is very low, generally will will differ from more than 1 order of magnitude with born of the same parents' inner mold.But but there is processing step complexity in the albumen producing born of the same parents' inner mold, foreign protein disturbs the shortcomings such as more.In addition, the human growth hormone adopting above-mentioned two kinds of methods to obtain also exist active low, purity is low etc. lacks.
Summary of the invention
In order to solve above-mentioned technical problem, an object of the present invention is to provide the preparation method of a kind of recombinant human somatropin, the method expression amount is relatively high, convenient operation, purity and the activity of active drug can be improved, purity and the activity of product can be increased under the condition not increasing cost, thus make medicine have higher activity.Another object of the present invention is to provide the preparation method of recombinant human somatropin's PEGization modifier, the method carrys out modified medicaments by Pegylation, polyoxyethylene glycol and human growth hormone are formed by covalently bound, increases the transformation period of medicine and reduce the toxic side effect of pharmaceutical protein.
In order to realize first above-mentioned object, present invention employs following technical scheme:
For a gene prepared by recombinant human somatropin, the sequence of this gene is as shown in SEQ ID NO:2.
A kind of expressing gene segment prepared for recombinant human somatropin, this gene segment is for increase by two fragment gene sequences successively at gene order 5` end according to claim 1, and two fragment gene sequences are respectively Factor Xa protease cutting site sequence FX and HIS sequence label HIS6; Obtain expressing gene segment HIS6-FX-HGH.
For an expressing gene segment prepared by recombinant human somatropin, this gene segment obtains troponin C-pp for cloning out by molecular chaperones troponin C full-length gene order SEQ ID NO:3 and adding Prescission Protease protease cutting site sequence pp structure at its 3` end; Then the 5` end of gene segment HIS6-FX-nHGH is connected to.
For a recombinant expression vector prepared by recombinant human somatropin, this recombinant expression vector enters PET-22b carrier by above-mentioned any one gene or expressing gene segment clone and obtains.
For a bacterial strain prepared by recombinant human somatropin, this bacterial strain is transferred in intestinal bacteria DE3 bacterial strain by above-mentioned recombinant expression vector and prepares.
A recombinant human somatropin's preparation method, the method comprises the following steps:
1) primer is designed according to human growth hormone HGH full length sequence SEQ ID NO:1, obtain the gene HGH optimized, the sequence of gene HGH is as shown in SEQ ID NO:2, gene order 5` end increases by two fragment gene sequences successively, be respectively Factor Xa protease cutting site sequence FX and HIS sequence label HIS6, obtain expressing gene segment HIS6-FX-HGH;
2) entered in PET-22b carrier and be recombinant expression vector PET-22b-HIS6-hgh by gene segment HIS6-FX-HGH clone, the corresponding recombinant protein given expression to is PET-22b-HIS6-FX-hgh;
3) then again molecular chaperones troponin C full-length gene order SEQ ID NO:3 is cloned out and add at its 3` end Prescission Protease protease cutting site sequence pp build obtain troponin C-pp; Then the 5` end of gene segment HIS6-FX-nHGH is connected to, eventually clone enters pET-22b carrier, namely be reassembled as expression vector pET-22b-troponin C-pp-HIS6-FX-HGH, the corresponding recombinant protein given expression to is troponin C-HIS6-FX-HGH;
4) by step 3) in the expression vector pET-22b-troponin C-HIS6-FX-HGH that builds be transferred in intestinal bacteria DE3 bacterial strain, with the expression of IPTG liquid storage induction recombination, collected by centrifugation thalline; Plasmid containing Prescission Protease is proceeded to Rosseta bacterial strain simultaneously, same with IPTG liquid storage abduction delivering, collected by centrifugation thalline; Then two kinds of thalline are mixed;
5) with the resuspended thalline obtained of PB damping fluid, ultrasonication, collected by centrifugation supernatant liquor; Adjustment pH value is 7.0-8.0, and low temperature vibration makes Prescission Protease be cut entirely by recombinant protein enzyme, discharges the HIS6-FX-nHGH albumen in intermediate stage, obtains the solution containing human growth hormone;
6) solution containing human growth hormone is carried out purification process, obtain the fine work containing human growth hormone.
