CN103113466B - Recombinant human interferon beta-1b modified by polyethylene glycol and preparation method of recombinant human interferon beta-1b - Google Patents
Recombinant human interferon beta-1b modified by polyethylene glycol and preparation method of recombinant human interferon beta-1b Download PDFInfo
- Publication number
- CN103113466B CN103113466B CN201310066150.8A CN201310066150A CN103113466B CN 103113466 B CN103113466 B CN 103113466B CN 201310066150 A CN201310066150 A CN 201310066150A CN 103113466 B CN103113466 B CN 103113466B
- Authority
- CN
- China
- Prior art keywords
- recombinant human
- interferon beta
- human interferon
- polyoxyethylene glycol
- modified
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 0 **NC(*(CO)N)=O Chemical compound **NC(*(CO)N)=O 0.000 description 2
- GUZHNHLBMZROSU-UHFFFAOYSA-N CNC(CC=O)=O Chemical compound CNC(CC=O)=O GUZHNHLBMZROSU-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Medicinal Preparation (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a recombinant human interferon beta-1b (IFN-beta-1b) modified by polyethylene glycol and preparation method of the recombinant human interferon beta-1b modified by polyethylene glycol. The preparation method comprises the following steps of: oxidizing serine (Ser) at a nitrogen terminal of the recombinant human interferon beta-1b into an aldehyde group, and then covalently coupling the aldehyde group with polyethylene glycol derivatives (PEG-NH2, PEG-O-NH2, PEG-HZ) capable of reacting with aldehyde group to obtain the recombinant human interferon beta-1b product subjected to site-specific modification of polyethylene glycol at the nitrogen terminal. The recombinant human interferon beta-1b modified by polyethylene glycol still has the physiological activity of the recombinant human interferon beta-1b, but is superior to original protein in aspects such as immunogenicity, pharmacology, pharmacokinetics, pharmacodynamics and the like.
Description
Technical field
The invention belongs to protein chemistry field, relate to a kind of polyethyleneglycol modified protein and preparation method, relate to site-PEGylation product and the preparation method of recombinant human interferon beta-1b nitrogen end more specifically.
Background technology
Along with the development of biotechnology, the biomacromolecule medicines such as peptide and protein continue to bring out, and obtain applying more and more widely in treatment of diseases.Proteins and peptides medicine has that high reactivity, hypotoxicity, biological function are clear and definite, the advantage of high specificity.But also also exist such as easily by enzymic hydrolysis, the shortcoming such as circulating half-life is short, immunogenicity is high and solubleness is low.Therefore, exploitation safety, the drug delivery system that effective, bioavailability is high have very important significance to the Clinical efficacy improving pharmaceutical grade protein.Through the development of more than 40 years, the polyethyleneglycol modified technology of protein was a kind of technology of improvement protein physiological property of maturation, can by the problems solving or alleviate proteins and peptides and exist in medicinal process.Through the protein after polyoxyethylene glycol covalent coupling compared with former albumen, its immunogenicity is more weak, and Half-life in vivo is longer, significantly can improve the pharmacology medicine of medicine for character.Up to now, there is multiple polyethyleneglycol modified medicine by FDA certification [Alconcel, S.N.S., Baas, A.S., Maynard H.D., waits people FDA-approved poly (ethylene glycol) protein conjugate drugs.Polymer Chemistry, 2011,2:1442 – 1448.].
Protein polyethyleneglycol modified, common decorating site is that the ε of lysine residue in protein sequence is amino, the amino of N-terminal, and the sulfydryl of halfcystine.Because amino free on protein structure is relatively many, multiple modified outcome be there will be to the modification of amino, even if obtain through separation and purification the situation that isomer also may appear in mono-modified product.Sulfydryl modification then has higher requirement to protein itself, needs protein itself to have free sulfhydryl groups or is introduced by engineered method.Polyethyleneglycol modified for protein, the mono-modified of fixed point is a kind of development trend, because what adopt such modification strategy to produce is homogeneous modified outcome, the separation and purification of product greatly simplifies, and the activity being conducive to protein drug retains.Specific to the protein that nitrogen end is Serine, the method for oxidation of high degree of specificity can be adopted to derive as aldehyde radical by protein N-terminal Serine, the activated polyethylene glycol that can react with aldehyde radical with end group carries out covalent coupling, reaches the object to such protein N-terminal pointed decoration.
