CN102229667A - Polyethylene glycol modified porcine alpha-interferon, preparation method and application thereof - Google Patents
Polyethylene glycol modified porcine alpha-interferon, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a polyethylene glycol modified porcine alpha-interferon and a preparation method thereof. The preparation method comprises the following steps: coupling polyethylene glycol with amino groups on lysine of porcine alpha-interferon under alkaline condition, and then performing cation exchange chromatography for separating and purifying, thereby acquiring the polyethylene glycol modified porcine alpha-interferon. The invention also discloses a long-acting injection containing the polyethylene glycol modified porcine alpha-interferon. The polyethylene glycol modified porcine alpha-interferon provided by the invention has the advantages that the modified rate is about 50%, the purity is above 97%, and the existing time in vivo is long.
Description
Technical field
The present invention relates to the biological medicine technology field, be specifically related to a kind of polyethyleneglycol modified porcine alpha-interferon and preparation method thereof, and the long-acting injection that contains this polyethyleneglycol modified porcine alpha-interferon.
Background technology
Porcine alpha-interferon is a kind of when organism infection when virus, by a kind of low-molecular-weight glycoprotein that the antiviral response reaction produces, has biological activitys such as antiviral, antitumor, immunomodulatory by white corpuscle.Porcine alpha-interferon is used for the treatment of various pig virus diseases, in clinical application, its short body-internal-circulation transformation period, have immunogenicity and antigenicity, easily be recycled System Cleaning, need problem such as multiple dosing, give sick pig bring huge stress, influence result of treatment.Also increase simultaneously labor cost, use inconvenience.
Polyoxyethylene glycol (PEG) is the neutral polymer with good biological compatibility, has the wetting ability of height, bigger ydrodynamics volume is arranged in the aqueous solution, and do not have immunogenicity.
At present in bibliographical information about the PEGization Interferon, rabbit, Roche Holding Ag adopts 40kD branch PEG to modify human interferon, its modifier molecular weight is big, protein active kept low, only be 7%, pharmacokinetic analysis is injected and was just occurred interferon activity in back 8 hours, the Plasma Concentration peak occurs in 80 hours, and the time length is 10-15 days.Such injection also is not suitable for pig interferon, because pig interferon belongs to when treatment pig acute viral infection disease, and the active Interferon, rabbit that need play a role fast, and drug effect can not be above 7 days.Because pig only can frequently be used viral lived vaccine (general 10 days at interval) in growth early stage, the plain active effect that can the viral interference living vaccine of the body internal interference of long time.
Also have method to use Interferon, rabbit N latter end as single PEGization site, Interferon, rabbit species specificity site has been sealed in such combination, has reduced the binding ability of Interferon, rabbit and acceptor, makes its activity compare much lower with the Interferon, rabbit of polylysine modification.Modification rate of the prior art is lower, is generally 30-40%.
Schering Plough company adopts the linear mono methoxy polyethylene glycol succinimide of 12kD carbonic ether to modify, the activity in vivo hold-time is too short, substantially detected in 5 days in vivo less than interferon activity, can not inject once in a week, and stability is not good, and is unstable in solution, can only make powder injection and preserve, increase cost, and use is made troubles to medicine.
Summary of the invention
In order to solve the problems of the technologies described above, one of purpose of the present invention provides the polyethyleneglycol modified porcine alpha-interferon of a kind of effectively prolong drug transformation period.
A further object of the present invention provides the preparation method of described polyethyleneglycol modified porcine alpha-interferon.
Another object of the present invention provides the long-acting injection that contains described polyethyleneglycol modified porcine alpha-interferon.
The chemical structural formula of polyethyleneglycol modified porcine alpha-interferon of the present invention is as follows:
Molecular weight is 20KD-80KD.Wherein, the value of n and n ' can be arbitrarily guaranteeing that total molecular weight is under the prerequisite of 20KD-80KD, and preferably, the value of n makes (OCH
2CH
2)
nMolecular weight between 10KD-40KD; The value of n ' makes (OCH
2CH
2)
N 'Molecular weight between 10KD-40KD.
Polyethyleneglycol modified porcine alpha-interferon of the present invention, by under alkaline condition with the amino coupled on the Methionin of polyoxyethylene glycol and porcine alpha-interferon, obtain through the cation-exchange chromatography separation and purification.