As preferably, in order to obtain the on all four HGH albumen with native sequences, the present invention utilizes Factor Xa proteolytic enzyme to remove the label of HIS6, expose the aminoterminal that it is original, antibody can not be produced after entering human body like this, step 5) obtain to adjust to pH containing the solution of HIS6-FX-nHGH albumen be 8.0, add the further enzyme of Factor Xa proteolytic enzyme and cut, finally discharge and the natural on all four albumen nHGH of HGH protein sequence in human body.
For the method for the solution containing human growth hormone being carried out pure reason, have in the prior art a lot, but mostly there is the shortcomings such as purifying rate is low, purification procedures is complicated, the invention provides the simplest a kind of, the best approach that purifying rate is higher, specific as follows: step 6) in the concrete steps that the solution containing human growth hormone carries out purifying be, the pH value adjusting above-mentioned solution is 7.0-8.0, then nickel ion affinity column is gone up, with PB buffered soln upper prop, 50mM imidazoles wash-out foreign protein, 200mM imidazoles wash-out target protein; Dialysed overnight, then carry out DEAE purifying, with PB buffered soln upper prop, use 0.2MNaCl wash-out, namely obtain highly purified recombinant human somatropin.Purity reaches more than 95%.
Compared with prior art, utilize DNA recombinant expression method of the present invention, first, adopt optimized gene sequence to remove rare codon and improve protein yield, second adds molecular chaperone protein helps the correction method of goal gene in born of the same parents; 3rd, add the label aided purification of HIS6, ensure target protein separation rapidly and efficiently; 4th uses Factor Xa proteolytic enzyme to remove the label of HIS6, obtains the on all four albumen with native sequences.Method of the present invention can take into account expression output in a word, and purifying process, its lytic activity and final amino acid form, and have very high using value.
Although Protocols in Molecular Biology has significantly increased the availability of a lot of protein and/or polypeptide (hereinafter referred to as protein), but the therepic use of described protein is subject to the obstruction of other factors often, the short plasma half-life such as caused due to kidney and receptor-mediated removing, gathering, protein degradation, poor bioavailability and physical properties (getting rid of effective preparation in advance).
Remove in the body of rhGH and be considered to be occurred by three kinds of mechanism below: the first is that kidney is removed, and wherein rhGH is removed by glomerular filtration from circulation; The second mechanism is liver removing; The third mechanism is the receptor-mediated removing in other tissue (chondrocyte of such as cartilage).
Different PEG, each different on their molecular weight and linking group, the impact of PEG on the antigenicity of albumen, immunogenicity and circulating half-life can change.
PEG can be side chain also can be straight chain.In some scheme, PEG is straight chain, and the molecular weight in general increasing PEG tends to reduce the immunogenicity of rhGH and increases circulating half-life.
PEGization
The principle increasing protein utilization degree is by protein and PEG are carried out covalent attachment, forms PEGization pharmaceutical grade protein.PEGization pharmaceutical grade protein advantage comprises: reduce immunogenicity and antigenicity, increasing action time length and change pharmacokinetic property.
RhGH can via biocompatible linking group and PEG covalent bonding, and the method for use is known in the art, and being used for the linking group of PEG and rhGH of covalent bonding can be any compatible linking group.In some embodiment, linking group is biocompatible linking group.PEG can pass through ester bond, ehter bond, mercaptan key or amido linkage and be connected with linking group.Suitable Bc linking group comprises, such as, ester group, amide group, sub-amide group, carbohydrate, maleimide groups (comprises, such as, succinimidyl succinate SS, succinimidyl propionate SPA, carboxylic formic acid succinimide ester SCM, succinimidyl succinamide SSA, N-hydroxy-succinamide NHS), epoxy group(ing), oxygen base carbonylic imidazole base (comprises, such as, carbonyl dimidazoles base CDI), nitre benzene phenyl (comprises, such as, to nitrophenylcarbonates ester NPC or carbonic acid trichlorine phenyl ester TPC), trysylate base, aldehyde radical, isocyanate group, ethene sulfuryl, tyrosine-based, halfcystine base, Histidine base or uncle's base.In some embodiment, linking group is ester group or succinimido.In some embodiment, linking group is SS, SPA, SCM, SSA or NHS.