Human interferon (IFN) is the cytokine class pharmaceutical grade protein produced by the inoblast activated and some epithelial cell, has anti-virus infection, antitumor and stronger immunoregulation effect.Interferon, rabbit in the application great majority all to there is the transformation period short, the shortcoming that immunogenicity is strong.For these problems, the polyethylene glycol modified product Pegintron(PEG-IFN-α-2b of interferon alpha) and Pegasys(PEG-IFN-α-2a) go on the market respectively at 2000 and 2002 through FDA approval, compared with the former albumen of unmodified, the interferon alpha that PEG modifies has longer transformation period and better pharmacology medicine for character, demonstrate great medicinal advantage [Yagyu clinically, H., Okada, K., Sato, S., Deng people .Pegylated interferon-α 2b and ribavirin combination therapy induces Hashitoxicosis followed by type 1 diabetes mellitus.Diabetes research and clinical practice, 2012, 95:52-54.].Be proved the recurrent number that can reduce multiple sclerosis, extend the interval of recurrence, alleviate ailing and disabled degree, although it is short equally to there is Half-life in vivo in the interferon beta of the generation postponing deformity and the histo pathological change improving central nervous system brain and spinal cord, there is immunogenicity, there are the problems such as certain side effect, but due to the albumen that it is a kind of strong-hydrophobicity, dissolving in water needs to add chaotropic agent as auxiliary, easily aggregate is formed under solution state, it is caused to analyze and quantitatively have difficulties, and the product separation purifying after PEG modifies also is difficult to effectively carry out because of the existence of aggregate.For many years scientific worker has carried out certain exploration for the polyethyleneglycol modified of interferon beta.Such as, Katre etc. have carried out polyethyleneglycol modified (see U.S. Patent number 4766106 and 4917888 and international publication number WO87/00056) to the amino of interferon beta-1a, and Alessandra is to the sulfydryl (Cys of interferon beta-1a
17) carry out modifying (see U.S. Patent number 0239529).Concerning recombinant human interferon beta-1b, there is not free sulfhydryl groups in its sequence, its modification is mainly concentrated on amino.As Basu etc. uses multiple PEG modifier to carry out chemically modified to recombinant human interferon beta-1b, and [Basu is compared to quality each other, A., Yang, K., Wang, M.L., Deng people .Structure-function engineering of interferon-β-1b for improving stability, solubility, potency, immunogenicity, and pharmacokinetic properties by site-selective mono-PEGylation.Bioconjugate chemistry, 2006, 17 (3): 618-630.].There is again new exploration recombinant human interferon beta-1b being carried out to chemically modified in organic phase solution recently, use polyoxyethylene glycol nitrogen N-Hydroxysuccinimide carbonic ether (mPEG-SC) modified recombinant human interferon β-1b, the modification be modified in organic phase relative to aqueous phase can improve modification rate [Su effectively, Z.G., Peng, F., Wang, Y.J., Deng people .PEGylation of Proteins in Organic Solution:a Case Study for Interferon beta-1b.Bioconjugate chemistry, 2012,23:1812-1820.].But these explorations are all modify for the amino of recombinant human interferon beta-1b, there is the problems such as such as many modification, separation difficulty, finally do not obtain the mono-modified product of homogeneous fixed point.By controlling solution ph, the amido modified method of N-terminal (see Japanese patent application publication No. 9-25298A) is carried out to protein for what extensively adopt now, also this method can produce the product [Yu modified equally to have report to point out in the recent period, D.Q., Ghosh.R., Purification of pegylated protein using membrane chromatograph.Journal of Pharmaceutical Sciences, 2010,99 (8): 3326-3333].Traditional amido modified its stability of the product obtained and physiologically active can not ensure, difficult on the road that these products are declared at new drug, also do not have polyethyleneglycol modified interferon beta medicine listing at present both at home and abroad.
Therefore, need stable in this area and the polyethyleneglycol modified recombinant human interferon beta-1b of pharmacology medicine for character of recombinant human interferon beta-1b can be improved.
Summary of the invention
The N-terminal that the present invention is based on recombinant human interferon beta-1b is the structure of Serine, be aldehyde radical by protein N-terminal serine oxidation, the activated polyethylene glycol that can react with aldehyde radical with end group carries out covalent coupling, obtains the recombinant human interferon beta-1b that N-terminal is mono-modified.
Concrete technical scheme of the present invention is as follows:
In first aspect, the invention provides the recombinant human interferon beta-1b that a kind of polyoxyethylene glycol (PEG) is modified, it is characterized in that, described modified outcome structure is such as formula shown in I:
PEG-IFN-β-1b (I)
Wherein, the N-terminal of described recombinant human interferon beta-1b is Serine, the N-terminal covalent coupling of described polyoxyethylene glycol and described recombinant human interferon beta-1b;
Preferably the end-group structure of described polyoxyethylene glycol is such as formula shown in II:
PEG-A (II)
Wherein, A is amino (-NH
2), oxygen amino (-O-NH
2) or hydrazide group (-CO-NH-NH
2);
Preferably described polyoxyethylene glycol is mono methoxy polyethylene glycol (mPEG).