Polyethyleneglycol modified porcine alpha-interferon of the present invention can prepare as follows:
1) porcine alpha-interferon that is dissolved in the PBS damping fluid is dialysed, concentrated, the displacement damping fluid is made the borate buffer solution that contains porcine alpha-interferon;
2) polyethyleneglycol modified dose is dissolved among the HCl, joins in the described borate buffer solution that contains porcine alpha-interferon of step 1), carry out modification reaction under stirring;
3) add Glacial acetic acid, the pH value is transferred to 4.5-4.8, termination reaction obtains reaction solution;
4) utilize cation-exchange chromatography that step 3) gained reaction solution is carried out separation and purification, obtain single polyethyleneglycol modified porcine alpha-interferon.
Wherein:
The concentration of porcine alpha-interferon is 2-10mg/ml in the borate buffer solution of step 1), and preferred concentration is 10mg/ml; The pH value of borate buffer solution is 8.4-9.0, and preferred pH value is 9.0;
Step 2) the polyethyleneglycol modified dose of mol ratio with porcine alpha-interferon is 2-4 described in: 1; The concentration of HCl is 0.01-0.1M, and preferred concentration is 0.1M; The rotating speed that stirs is 500-1000 rev/min, and preferred 1000 rev/mins, the time of stirring is 2-4 hour, preferred 4 hours;
The cation-exchange chromatography of step 4) adopts gradient elution, and moving phase is the A phase: 20mmol/L acetate-sodium acetate buffer, the B phase: A mutually in adding 1mol/L sodium-chlor; The concentration gradient of B phase is 0-50%, and elution time is 28-35min; Flow velocity 3ml/min.
Preferably, polyethyleneglycol modified dose of the present invention is selected from methoxy poly (ethylene glycol) amine (mPEG-NH
2), a kind of in methoxy poly (ethylene glycol)-succinimdyl carbonate (mPEG-SC), methoxy poly (ethylene glycol) propionic aldehyde (mPEG-ALD), methoxy poly (ethylene glycol) mercapto alcohol (mPEG-SH), methoxy poly (ethylene glycol) maleimide (mPEG-MAL), methoxy poly (ethylene glycol) carboxylic acid (mPEG-COOH), methoxy poly (ethylene glycol)-acid amides-propionic acid succinimide ester (mPEG-NHS) and the methoxy poly (ethylene glycol)-propenyl (mPEG-AC); Described polyethyleneglycol modified dose molecular weight ranges is between 10KD-40KD.
More preferably, described polyethyleneglycol modified dose is mPEG-NHS.
The present invention also provides the preparation method of described polyethyleneglycol modified porcine alpha-interferon, and this method is included under the alkaline condition with the amino coupled on the Methionin of polyoxyethylene glycol and porcine alpha-interferon, through the step of cation-exchange chromatography separation and purification.
Particularly, the preparation method of polyethyleneglycol modified porcine alpha-interferon of the present invention comprises the steps:
1) porcine alpha-interferon that is dissolved in the PBS damping fluid is dialysed, concentrated, the displacement damping fluid is made the borate buffer solution that contains porcine alpha-interferon;
2) polyethyleneglycol modified dose is dissolved among the HCl, joins in the described borate buffer solution that contains porcine alpha-interferon of step 1), carry out modification reaction under stirring;
3) add Glacial acetic acid, the pH value is transferred to 4.5-4.8, termination reaction obtains reaction solution;
4) utilize cation-exchange chromatography that step 3) gained reaction solution is carried out separation and purification, obtain single polyethyleneglycol modified porcine alpha-interferon.
Wherein:
The concentration of porcine alpha-interferon is 2-10mg/ml in the borate buffer solution of step 1), and preferred concentration is 10mg/ml; The pH value of borate buffer solution is 8.4-9.0, and preferred pH value is 9.0;
Step 2) the polyethyleneglycol modified dose of mol ratio with porcine alpha-interferon is 2-4 described in: 1; The concentration of HCl is 0.01-0.1M, and preferred concentration is 0.1M; The rotating speed that stirs is 500-1000 rev/min, and preferred 1000 rev/mins, the time of stirring is 2-4 hour, preferred 4 hours;
The cation-exchange chromatography of step 4) adopts gradient elution, and moving phase is the A phase: 20mmol/L acetate-sodium acetate buffer, the B phase: A mutually in adding 1mol/L sodium-chlor; The concentration gradient of B phase is 0-50%, and elution time is 28-35min; Flow velocity 3ml/min.