RhGH can the direct and PEG coupling by amino, sulfhedryl, hydroxyl or carbonyl.In some embodiment, PEG is coupled on the bad ammonia residue of rhGH.
rhGH-PEG
For medical usage, can by increasing its Half-life in vivo (thus realize dosage to reduce or administration frequency reduces), improving its stability and reducing its antigenicity or their combination improves molecular characterization by the rhGH of PEGization.
RhGH upper connection PEG adds the circulating half-life of rhGH.PEG molecular amounts on rhGH shows relevant with the circulating half-life of enzyme.The quantity increasing the PEG unit on rhGH reduces the activity of enzyme.Because of a little, need to reach balance in some embodiment between both.
The PEG modifier of rhGH, PEG with rhGH is connected with covalent linkage, and each rhGH molecule can connect 2-5 PEG molecule, and PEG molecular weight is 5kd-40kd.Particularly preferred rhGH modifier of the present invention, the molecular-weight average of PEG is 20kd.
The present invention prepares rhGH carbowax modifier method, and activated PEG molecules or monomethoxyPEG molecule are coupled on urico-oxidase molecule by amine ester covalent linkage.
The preparation method of rhGH carbowax modifier of the present invention, activated PEG is PEG nitro phenyl ester, PEG succinimide ester, PEG propionic ester, PEG acetic ester and PEG carbonic ether, PEG carboxylic formic acid succinimide ester, PEG succinimidyl succinamide ester, PEG N-hydroxy-succinamide ester.
The preparation method of rhGH carbowax modifier of the present invention, in preferred activated PEG succinimide ester, PEG carboxylic formic acid succinimide ester, PEG succinimidyl succinamide ester, PEG N-hydroxy-succinamide ester any one.
The method realizes by the following technical solutions: the rhGH albumen that purity is greater than 95%, employing molecular-weight average is that the activated polyethylene glycol molecule of 5kd-40kd was modified in pH value 5 ~ 10, temperature 4 ~ 37 DEG C, 0.1 ~ 10 hour time, and the mass ratio of peg molecule and rhGH is 1:1 ~ 7:1.
The present invention prepares rhGH carbowax modifier method, and the pH value of preferred modification reaction controls at 8.0-9.2.
The present invention prepares rhGH carbowax modifier method, and the temperature of preferred modification reaction is 4-25 DEG C.
The present invention prepares rhGH carbowax modifier method, and the preferred modification reaction time is 0.5-4 hour.
The present invention prepares rhGH carbowax modifier method, and the mass ratio of preferred peg molecule and rhGH is 2:1 ~ 5:1.
The purification process of rhGH carbowax modifier:
First reacted modified outcome is carried out desalination chromatography, as Sephadex G25, carries out buffer-exchanged, applicable buffer solution system PB damping fluid, ph value of buffer solution is 7.4, and concentration is 20mM.The reaction mixture of desalination is carried out purifying on Q anion-exchange column (as Mono-O or Q-Sepharose), and buffering liquid is PB damping fluid, and pH value is 7.4, and concentration is 20mM.Free polyoxyethylene glycol is in loading with just separated when washing with balance liquid, and polyoxyethylene glycol tethelin conjugate and free tethelin are then combined on pillar.After loading, with balance liquid washing, then can cross increase salt ionic concentration to be separated polyoxyethylene glycol tethelin conjugate and free tethelin.The adsorptive power of polyoxyethylene glycol tethelin conjugate and anion-exchange column is more weak, is first separated by washing, obtains highly purified polyoxyethylene glycol tethelin conjugate.
Gordian technique problem to be solved by this invention is to provide and is a kind ofly conducive to suitability for industrialized production, reduces to wanting number of times, PEG-rhGH medicine for the clinical treatment of life hormone deficiency of recombinating.Realize above-mentioned purpose, one aspect of the present invention can improve purity and the activity of active drug, purity and the activity of product can be increased under the condition not increasing cost, thus make medicine have higher activity, modified medicaments is carried out on the other hand by Pegylation, polyoxyethylene glycol and human growth hormone are formed by covalently bound, increases the transformation period of medicine and reduce the toxic side effect of pharmaceutical protein.