In polyethyleneglycol modified recombinant human interferon beta-1b of the present invention, the connecting key of described polyoxyethylene glycol and described recombinant human interferon beta-1b can be imines
oxime
and acylhydrazone
in one, or amine
oxygen amine
and acid amides
in one.
In polyethyleneglycol modified recombinant human interferon beta-1b of the present invention, described polyoxyethylene glycol can be straight chain, side chain or multi-arm polyethylene glycol, is preferably straight chain polyoxyethylene glycol.
In polyethyleneglycol modified recombinant human interferon beta-1b of the present invention, the molecular weight ranges of polyoxyethylene glycol can be 2000Da-80000Da, is preferably 5000Da-50000Da, is more preferably 10000Da-30000Da.
In second aspect, the invention provides the preparation method of polyethyleneglycol modified recombinant human interferon beta-1b as described in relation to the first aspect, it is characterized in that, it is aldehyde radical that described method comprises the serine oxidation of the N-terminal of recombinant human interferon beta-1b, is the polyoxyethylene glycol covalent coupling of ammonia, oxygen ammonia or hydrazides afterwards with end group.
The preparation method of polyethyleneglycol modified recombinant human interferon beta-1b of the present invention can comprise following two steps:
(A) be aldehyde radical by the serine oxidation of the N-terminal of recombinant human interferon beta-1b, as the formula (1):
with
(B1) be ammonia by end group, the product that obtains of the polyoxyethylene glycol of oxygen ammonia or hydrazides and step (A) reacts, and carries out covalent coupling and obtain polyoxyethylene glycol and to fix a point the recombinant human interferon beta-1b that mono-modified N holds, react such as formula shown in (2)-(4):
or
(A) be aldehyde radical by the serine oxidation of the N-terminal of recombinant human interferon beta-1b, as the formula (1):
with
(B2) be ammonia by end group, the product that obtains of the activated polyethylene glycol of oxygen ammonia or hydrazides and step (A) reacts, reductive agent is added in reaction process, obtain polyoxyethylene glycol fix a point mono-modified N hold recombinant human interferon beta-1b reduzate, reaction formula is as shown in (5)-(7):
In the preparation method of polyethyleneglycol modified recombinant human interferon beta-1b of the present invention, the oxygenant used in step (A) can be ammonium chlorochromate, Manganse Dioxide, Periodic acid and periodate, is preferably periodate, is more preferably sodium periodate.
In the preparation method of polyethyleneglycol modified recombinant human interferon beta-1b of the present invention, the reductive agent used in step (B) can be sodium borohydride, acetic acid sodium borohydride and sodium cyanoborohydride, is preferably sodium cyanoborohydride.
In the third aspect, the invention provides the pharmaceutical composition of the polyethyleneglycol modified recombinant human interferon beta-1b comprised as described in relation to the first aspect.
In fourth aspect, polyethyleneglycol modified recombinant human interferon beta-the 1b that the invention provides as described in relation to the first aspect is preparing the application in medicine, described medicine is used for acute, the chronic and recurrent viral infection disease of prevention and therapy and neural system inflammatory immunological disease, alleviate and treat relapsive sclerosis disease, and treatment malignant tumour is as cervical intraepithelial neoplasia, neoplastic pleural effusion, hairy cell, breast tumor or endometrial tumors.
Compared with the prior art in this area, the invention has the advantages that:
(1) adopt the method for oxidation of high degree of specificity to obtain the N-terminal aldehyde radical only of recombinant human interferon beta 1b, the product then obtained with polyoxyethylene glycol covalent coupling is the mono-modified recombinant human interferon beta-1b of N-terminal, and product is single, is separated easy;
(2) polyethyleneglycol modified dose that adopts has very high reactive behavior with aldehyde radical, modification reaction productive rate is higher than the modification in organic phase, drastically increase the service efficiency of protein and polyethyleneglycol modified dose, the modified outcome obtained significantly can retain the antiviral anti-tumor activity of former recombinant human interferon beta-1b, can also reach simultaneously improve recombinant human interferon beta-1b pharmacology medicine for the object of character.
Accompanying drawing explanation
Fig. 1 detects mPEG for using SDS-PAGE
5kthe electrophorogram of-HZ modified recombinant human interferon β-1b.Use 12% separation gel, 4.5% concentrated glue.
Wherein:
1. standard protein sample, molecular weight is followed successively by 14.4KDa, 20.1KDa, 29.0KDa, 43.1KDa, 66.4KDa, 97.2KDa.