Preferably, polyethyleneglycol modified dose of the present invention is selected from methoxy poly (ethylene glycol) amine (mPEG-NH
2), a kind of in methoxy poly (ethylene glycol)-succinimdyl carbonate (mPEG-SC), methoxy poly (ethylene glycol) propionic aldehyde (mPEG-ALD), methoxy poly (ethylene glycol) mercapto alcohol (mPEG-SH), methoxy poly (ethylene glycol) maleimide (mPEG-MAL), methoxy poly (ethylene glycol) carboxylic acid (mPEG-COOH), methoxy poly (ethylene glycol)-acid amides-propionic acid succinimide ester (mPEG-NHS) and the methoxy poly (ethylene glycol)-propenyl (mPEG-AC); Described polyethyleneglycol modified dose molecular weight ranges is between 10KD-40KD.
More preferably, described polyethyleneglycol modified dose is mPEG-NHS.
The present invention also provides a kind of long-acting injection that contains aforementioned any polyethyleneglycol modified porcine alpha-interferon.
The moiety that the present invention contains the long-acting injection of above-mentioned polyethyleneglycol modified porcine alpha-interferon is: IFN-PEG:250-500mg; The PBS:50-100mM of pH6.5; Glycine: 20-25g; Water for injection adds to 1000ml.
In a preferred embodiment, the present invention contains the moiety of the long-acting injection of above-mentioned polyethyleneglycol modified porcine alpha-interferon and is: IFN-PEG:500mg; The PBS:100mM of pH6.5; Glycine: 23g; Water for injection adds to 1000ml.
Beneficial effect of the present invention is as follows:
1. the modification rate of polyethyleneglycol modified porcine alpha-interferon of the present invention is about 50%, and purity is more than 97%.Porcine alpha-interferon conjugates through PEG modifies can make porcine alpha-interferon have long lasting feature.
2. polyethyleneglycol modified porcine alpha-interferon long-acting injection of the present invention can be injected once in 7 days, and remaining time is long in the body.The present invention has also studied the external activity of this long-acting injection, and its external activity keeps can reach 28%.
3. the present invention adopts polyoxyethylene glycol that porcine alpha-interferon is carried out chemically modified, and effectively the prolong drug transformation period is a kind of important effective way that addresses the above problem.
The present invention with PEG and porcine alpha-interferon coupling after, can improve the stability of medicine in blood plasma, reduce the enzymolysis of medicine, avoid identification and the removing of immunity system, thereby prolong the porcine alpha-interferon transformation period in vivo medicine.
5. preparation method of the present invention is simple to operate, and mild condition is suitable for suitability for industrialized production.
Description of drawings
Fig. 1 is the protein electrophoresis figure of modification reaction product.Wherein 1 is natural porcine alpha-interferon, and 2 is the standard protein marker, and 3 is the modification reaction product.The 20kD place is the unmodified porcine alpha-interferon in the Reference numeral 3, and the 60kD place is mono-modified porcine alpha-interferon, and the 90kD place is above to be the porcine alpha-interferons of modifying more.
Fig. 2 is the porcine alpha-interferon collection of illustrative plates that the isolating PEG of cation-exchange chromatography modifies.Wherein, P0 is free PEG, and P1 is the porcine alpha-interferons of modifying more, and P2 is mono-modified porcine alpha-interferon (13.71-15.08ms/cm), and P3 is unmodified porcine alpha-interferon (17.20-19.26ms/cm).
Fig. 3 is that natural porcine alpha-interferon and cation-exchange chromatography separate the efficient gel filtration collection of illustrative plates that PEG modifies the P2 peak (mono-modified product) of porcine alpha-interferon.