The purity of protein of recombinant human somatropin is up to more than 95%, and the polyoxyethylene glycol tethelin purity after modification also reaches more than 95%.
Recombinant human somatropin (rhGH) is the medicine of the various diseases such as lipodystrophy being widely used in treating cretinism, serious burn, AIDS patient clinically, transformation period in body short (0.5 hour), the transformation period of present PEGization recombinant human somatropin (rhGH) substantially prolongs.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE electrophorogram of the modification reaction result of recombinant human somatropin and polyoxyethylene glycol different ratios, and the 1st sample is recombinant human somatropin's albumen; 2nd sample is the ratio 1:1 of peg molecule and rhGH; 3rd sample is the ratio 3:1 of peg molecule and rhGH; 4th sample is the ratio 5:1 of peg molecule and rhGH; 5th sample is the ratio 7:1 of peg molecule and rhGH.
Fig. 2 is intravenous injection retention time figure.
Fig. 3 is subcutaneous injection retention time figure.
Embodiment
Embodiment 1
The purity of the recombinant human somatropin used in the present embodiment is greater than 95%, and maintains higher biological activity.
Recombinant human somatropin (rhGH) is dissolved in 100Mm phosphate buffered saline buffer (PH10), in recombinant human somatropin (rhGH): the ratio of polyoxyethylene glycol (PEG)=1:3 (W/W) adds PEG succinimidyl succinamide ester (m PEG-SS), stirring and dissolving, 4 degree of reactions 2 hours.
Embodiment 2
The purity of the recombinant human somatropin used in the present embodiment is greater than 95%, and maintains higher biological activity.
Recombinant human somatropin (rhGH) is dissolved in 100Mm phosphate buffered saline buffer (PH8), in recombinant human somatropin (rhGH): the ratio of polyoxyethylene glycol (PEG)=1:6 (W/W) adds PEG succinimidyl succinamide ester (m PEG-SS), stirring and dissolving, 4 degree of reactions 2 hours.
The separation purifying technique of PEG-rhGH medicine: the product that recombinant human somatropin (rhGH) is obtained by reacting with polyoxyethylene glycol, after anionite-exchange resin or cationic exchange resin adsorption, with eluting salt, collect PEG-rhGH medicine, upper molecular sieve gel chromatographic separation and purification after concentration, the purity of the polyoxyethylene glycol recombinant human somatropin medicine obtained can be greater than 95%, after too small filtering bacterium, lyophilize, be product, following examples 3-4 is specific embodiment.
Embodiment 3
The product that recombinant human somatropin (rhGH) and polyoxyethylene glycol are obtained by reacting is dissolved in 100Mm phosphate buffered saline buffer (PH8) and dialyses, after anionite-exchange resin or cationic exchange resin adsorption, with eluting salt, collect PEG-rhGH medicine, upper molecular sieve gel chromatographic separation and purification after concentration, the purity of the polyoxyethylene glycol recombinant human somatropin medicine obtained can be greater than 95%, after too small filtering bacterium, lyophilize, be product.
Embodiment 4
Below the retention time experiment of PEG-rhGH medicine in mouse blood.
This research is carried out in mouse, adopts vein and subcutaneous two kinds of approach injection PEG-rhGH, and with recombinant human somatropin in contrast, evaluates its retention time in mouse blood.
Get 48 mouse, be divided into two groups, respectively through vein and subcutaneous two kinds of administrations, every injected in mice 30.0ug, 0.2ml, by 5min, 20min, 40min, 60min, 2h, 4h, 6h, 12h, 18h, 24h, 36h, 48h time point, puts to death animal and gets blood, add heparin sodium anti-freezing, centrifuging and taking goes out blood plasma, and freezen protective is to be measured.
Adopt double-antibody method to measure the content of polyoxyethylene glycol restructuringization human growth hormone and rhGH in blood plasma, measure wavelength 490nm, take time as X-coordinate, OD490 is ordinate zou, draws the retention time curve of mPEG-rhGH and rhGH in mice serum.