2.mPEG
5kthe reaction mixture of-HZ modified recombinant human interferon β-1b.
3. the former albumen of recombinant human interferon beta-1b.
4.mPEG
5krecombinant human interferon beta-1b(Mono HZ-5k-PEG-IFN-β-the 1b that-HZ modifies, product before reduction).
Fig. 2 detects mPEG for using SDS-PAGE
20kthe electrophorogram of-HZ modified recombinant human interferon β-1b.Use 15% separation gel, 4.5% concentrated glue.
Wherein:
1. standard protein sample, molecular weight is followed successively by 14.4KDa, 20.1KDa, 29.0KDa, 43.1KDa, 66.4KDa, 97.2KDa.
2. the former albumen of recombinant human interferon beta-1b.
3.mPEG
20kthe reaction mixture of-HZ modified recombinant human interferon β-1b.
4.mPEG
20krecombinant human interferon beta-1b(Mono HZ-20k-PEG-IFN-β-the 1b that-HZ modifies, product before reduction).
Fig. 3 detects mPEG for using SDS-PAGE
20k-NH
2, mPEG
20k-O-NH
2the electrophorogram of modified recombinant human interferon β-1b.Use 15% separation gel, 4.5% concentrated glue.
Wherein:
1. standard protein sample, molecular weight is followed successively by 14.4KDa, 20.1KDa, 29.0KDa, 43.1KDa, 66.4KDa, 97.2KDa.
2.mPEG
20k-NH
2recombinant human interferon beta-1b(Mono the NH modified
2-20k-PEG-IFN-β-1b, product before reduction).
3.mPEG
20k-O-NH
2recombinant human interferon beta-the 1b modified
(Mono ONH
2-20k-PEG-IFN-β-1b, product before reduction).
Embodiment
Illustrate the present invention below with reference to embodiment, but embodiment is only the object illustrated, but not limitation of the invention.
The nitrogen of embodiment 1 recombinant human interferon beta-1b is terminal oxidized
Take IFN-β-1b lyophilized powder 2mg, be dissolved in the 50mM PB of 2mL, in pH4.0,0.1%SDS buffered soln.Add the 1mg/mLNaIO of 200 μ L
4the aqueous solution, NaIO
4be 10:1 with the molar feed ratio of IFN-β-1b.1h is reacted under 25 DEG C of conditions.Then in reaction system, 100 μ L ethylene glycol termination reactions are added, room temperature reaction 10min.Collect protein peak through gel filtration chromatography, sampling adds the colour developing of Decolor Fuchsin colouring reagents.Colour developing result is that the IFN-β-1b sample after oxidation shows bright red, and for Decolor Fuchsin detects the characteristic color of aldehyde radical, non-oxidation sample colour developing result is colourless, shows NaIO
4oxidation IFN-β-1b generates aldehyde radical product.
Embodiment 2 polyoxyethylene glycol hydrazides 5000(mPEG
5k-HZ) preparation of the mono-modified recombinant human interferon beta-1b that fixes a point
Get the recombinant human interferon beta-1b solution after oxidation, add polyoxyethylene glycol hydrazides powder, molecular weight 5000Da, the molar feed ratio of polyoxyethylene glycol hydrazides and recombinant human interferon beta-1b is 10:1.In pH value 4.5, oscillatory reaction 24h under 25 DEG C of conditions.
After having reacted, extract reaction solution and carry out chromatographic separation through Sephacryl S-200HR post, moving phase is for containing 20mM PB, 0.1M Na
2sO
4buffer system (pH7.4).Flow velocity 0.8mL/min, single applied sample amount is 500 μ L.Determined wavelength 280nm, collects out peak, by the sample of collection at 20mM PB, and dialysed overnight under 5% N.F,USP MANNITOL system, and ultrafiltration and concentration.
Through SDS-PAGE detection reaction and separating resulting, result shows that polyoxyethylene glycol hydrazides 5000 is modified and separation and purification obtains mono-modified recombinant human interferon beta-1b.See Fig. 1.
Embodiment 3 polyoxyethylene glycol hydrazides 20000(mPEG
20k-HZ) preparation of the mono-modified recombinant human interferon beta-1b that fixes a point
Get the recombinant human interferon beta-1b solution after oxidation, add polyoxyethylene glycol hydrazides powder, molecular weight 20000Da, the molar feed ratio of polyoxyethylene glycol hydrazides and recombinant human interferon beta-1b is 10:1.In pH value 4.5, oscillatory reaction 24h under 25 DEG C of conditions.