Fig. 4 is the transformation period of the mono-modified porcine alpha-interferon of natural porcine alpha-interferon and PEG in the rat body-internal-circulation.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
The preparation method of the porcine alpha-interferon that embodiment 1 is polyethyleneglycol modified
1) preparation of porcine alpha-interferon borate solution
Get commercial porcine alpha-interferon (being dissolved in the PBS damping fluid), under 4 ℃ available from Beijing Lv Yuan creation Bioisystech Co., Ltd, dialyse with the PBS damping fluid, concentrate, adopt the mode of displacement damping fluid then, porcine alpha-interferon is replaced in the borate buffer (pH 9.0).4 ℃ of preservations.Measure protein concentration with the LOWRY method, adjust protein concentration to 10mg/ml.
2) preparation of PEG hydrochloric acid soln
Get 100mg methoxy poly (ethylene glycol)-acid amides-propionic acid succinimide ester (mPEG-NHS, molecular weight 20kD is available from the Jiankai Science and Technology Co., Ltd., Beijing), be dissolved in the 0.1M hydrochloric acid soln of 0.5ml, standby.
3) modification reaction of PEG
The PEG hydrochloric acid soln is joined 4ml contain in the borate solution of porcine alpha-interferon, stirred 4 hours with 1000 rev/mins under 4 ℃ of conditions.Add Glacial acetic acid and the pH value is adjusted to 4.5, termination reaction.Add the 50ml sodium chloride solution.
SDS-PAGE electrophoresis detection, concentrated glue are 4.5-4.8%, and separation gel is 13.5%.The result as shown in Figure 1.
4) separation and purification of modified outcome
SP.Sepharose Fast Flow chromatographic column is connected to AKTA explorer 100 liquid chromatographic systems, the A phase: acetate-sodium acetate buffer (20mmol/L), B phase: add 1mol/L sodium-chlor in mutually at A.Gradient elution, B phase concentration 0-50%, elution time 30min, flow velocity 3ml/min.
The result as shown in Figure 2.Collect respectively and penetrate peak p0, elution peak p1, p2, p3 carry out electrophoresis detection, determine the porcine alpha-interferon that single PEG modifies.P0 is a free modifier of not participating in reaction, and p1 is that many PEG modify porcine alpha-interferon, and p2 is mono-modified porcine alpha-interferon, and p3 is not adorned porcine alpha-interferon.Each elution peak purity is all higher, shows that cation-exchange chromatography can well separate each component.
The preparation method of the porcine alpha-interferon that embodiment 2 is polyethyleneglycol modified
With the method operation, difference only is according to embodiment 1:
Step 1) mesoboric acid pH of buffer is 8.4, and adjusting the porcine alpha-interferon protein concentration is 2.5mg/ml.
Step 2) gets 100mg methoxy poly (ethylene glycol) carboxylic acid (mPEG-COOH, molecular weight 30kD is available from the Jiankai Science and Technology Co., Ltd., Beijing) in, be dissolved in the 0.05M hydrochloric acid soln of 1ml.
In the step 3) PEG hydrochloric acid soln is joined 10ml and contain in the borate solution of porcine alpha-interferon, stirred 4 hours with 500 rev/mins under 4 ℃ of conditions.Add Glacial acetic acid and the pH value is adjusted to 4.8, termination reaction.Add the 50ml sodium chloride solution.
SDS-PAGE electrophoresis detection result is identical with Fig. 1.
Behind cation-exchange chromatography, obtain the porcine alpha-interferon that single PEG modifies, result and Fig. 2 are basic identical.
The preparation method of the porcine alpha-interferon that embodiment 3 is polyethyleneglycol modified
With the method operation, difference only is according to embodiment 1:
Step 1) mesoboric acid pH of buffer is 8.7, and adjusting the porcine alpha-interferon protein concentration is 5mg/ml.
Step 2) gets 100mg methoxy poly (ethylene glycol) amine (mPEG-NH in
2, molecular weight 40kD is available from the Jiankai Science and Technology Co., Ltd., Beijing), be dissolved in the 0.1M hydrochloric acid soln of 0.5ml.
In the step 3) PEG hydrochloric acid soln is joined 6ml and contain in the borate solution of porcine alpha-interferon, stirred 4 hours with 800 rev/mins under 4 ℃ of conditions.Add Glacial acetic acid and the pH value is adjusted to 4.5, termination reaction.Add the 50ml sodium chloride solution.
SDS-PAGE electrophoresis detection result is identical with Fig. 1.