This test-results shows, rhGH is after intravenous injection, and 20min peaks in mouse blood, declines rapidly subsequently, and 2h is eliminated in blood substantially; And polyoxyethylene glycol restructuringization human growth hormone (mPEG-rhGH) is through intravenous injection, 20min peaks in mouse blood, is substantially eliminated in blood after continuing to maintain 6h, 18h.
After rhGH subcutaneous injection, 20min peaks in mouse blood, can continue to maintain 60min, 3h and substantially be eliminated in blood; And polyoxyethylene glycol restructuringization human growth hormone (mPEG-rhGH) subcutaneous injection, 20min peaks in mouse blood, is substantially eliminated in blood after continuing to maintain 18h, 24h.
This illustrates compared with reference substance rhGH, and mPEG-rhGH, through subcutaneous and Intravenous administration route administration, all can extend retention time in blood significantly, postpones the time be eliminated, has long-acting effect.
Claims (12)
1., for a gene prepared by recombinant human somatropin, the sequence of this gene is as shown in SEQ ID NO:2.
2. the expressing gene segment prepared for recombinant human somatropin, this gene segment is for increase by two fragment gene sequences successively at gene order 5` end according to claim 1, two fragment gene sequences are respectively Factor Xa protease cutting site sequence FX and HIS sequence label HIS6, obtain expressing gene segment HIS6-FX-HGH.
3., for an expressing gene segment prepared by recombinant human somatropin, this gene segment obtains troponin C-pp for cloning out by molecular chaperones troponin C full-length gene order SEQ ID NO:3 and adding Prescission Protease protease cutting site sequence pp structure at its 3` end; Then the 5` end of gene segment HIS6-FX-nHGH according to claim 2 is connected to.
4., for a recombinant expression vector prepared by recombinant human somatropin, this recombinant expression vector enters PET-22b carrier by the gene clone described in claim 1 or 2 or 3 and obtains.
5., for a bacterial strain prepared by recombinant human somatropin, this bacterial strain is transferred in intestinal bacteria DE3 bacterial strain by recombinant expression vector according to claim 4 and prepares.
6. a recombinant human somatropin's preparation method, is characterized in that the method comprises the following steps:
1) primer is designed according to human growth hormone HGH full length sequence SEQ ID NO:1, obtain the gene HGH optimized, the sequence of gene HGH is as shown in SEQ ID NO:2, gene order 5` end increases by two fragment gene sequences successively, be respectively Factor Xa protease cutting site sequence FX and HIS sequence label HIS6, obtain expressing gene segment HIS6-FX-HGH;
2) entered in PET-22b carrier and be recombinant expression vector PET-22b-HIS6-hgh by gene segment HIS6-FX-HGH clone, the corresponding recombinant protein given expression to is PET-22b-HIS6-FX-hgh;
3) then again molecular chaperones troponin C full-length gene order SEQ ID NO:3 is cloned out and add at its 3` end Prescission Protease protease cutting site sequence pp build obtain troponin C-pp; Then the 5` end of gene segment HIS6-FX-nHGH is connected to, eventually clone enters pET-22b carrier, namely be reassembled as expression vector pET-22b-troponin C-pp-HIS6-FX-HGH, the corresponding recombinant protein given expression to is troponin C-HIS6-FX-HGH;
4) by step 3) in the expression vector pET-22b-troponin C-HIS6-FX-HGH that builds be transferred in intestinal bacteria DE3 bacterial strain, with the expression of IPTG liquid storage induction recombination, collected by centrifugation thalline; Plasmid containing Prescission Protease is proceeded to Rosseta bacterial strain simultaneously, same with IPTG liquid storage abduction delivering, collected by centrifugation thalline; Then two kinds of thalline are mixed;
5) with the resuspended thalline obtained of PB damping fluid, ultrasonication, collected by centrifugation supernatant liquor; Adjustment pH value is 7.0-8.0, and low temperature vibration makes Prescission Protease be cut entirely by recombinant protein enzyme, discharges the HIS6-FX-nHGH albumen in intermediate stage, obtains the solution containing human growth hormone;
6) solution containing human growth hormone is carried out purification process, obtain the fine work containing human growth hormone.