After having reacted, extract reaction solution and carry out chromatographic separation through Sephacryl S-200HR post, moving phase is for containing 20mM PB, 0.1M Na
2sO
4buffer system (pH7.4).Flow velocity 0.8mL/min, single applied sample amount is 500 μ L.Determined wavelength 280nm, collects out peak, will collect sample at 20mM PB, dialysed overnight under 5% N.F,USP MANNITOL system, and ultrafiltration and concentration.
Through SDS-PAGE detection reaction and separating resulting, result shows that polyoxyethylene glycol hydrazides 20000 is modified and separation and purification obtains mono-modified recombinant human interferon beta-1b.See Fig. 2.
Embodiment 4 polyoxyethylene glycol hydrazides 20000(mPEG
20k-HZ) preparation of mono-modified recombinant human interferon beta-1b reduzate of fixing a point
Get the recombinant human interferon beta-1b solution after oxidation, add polyoxyethylene glycol hydrazides powder, molecular weight 20000Da, the molar feed ratio of polyoxyethylene glycol hydrazides and recombinant human interferon beta-1b is 10:1, adds reductive agent sodium cyanoborohydride and makes final concentration be 10mM.In pH value 4.5, oscillatory reaction 24h under 25 DEG C of conditions.
After having reacted, extract reaction solution and carry out chromatographic separation through Sephacryl S-200HR post, moving phase is for containing 20mM PB, 0.1M Na
2sO
4buffer system (pH7.4).Flow velocity 0.8mL/min, single applied sample amount is 500 μ L.Determined wavelength 280nm, collects out peak, will collect sample at 20mM PB, dialysed overnight under 5% N.F,USP MANNITOL system, and ultrafiltration and concentration.
Through SDS-PAGE detection reaction and separating resulting, result shows that polyoxyethylene glycol hydrazides 20000 is modified and separation and purification obtains mono-modified recombinant human interferon beta-1b reduzate.
Embodiment 5 polyoxyethylene glycol hydrazides 40000(mPEG
40k-HZ) preparation of the mono-modified recombinant human interferon beta-1b that fixes a point
Get the recombinant human interferon beta-1b solution after oxidation, add polyoxyethylene glycol hydrazides powder, molecular weight 40000Da, the molar feed ratio of polyoxyethylene glycol hydrazides and recombinant human interferon beta-1b is 10:1, in pH value 4.5, and oscillatory reaction 24h under 25 DEG C of conditions.
After having reacted, extract reaction solution and carry out chromatographic separation through Sephacryl S-200HR post, moving phase is for containing 20mM PB, 0.1M Na
2sO
4buffer system (pH7.4).Flow velocity 0.8mL/min, single applied sample amount is 500 μ L.Determined wavelength 280nm, collects out peak, will collect sample at 20mM PB, dialysed overnight under 5% N.F,USP MANNITOL system, and ultrafiltration and concentration.
Through SDS-PAGE detection reaction and separating resulting, result shows that polyoxyethylene glycol hydrazides 40000 is modified and separation and purification obtains mono-modified recombinant human interferon beta-1b.
Embodiment 6 polyoxyethylene glycol ammonia 20000(mPEG
20k-NH
2) preparation of the mono-modified recombinant human interferon beta-1b that fixes a point
Get the recombinant human interferon beta-1b solution after oxidation, add polyoxyethylene glycol ammonia powder, molecular weight 20000Da, the molar feed ratio of polyoxyethylene glycol ammonia and recombinant human interferon beta-1b is 10:1, in pH value 4.5, and oscillatory reaction 24h under 25 DEG C of conditions.
After having reacted, extract reaction solution and carry out chromatographic separation through Sephacryl S-200HR post, collect sample through SDS-PAGE detected result, result shows that polyoxyethylene glycol ammonia 20000 is modified and separation and purification obtains mono-modified recombinant human interferon beta-1b.See Fig. 3.
Embodiment 7 polyoxyethylene glycol oxygen ammonia 20000(mPEG
20k-O-NH
2) preparation of the mono-modified recombinant human interferon beta-1b that fixes a point
Get the recombinant human interferon beta-1b solution after oxidation, add polyoxyethylene glycol oxygen ammonia powder, molecular weight 20000Da, the molar feed ratio of polyoxyethylene glycol oxygen ammonia and recombinant human interferon beta-1b is 10:1, in pH value 4.5, and oscillatory reaction 24h under 25 DEG C of conditions.
After having reacted, extract reaction solution and carry out chromatographic separation through Sephacryl S-200HR post, collect sample through SDS-PAGE detected result, result shows that polyoxyethylene glycol oxygen ammonia 20000 is modified and separation and purification obtains mono-modified recombinant human interferon beta-1b.See Fig. 3.