Behind cation-exchange chromatography, obtain the porcine alpha-interferon that single PEG modifies, result and Fig. 2 are basic identical.
The preparation method of embodiment 4 injections
Adopt polyethyleneglycol modified porcine alpha-interferon of the present invention to prepare long-acting injection respectively, the concentration of porcine alpha-interferon can be between 250-500 μ g/ml, but can suitably adjust according to concrete purposes, is not limited thereto.
The present invention contains the long-acting injection of polyethyleneglycol modified porcine alpha-interferon according to different size, and following prescription can be arranged:
(1)250μg/ml
IFN-PEG:250mg; The PBS:50mM of pH6.5; Glycine: 25g; Water for injection adds to 1000ml.
(2)300μg/ml
IFN-PEG:300mg; The PBS:75mM of pH6.5; Glycine: 20g; Water for injection adds to 1000ml.
(3)400μg/ml
IFN-PEG:400mg; The PBS:80mM of pH6.5; Glycine: 23g; Water for injection adds to 1000ml.
(4)500μg/ml
IFN-PEG:500mg; The PBS:100mM of pH6.5; Glycine: 23g; Water for injection adds to 1000ml.
Experimental example 1 single PEG modifies porcine alpha-interferon purity and external activity detects
The porcine alpha-interferon that single PEG of the porcine alpha-interferon of unmodified and embodiment 1 is modified is through high-efficient gel filtration chromatography, and color atlas is seen Fig. 3.The 19.3mL place goes out the porcine alpha-interferon that the peak is a unmodified among Fig. 3, and it is the porcine alpha-interferon that single PEG modifies that the 13.5mL place goes out the peak.By calculating peak area as can be known, through the mono-modified product of porcine alpha-interferon that the isolating poly-ethanol of ion-exchange is modified, its purity is more than 97%, and the modification rate is 50%.
Adopt cytopathic-effect inhibition assay to measure external activity, the activity reservation of the mono-modified product of gained has reached 28%.
Polyethyleneglycol modified porcine alpha-interferon product to embodiment 2 and 3 is tested with method, and the result is the same.
The transformation period is measured in experimental example 2 bodies
Adopt 10 of SD rats, be divided into two groups at random, 6 of the mono-modified porcine alpha-interferon groups of embodiment 1, intramuscular injection contains the mono-modified Interferon, rabbit of porcine alpha-interferon 50ug/kg body weight; 4 of common porcine alpha-interferon groups, the common porcine alpha-interferon of intramuscular injection 50ug/kg body weight, different time points sampling blood sampling, with 2500 rev/mins of centrifugal removal serum,-20 ℃ of preservations, treat all serum collect finish after, measure the wherein biological activity of porcine alpha-interferon with cytopathic-effect inhibition assay, transformation period the results are shown in Figure 4 for the blood biological activity is reduced to half time of maximum.
As seen from Figure 4, after the injection, the biologic activity of unmodified porcine alpha-interferon reaches maximum value rapidly, reduces to half at 1h; And the biologic activity of mono-modified porcine alpha-interferon reaches maximum value at 8h, and 24h reduces to half.This shows that mono-modified porcine alpha-interferon albumen circulating half-life has in animal body obtained prolonging significantly, is proteic about 16 times of unmodified porcine alpha-interferon.
Polyethyleneglycol modified porcine alpha-interferon product to embodiment 2 and 3 is tested with method, and the result is the same.
Experimental example 3 stability tests
Get the mono-modified porcine alpha-interferon of embodiment 1-3, be made into solution in different concentration, concentration is respectively 500 μ g/ml, 600 μ g/ml, 700 μ g/ml, 800 μ g/ml, each 5 batches, 4 ℃ place 1,2,3,6 month after, through high-efficient gel filtration chromatography and electrophoresis detection, do not see and assemble or degraded; Measure through cytopathic-effect inhibition assay, it still is retained in 98% than living.As seen mono-modified porcine alpha-interferon of the present invention has good aqueous stability.
Claims (10)
1. polyethyleneglycol modified porcine alpha-interferon, its chemical structural formula is as follows:
Molecular weight is 20KD-80KD.