7. the preparation method of a kind of recombinant human somatropin according to claim 6, it is characterized in that step 5) obtain to adjust to pH containing the solution of HIS6-FX-nHGH albumen be 8.0, add the further enzyme of Factor Xa proteolytic enzyme to cut, finally discharge and the natural on all four albumen nHGH of HGH protein sequence in human body.
8. the preparation method of a kind of recombinant human somatropin according to claim 6, it is characterized in that step 6) in the concrete steps that the solution containing human growth hormone carries out purifying be, the pH value adjusting above-mentioned solution is 7.0-8.0, then nickel ion affinity column is gone up, with PB buffered soln upper prop, 50mM imidazoles wash-out foreign protein, 200mM imidazoles wash-out target protein; Dialysed overnight, then carry out DEAE purifying, with PB buffered soln upper prop, use 0.2MNaCl wash-out, namely obtain highly purified recombinant human somatropin.
9. the preparation method of the PEG modifier of recombinant human somatropin, the method adopts the method described in claim 6 ~ 8 any one claim to prepare recombinant human somatropin, and the recombinant human somatropin of preparation is connected with covalent linkage with PEG, each rhGH molecule can connect 2-5 PEG molecule, and PEG molecular weight is 5kd-40kd; Molecular-weight average as most preferably described PEG is 20kd.
10. preparation method according to claim 9, it is characterized in that the method adopts molecular-weight average to be that the PEG of 5kd-40kd modified in pH value 5 ~ 10, temperature 4 ~ 37 DEG C, 0.1 ~ 10 hour time, the mass ratio of PEG and rhGH is 1:1 ~ 7:1; The pH value of preferred modification reaction controls at 8.0-9.2, and the temperature of modification reaction is 4-25 DEG C, and the modification reaction time is the mass ratio of 0.5-4 hour, PEG and rhGH is 2:1 ~ 5:1.
11. preparation methods according to claim 9, is characterized in that PEG selects activated PEG molecules or monomethoxyPEG molecule to be coupled on urico-oxidase molecule by amine ester covalent linkage; Preferably, described activated PEG is PEG nitro phenyl ester, PEG succinimide ester, PEG propionic ester, PEG acetic ester and PEG carbonic ether, PEG carboxylic formic acid succinimide ester, PEG succinimidyl succinamide ester, PEG N-hydroxy-succinamide ester.
12. preparation methods according to claim 9, it is characterized in that the method also comprises the purification process of the PEG modifier of recombinant human somatropin, described purification process comprises the following steps:
1) first reacted modified outcome is carried out desalination chromatography, carry out buffer-exchanged, applicable buffer solution system PB damping fluid, ph value of buffer solution is 7.4, and concentration is 20mM; The reaction mixture of desalination is carried out purifying on Q anion-exchange column, and buffering liquid is PB damping fluid, and pH value is 7.4, and concentration is 20mM; Free polyoxyethylene glycol is in loading with just separated when washing with balance liquid, and PEG modifier and the free tethelin of recombinant human somatropin are then combined on pillar;
2), after loading, with balance liquid washing, then can cross and increase salt ionic concentration to be separated the PEG modifier of recombinant human somatropin and free tethelin; The PEG modifier of recombinant human somatropin and the adsorptive power of anion-exchange column more weak, first separated by washing, obtain the PEG modifier of highly purified recombinant human somatropin.
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| CN109153712A (en) * | 2016-04-19 | 2019-01-04 | 格里芬制药有限公司 | PEGylated biologically active peptide and application thereof |
| CN109153712B (en) * | 2016-04-19 | 2022-09-16 | 格里芬制药国际公司 | PEG biological active peptide and its use |
| CN115845079A (en) * | 2022-11-24 | 2023-03-28 | 武汉禾元生物科技股份有限公司 | Conjugate of recombinant human serum albumin and recombinant human growth hormone and preparation method thereof |
| CN115845079B (en) * | 2022-11-24 | 2023-07-18 | 武汉禾元生物科技股份有限公司 | Conjugate of recombinant human serum albumin and recombinant human growth hormone and preparation method thereof |
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