Embodiment 8 polyoxyethylene glycol oxygen ammonia 20000(PEG
20k-O-NH
2) preparation of the mono-modified recombinant human interferon beta-1b that fixes a point
Get the recombinant human interferon beta-1b solution after oxidation, add polyoxyethylene glycol oxygen ammonia powder, molecular weight 20000Da, the molar feed ratio of polyoxyethylene glycol oxygen ammonia and recombinant human interferon beta-1b is 10:1, in pH value 4.5, and oscillatory reaction 24h under 25 DEG C of conditions.
After having reacted, extract reaction solution and carry out chromatographic separation through Sephacryl S-200HR post, collect sample through SDS-PAGE detected result, result shows that polyoxyethylene glycol ammonia 20000 is modified and separation and purification obtains mono-modified recombinant human interferon beta-1b.
Embodiment 9 polyoxyethylene glycol hydrazides 20000(PEG
20k-HZ) preparation of mono-modified recombinant human interferon beta-1b reduzate of fixing a point
Get the recombinant human interferon beta-1b solution after oxidation, add polyoxyethylene glycol hydrazides powder, molecular weight 20000Da, the molar feed ratio of polyoxyethylene glycol hydrazides and recombinant human interferon beta-1b is 10:1, adds reductive agent sodium cyanoborohydride and makes final concentration be 10mM.In pH value 4.5, oscillatory reaction 24h under 25 DEG C of conditions.
After having reacted, extract reaction solution and carry out chromatographic separation through Sephacryl S-200HR post, moving phase is for containing 20mM PB, 0.1M Na
2sO
4buffer system (pH7.4).Flow velocity 0.8mL/min, single applied sample amount is 500 μ L.Determined wavelength 280nm, collects out peak, will collect sample at 20mM PB, dialysed overnight under 5% N.F,USP MANNITOL system, and ultrafiltration and concentration.
Through SDS-PAGE detection reaction and separating resulting, result shows that polyoxyethylene glycol hydrazides 20000 is modified and separation and purification obtains mono-modified recombinant human interferon beta-1b reduzate.
Embodiment 10 polyoxyethylene glycol ammonia 20000(PEG
20k-NH
2) preparation of the mono-modified recombinant human interferon beta-1b that fixes a point
Get the recombinant human interferon beta-1b solution after oxidation, add polyoxyethylene glycol ammonia powder, molecular weight 20000Da, the molar feed ratio of polyoxyethylene glycol ammonia and recombinant human interferon beta-1b is 10:1, in pH value 4.5, and oscillatory reaction 24h under 25 DEG C of conditions.
After having reacted, extract reaction solution and carry out chromatographic separation through Sephacryl S-200HR post, collect sample through SDS-PAGE detected result, result shows that polyoxyethylene glycol ammonia 20000 is modified and separation and purification obtains mono-modified recombinant human interferon beta-1b.
Recombinant human interferon beta-1b the Anti-viral activity in vitro that embodiment 11 modifies front and back detects
Anti-viral activity in vitro determination experiment selects WISH-VSV system.
Adopt the suppression micromethod based on the suppression of cytopathicity effect.Pathology metering (TCID is there is in the venomous scheme amount of attacking of VSV virus for making cell half
50) 100 times.Get the VSV virus of preserving in laboratory and be diluted to working concentration with attacking malicious nutrient solution.Cell counting is chosen as CCK-8 test kit, detects light absorption value at 450nm place.Determination of activity the results are shown in Table 1.
The antiviral activity of front and back modified by table 1. recombinant human β-1b Interferon, rabbit
Note: * 1 is non-reduced product, * 2 is reduction after product.
Applicant states, the present invention illustrates detailed features of the present invention and method by above-described embodiment, but the present invention is not limited to above-mentioned detailed features and method, does not namely mean that the present invention must rely on above-mentioned detailed features and method could be implemented.Person of ordinary skill in the field should understand; any improvement in the present invention; to equivalence replacement and the increase of subsidiary material and step, the concrete way choice etc. of material selected by the present invention and step, all drop within protection scope of the present invention and open scope.