2. polyethyleneglycol modified porcine alpha-interferon according to claim 1 is characterized in that, by under alkaline condition with the amino coupled on the Methionin of polyoxyethylene glycol and porcine alpha-interferon, obtain through the cation-exchange chromatography separation and purification.
3. a method for preparing claim 1 or 2 described polyethyleneglycol modified porcine alpha-interferons is characterized in that, comprises the steps:
1) porcine alpha-interferon that is dissolved in the PBS damping fluid is dialysed, concentrated, the displacement damping fluid is made the borate buffer solution that contains porcine alpha-interferon;
2) polyethyleneglycol modified dose is dissolved among the HCl, joins in the described borate buffer solution that contains porcine alpha-interferon of step 1), carry out modification reaction under stirring;
3) add Glacial acetic acid, the pH value is transferred to 4.5-4.8, termination reaction obtains reaction solution;
4) utilize cation-exchange chromatography that step 3) gained reaction solution is carried out separation and purification, obtain single polyethyleneglycol modified porcine alpha-interferon.
4. method according to claim 3 is characterized in that, the concentration of porcine alpha-interferon is 2-10mg/ml in the borate buffer solution of step 1), and the pH value of borate buffer solution is 8.4-9.0.
5. method according to claim 3 is characterized in that step 2) described in polyethyleneglycol modified dose of mol ratio with porcine alpha-interferon be 2-4: 1.
6. method according to claim 3 is characterized in that step 2) in the concentration of HCl be 0.01-0.1M.
7. method according to claim 3 is characterized in that step 2) in the rotating speed that stirs be 500-1000 rev/min, the time of stirring is 2-4 hour.
8. method according to claim 3 is characterized in that, the cation-exchange chromatography of step 4) adopts gradient elution, and moving phase is the A phase: 20mmol/L acetate-sodium acetate buffer, the B phase: A mutually in adding 1mol/L sodium-chlor; The concentration gradient of B phase is 0-50%, and elution time is 28-35min; Flow velocity 3ml/min.
9. according to each described method of claim 3-8, it is characterized in that, described polyethyleneglycol modified dose is selected from a kind of in methoxy poly (ethylene glycol) amine, methoxy poly (ethylene glycol)-succinimdyl carbonate, methoxy poly (ethylene glycol) propionic aldehyde, methoxy poly (ethylene glycol) mercapto alcohol, methoxy poly (ethylene glycol) maleimide, methoxy poly (ethylene glycol) carboxylic acid, methoxy poly (ethylene glycol)-acid amides-propionic acid succinimide ester and the methoxy poly (ethylene glycol)-propenyl, and molecular weight is 10KD-40KD.
10. long-acting injection that contains claim 1 or 2 described polyethyleneglycol modified porcine alpha-interferons.
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Cited By (2)
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| CN103751796A (en) * | 2013-10-29 | 2014-04-30 | 王明丽 | Straight-chain polyethylene glycol-recombinant porcine interferon alpha conjugate, and preparing method and application thereof |
| CN104262480A (en) * | 2014-09-28 | 2015-01-07 | 重庆理工大学 | Construction and modification method of recombinant porcine long-acting-alpha interferon and preparation method of lyophilized injection |
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| CN1634993A (en) * | 2004-10-29 | 2005-07-06 | 上海生物制品研究所 | Recombined human interferon-alpha 1b compound and process for preparation |
| CN102229944A (en) * | 2011-06-03 | 2011-11-02 | 北京伟嘉人生物技术有限公司 | Method for preparing recombinant porcine alpha-interferon |
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| CN1167777A (en) * | 1996-05-31 | 1997-12-17 | 弗·哈夫曼-拉罗切有限公司 | Interferon conjugates |
| CN1634993A (en) * | 2004-10-29 | 2005-07-06 | 上海生物制品研究所 | Recombined human interferon-alpha 1b compound and process for preparation |
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| CN103751796A (en) * | 2013-10-29 | 2014-04-30 | 王明丽 | Straight-chain polyethylene glycol-recombinant porcine interferon alpha conjugate, and preparing method and application thereof |
| CN104262480A (en) * | 2014-09-28 | 2015-01-07 | 重庆理工大学 | Construction and modification method of recombinant porcine long-acting-alpha interferon and preparation method of lyophilized injection |
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Application publication date: 20111102 |