Claims (13)
1. recombinant human interferon beta-the 1b that modifies of a polyoxyethylene glycol (PEG), it is characterized in that, described modified outcome structure is such as formula shown in I:
PEG-IFN-β-1b (I)
Wherein, the N-terminal of described recombinant human interferon beta-1b is Serine, the N-terminal covalent coupling of described polyoxyethylene glycol and described recombinant human interferon beta-1b;
The end-group structure of described polyoxyethylene glycol is such as formula shown in II:
PEG-A (II)
Wherein, A is hydrazide group (-CO-NH-NH
2);
Described polyoxyethylene glycol is mono methoxy polyethylene glycol (mPEG), and described polyoxyethylene glycol is straight chain or multi-arm polyethylene glycol, and the molecular weight ranges of described polyoxyethylene glycol is 5000Da-30000Da;
The connecting key of described polyoxyethylene glycol and described recombinant human interferon beta-1b is acylhydrazone
or acid amides
2. polyethyleneglycol modified recombinant human interferon beta-1b according to claim 1, is characterized in that, described polyoxyethylene glycol is straight chain polyoxyethylene glycol.
3. recombinant human interferon beta-1b polyethyleneglycol modified as claimed in claim 1, is characterized in that, the molecular weight ranges of described polyoxyethylene glycol is 10000Da-30000Da.
4. the preparation method of the polyethyleneglycol modified recombinant human interferon beta-1b according to any one of claim 1-3, it is characterized in that, it is aldehyde radical that described method comprises the serine oxidation of the N-terminal of recombinant human interferon beta-1b, is the polyoxyethylene glycol covalent coupling of hydrazides afterwards with end group.
5. preparation method according to claim 4, is characterized in that, said method comprising the steps of:
(A) be aldehyde radical by the serine oxidation of the N-terminal of recombinant human interferon beta-1b, shown in (1):
with
(B1) be that the product that the polyoxyethylene glycol of hydrazides and step (A) obtain reacts by end group, carry out covalent coupling and obtain polyoxyethylene glycol and to fix a point the recombinant human interferon beta-1b that mono-modified N holds, react such as formula shown in (4):
or
(A) be aldehyde radical by the serine oxidation of the N-terminal of recombinant human interferon beta-1b, shown in (1):
with
(B2) be that the product that the activated polyethylene glycol of hydrazides and step (A) obtain reacts by end group, reductive agent is added in reaction process, obtain polyoxyethylene glycol fix a point mono-modified N hold recombinant human interferon beta-1b reduzate, reaction formula is as shown in (7):
6. preparation method according to claim 5, is characterized in that, the oxygenant used in step (A) is ammonium chlorochromate, Manganse Dioxide, Periodic acid or periodate.
7. preparation method according to claim 6, is characterized in that, the oxygenant used in step (A) is periodate.
8. preparation method according to claim 7, is characterized in that, the oxygenant used in step (A) is sodium periodate.
9. the preparation method according to any one of claim 5-8, is characterized in that, the reductive agent used in step (B2) is sodium borohydride, acetic acid sodium borohydride or sodium cyanoborohydride.
10. preparation method according to claim 9, is characterized in that, the reductive agent used in step (B2) is sodium cyanoborohydride.
11. 1 kinds of pharmaceutical compositions, it comprises recombinant human interferon beta-1b polyethyleneglycol modified as claimed any one in claims 1 to 3.
12. polyethyleneglycol modified as claimed any one in claims 1 to 3 recombinant human interferon beta-1b are preparing the application in medicine, described medicine is used for acute, the chronic and recurrent viral infection disease of prevention and therapy and neural system inflammatory immunological disease, alleviate and treat relapsive sclerosis disease, and treating malignant tumour and neoplastic pleural effusion.
13. application according to claim 12, is characterized in that, described malignant tumour is cervical intraepithelial neoplasia, hairy cell, breast tumor or endometrial tumors.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310066150.8A CN103113466B (en) | 2013-03-01 | 2013-03-01 | Recombinant human interferon beta-1b modified by polyethylene glycol and preparation method of recombinant human interferon beta-1b |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310066150.8A CN103113466B (en) | 2013-03-01 | 2013-03-01 | Recombinant human interferon beta-1b modified by polyethylene glycol and preparation method of recombinant human interferon beta-1b |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103113466A CN103113466A (en) | 2013-05-22 |
| CN103113466B true CN103113466B (en) | 2015-06-03 |
Family
ID=48411888
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310066150.8A Active CN103113466B (en) | 2013-03-01 | 2013-03-01 | Recombinant human interferon beta-1b modified by polyethylene glycol and preparation method of recombinant human interferon beta-1b |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103113466B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104672248A (en) * | 2013-12-03 | 2015-06-03 | 深圳市艾瑞生物科技有限公司 | Covalent coupling agent, chemically modified covalent coupling solid phase carrier containing covalent coupling agent and preparation method and application of carrier |
| MX2017002140A (en) | 2014-08-19 | 2017-08-21 | Biogen Ma Inc | Pegylation method. |
| CN105777892A (en) * | 2014-12-17 | 2016-07-20 | 深圳翰宇药业股份有限公司 | Pramlintide polyethylene glycol derivative, and preparation method and application thereof |
| CN109400695B (en) * | 2018-10-31 | 2020-06-30 | 中南大学湘雅医院 | Polypeptide modification method and application |
| CN110124048B (en) * | 2019-04-23 | 2022-06-17 | 湖南华腾制药有限公司 | Multi-arm polyethylene glycol matrine conjugate, preparation method and application thereof |
| CN116023466B (en) * | 2023-03-30 | 2023-06-27 | 北京生物制品研究所有限责任公司 | Method for purifying PEG modified recombinant human interferon beta 1b protein |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101002944A (en) * | 2006-01-17 | 2007-07-25 | 中国科学院过程工程研究所 | Conjugate of branched chair polymacrogol-interferon, and its preparing method |
| EP2392594A1 (en) * | 1994-10-12 | 2011-12-07 | Amgen Inc. | N-terminally monopegylated polypeptides and process for their preparation |
| CN102453089A (en) * | 2010-10-25 | 2012-05-16 | 重庆富进生物医药有限公司 | Preparation and application of recombinant consensus interferon mutant polyethylene glycol conjugate |
-
2013
- 2013-03-01 CN CN201310066150.8A patent/CN103113466B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2392594A1 (en) * | 1994-10-12 | 2011-12-07 | Amgen Inc. | N-terminally monopegylated polypeptides and process for their preparation |
| CN101002944A (en) * | 2006-01-17 | 2007-07-25 | 中国科学院过程工程研究所 | Conjugate of branched chair polymacrogol-interferon, and its preparing method |
| CN102453089A (en) * | 2010-10-25 | 2012-05-16 | 重庆富进生物医药有限公司 | Preparation and application of recombinant consensus interferon mutant polyethylene glycol conjugate |
Non-Patent Citations (2)
| Title |
|---|
| Structure-Function Engineering of Interferon-β-1b for Improving Stability, Solubility, Potency, Immunogenicity, and Pharmacokinetic Properties by Site-Selective Mono-PEGylation;Amartya Basu，et al.;《Bioconjugate Chem.》;20060503;第17卷(第3期);摘要，第619页第3段，第621页右栏倒数第2段和第622页Chart 1，第623页表1 * |
| 蛋白质多肽药物聚乙二醇定点修饰的研究进展;付正明等;《军事医学科学院院刊》;20070430;第31卷(第2期);第181页左栏第1段，右栏第2段和方案6 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103113466A (en) | 2013-05-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103113466B (en) | Recombinant human interferon beta-1b modified by polyethylene glycol and preparation method of recombinant human interferon beta-1b | |
| CA2312976C (en) | Compositions comprising histidine-linked protein polymer conjugates | |
| CN1088721C (en) | Interferon conjugates | |
| RU2409389C2 (en) | Peg dendrimer with four branches for conjugation with proteins and peptides | |
| JP5325884B2 (en) | Polyethylene glycolated interferon α2b and method for producing and using the same | |
| CN107405384A (en) | The parts of IL 7 and the conjugate of polymer | |
| KR100694994B1 (en) | Human Granulocyte-Colony Stimulating Factor Isoforms | |
| JP2018024882A (en) | Glycopolysialylation of non-blood coagulation protein | |
| WO2016106941A1 (en) | Site-specific polyethylene glycol modified asparaginase, preparation method therefor and application thereof | |
| CN101636411B (en) | Interferon alpha 2a modified by polyethylene glycol, synthesis process and application thereof | |
| RU2488598C2 (en) | Double-stranded polyethylene glycol modified growth hormone, preparation method and use thereof | |
| CN101491682A (en) | PEG-IFN omega conjugate and preparation technique thereof | |
| CN100355784C (en) | Method for preparing polyethylene glycol-modified alpha-interferon 1b | |
| CN106749608B (en) | Interferon alpha conjugates | |
| CN101163716A (en) | Interleukin-6 polyethylene glycol conjugate and its preparing method and use | |
| CN107226858A (en) | Interferon macromolecule combination IFN-PMPC preparation and its application | |
| CN100589844C (en) | Mono-modified polyethyleneglycol-insulin complexes and preparation method thereof | |
| CN106674325A (en) | Method of preparing interferon macromolecular combination IFN-POEGMA | |
| KR102039493B1 (en) | Novel exenatide variants-polymer complex | |
| JP6367952B2 (en) | Complexes containing erythropoietin and branched polymer structures | |
| CN117164717A (en) | Polyethylene glycol modified antibody fragment and preparation method thereof | |
| CN117427179A (en) | Chemical modifier of human urinary kallidinogenase | |
| TW201249874A (en) | An interferon conjugate modified by PEG |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |