CN1673230A - Prepn of recombinant composite interferon-polyglycol conjugate and conjugate product - Google Patents
Prepn of recombinant composite interferon-polyglycol conjugate and conjugate product Download PDFInfo
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Abstract
The present invention relates to one kind of recombinant composite interferon-polyglycol conjugate, which includes polypeptide and non-polypeptide components with the physiological activity of recombinant human composite interferon. The present invention prepares high quality protein-polyglycol conjugate via controllable modification reaction, and separates and purifies the protein-polyglycol conjugate via chromatographic separation process. The polyglycol modified recombinant human composite interferon has the antiviral activity of natural human interferon maintained, heat stability and enzymolysis resisting stability higher that of natural human interferon, lower serum eliminating rate and longer intracorporeal circulation half time.
Description
Technical field
The invention belongs to the protein chemistry field, particularly proteinic polyoxyethylene glycol chemistry modifying method and modified outcome.
Background technology
The fast development of modern biotechnology makes people become a reality polypeptide and the protein dream as drug use
[1]Since nineteen eighty-two FDA ratifies the bio-pharmaceutical that first is used for the treatment of human diabetes---there has been the biological medicine of hundreds of kind to be applied in the clinical treatment since the recombinant human insulin (rhInsulin), comprised somatomedin, G CFS, hormone, Interferon, rabbit, interleukin-, monoclonal antibody or the like.According to estimates, in 2000, have about 500 bio-pharmaceutical goods carrying out clinical trial, and the annual growth of protein articles (glycoprotein, non-glycoprotein, and antibody) approximately will be at 10-35%
[2]Although compare than before, there is more biologics to develop, the typical problem that all has polypeptide drug when but all these protein drugs are used for human body: tangible immunogenicity and antigenicity, degraded by the body endoproteinase easily, can be removed by kidney, intravital circulation half life weak point, poor stability and solubleness are low etc.Therefore the formulating of recipe (formulation) of the biological medicine of polypeptide class and administering mode (Administration) become the key of the biological medicine development of restriction.Clinical Experience for many years finds that there is following shortcoming in the most existing bio-pharmaceutical
[3]:
1. route of administration is single, and most of biological medicine carries out administration by intravenous injection or hypodermic mode, and oral biological medicine is considerably less.
Circulation half life in vivo too short, this mainly is because by the degraded of body endoproteinase, and reticuloendothelial system is removed and reasons such as kidney removing cause.
3. some medicine has tangible antigen-antibody reaction in vivo.
4. owing to above reason causes its pharmacokinetics in vivo relatively poor, thereby influenced the performance of drug effect.
For pharmacokinetics and the pharmacodynamic properties people that improve biological agents such as protein have adopted several strategies to solve these problems:
1) changes the amino acid whose order of native protein to reduce its immunogenicity and antigenicity and to increase the ability that its protease inhibitor is degraded by genetic engineering means.
2) by chemistry or genetic engineering means with itself and immunoglobulin (Ig) or serum albumin coupling or fusion.
3) realize the protection and the slowly-releasing of pharmaceutical protein by drug release carrier.
4) by chemical means with pharmaceutical protein and natural or synthetic polymer coupling mutually.Increase proteinic apparent molecular weight with polymer, cover protein surface and reduce its immunogenicity and antigenicity to reach, and the ability that increases its protease inhibitor degraded.
But successful method is an employing method 4 the most up to now), by what polyoxyethylene glycol and pharmaceutical protein covalent coupling were realized, i.e. PEGYLATION OF PROTEINS technology (PEGylation Technology)
[4-5]
(IFN is antiviral, antiproliferative and the immunomodulatory that a class has wide spectrum Interferon) to Interferon, rabbit, can activate the general name of the cytokine of natural killer cell multiple functions such as (NK cell).It is that the similar, the function that are produced by multiple inductor inducing cell are close, the low molecular saccharides albumen of biologically active, is a class important cytokine.Interferon, rabbit generally is divided into I type and II type, and the I type comprises IFN-α, IFN-β and IFN-ω, and wherein IFN-α can be divided into the hypotype that primary structure there are differences more than 25 kinds again; The II type comprises IFN-γ.These Interferon, rabbit are produced by different genes encodings respectively, and biological activity is different.The Interferon, rabbit that uses clinically mostly is to obtain by gene recombination technology production at present.The advantage that the DNA recombinant technology is produced Interferon, rabbit be this method can high purity, produce Interferon, rabbit in large quantity.
1981, the researchist that (Amgen) company is pacified into by the U.S. synthesized a kind of non-natural I type Interferon, rabbit (Consensus interferon, IFN-α-conl, Interferon alfacon-1) first.Close on this Interferon alfacon-1 structure with common Interferon, rabbit, have 166 amino-acid residues, wherein about 30% locational amino acid is identical with IFN-β, and the amino acid more than 60% is identical with IFN-γ, has only 20 the locational amino acid and the IFN-α of normal use clinically
2aThe amino acid difference of corresponding position.But the biological activity of Interferon alfacon-1 but is 2-20 times of common Interferon, rabbit.Thereby become the focus of research.United States Patent (USP) 4695623,4897471,5372808 discloses the interferon activity that Interferon alfacon-1 has wide spectrum, has stronger antivirally and antitumor and activate the NK cell activity, can be used for the treatment of disease.Chinese patent 97193506 discloses the method for using composite interference extract for treating hepatitis C, and Chinese patent 98114663 discloses the preparation method of recombinant consensus interferon and the purposes of treatment hepatitis B and hepatitis C.These existing researchs show that all composite interference extract for treating chronic hepatitis C effect is remarkable, particularly aspect reducing the serum-virus load and treating the patient of the invalid or recurrence of other interferon therapy, are better than the Interferon, rabbit of other type.
But Interferon alfacon-1 is identical with other pharmaceutical grade protein, has poor stability, the transformation period is short in the body, shortcomings such as pharmacokinetics difference, therefore modify Interferon alfacon-1 with PEG, improve its thermostability, and by improving its apparent molecular weight and resistance to enzymolysis ability in vivo, improve its transformation period in vivo, the long-acting that reaches Interferon alfacon-1 has great importance.
Protein PEGization principle
Phase late 1950s is with the structure of the method research protein molecule of chemically modified and the focus that function relationship becomes biological chemistry and biology field.Since late 1970s, about the research report that carries out the protein chemistry modification with synthetic macromolecule polyoxyethylene glycol (PEG) and derivative thereof gets more and more.Protein PEG chemically modified is mainly used in biomedicine and biological technical field.At biomedical aspect, the PEG chemically modified can reduce immunogenic immunoreactivity, suppress the generation of immunoglobulin (Ig), prolong drug albumen circulation half life in vivo etc.; At biological technical field, enzyme can be brought into play katalysis efficiently through after the chemically modified in organic solvent, and shows novel catalytic performance.Proteinic immunogenicity is by the antigenic determinant decision on protein molecule surface.PEG is a kind of linearity, hydrophilic, softness and uncharged molecules, when it and proteinic nonessential group covalent attachment, can be used as a kind of barrier, cover the antigenic determinant of protein surface, this albumen can not be identified as xenobiotic, avoid the generation of corresponding antibodies, thereby perhaps stop antibody to suppress corresponding immune response with antigenic the combination.Can eliminate proteinic immunogenicity because PEG modifies, thereby can not be eliminated by immune response; Also because PEG in the bridging effect of protein surface, makes protein be difficult for by proteasome degradation; Also, be difficult for by glomerular filtration because after protein modified, its molecular weight increased greatly.The result of above-mentioned Several Factors effect obviously prolongs the protein circulation half life in vivo after modifying.
The chemical property of PEG
PEG is the hydroxyl simple linear polymers in a kind of two ends, and structural formula is as follows:
PEG is the ethylene oxide,1,2-epoxyethane anionic ring-opening polymerization reaction synthetic by being caused by hydroxide ion nucleophillic attack epoxide.The most frequently used PEG is mono methoxy PEG (mPEG) in the PEGization technology, and structural formula is as follows:
MPEG is the ethylene oxide,1,2-epoxyethane anionic ring-opening polymerization reaction synthetic by being caused by the methoxy ion.Some characteristics of PEG can simply be summarized as follows:
1) amphiphilic---not only water-soluble but also be dissolved in most of organic solvent, be insoluble to ether and aliphatic hydrocarbon;
2) nontoxicity (MW＞1000), the FDA approval can be taken orally;
3) extremely weak immunogenicity;
4) can impel cytogamy during high density;
5) with other polymers, for example dextran forms double-aqueous phase system;
6) can be as the precipitation agent of protein and nucleic acid.
Compare with other high molecular polymer, (Mw/Mn) is narrow for the polymolecularity of PEG: for lower molecular weight PEG (＜5kd) Mw/Mn is less than 1.01; For high molecular weight PEGs (＞50kd) Mw/Mn is less than 1.1.Because PEG good solubility in water and most of organic solvent is generally considered to be amphiphilic.Each ethyl oxygen unit can be in conjunction with 2～3 water moleculess in the aqueous solution to studies show that PEG.Because the PEG chain has the snappiness of height and the ability of bound water molecule, therefore, PEG compares with the globular protein of same molecular amount in solution, and its hydraulic radius is 5～10 times of the latter.This characteristic is considered to the PEG precipitating proteins; Exclusion protein and cell; Reduce proteinic immunogenicity and antigenicity; Increase the major cause of protein resistance to enzymolysis ability.
The molecular weight ranges of PEG is very wide, is suitable as the set out relative molecular mass of PEG of raw material of modifier or modifier and is generally 300-100,000.PEG does not have immunogenicity and toxicity usually, can not destroy the activity of biomolecules, and its biocompatibility has passed through the authentication of food and drug administration (FDA).Particularly after protein was modified through PEG class modifier, many advantageous properties of modifier also can obtain embodying in modified protein.The PEG molecular end has two activable-OH groups, and then adopt the derivative of mono methoxy polyethylene glycols (mPEG) during chemically modified is modifier more.Except linear mPEG, also have mPEG of branch (promptly two or more PEG chains being linked together) and star mPEG by Methionin, triazine etc.In most cases PEG derivative and proteinic covalent attachment be with the amino on the protein molecule and sulfydryl as decorating site, this mainly is because amino and sulfydryl has nucleophilicity, and is present in the surface of protein molecule mostly.
In the PEGYLATION OF PROTEINS process, the hydroxyl of PEG end is the functional group of its chemical reaction.But its reactive behavior is poor, can only be under fierce condition and other group generation chemical reaction, and such condition usually is impatient at by protein.Therefore, PEG must be transformed into various activated intermediates, so that can be with higher speed of reaction and protein coupling mutually under the condition of gentleness, this be the activation of PEG, promptly connects the functional group that can react on the end of PEG or two ends.The selection of functional group decides according to available reactive group on the connected molecule of desire.For protein, typical reactive amino acid comprises Methionin, halfcystine, Histidine, arginine, aspartic acid, L-glutamic acid, Serine, Threonine, tyrosine, N-terminal amino group and C-terminal carboxyl(group).If glycoprotein, the adjacent hydroxyl can be formed two formaldehyde compositions that can react by periodate oxidation.The most general path of PEG and proteinic coupling is to react with the amino that functional group's activated PEG makes it to be fit to Methionin and N-end.About the PEG activation method, Samuel Zalipsky
6,7And J.M.Harris
8Made more detailed summary.
The influence factor of modification reaction
Protein and the desired reaction conditions of modifier effect, except that modification can be carried out smoothly, also must satisfy following requirement: the one, do not cause proteinic irreversible denaturation, the 2nd, help optionally modifying protein.For this reason, reactant ratio, pH, temperature of reaction and reaction medium etc. all will be controlled at certain scope.In the condition of chemical modification reaction of influential protein molecule, the pH value is a most important condition, because it has determined to have the residing ionic condition of reacting and can not react of functional group of potential reaction ability.Generally speaking, increase pH and can improve speed of reaction, reduce pH and can reduce speed of reaction.The higher modifier of reactive behavior can be coupled with protein under physiological pH; And the lower modifier of reactive behavior needs higher pH usually.The specificity of modification reaction is then optimized the pH value corresponding to one.Can promptly provide experiment condition by changing modifier and proteinic mol ratio and change pH corresponding to specific modification degree.Temperature also is the condition that must note, because temperature can have influence on the microenvironment of sulfhydryl-group activity.The appropriate radical reaction of selecting temperature can reduce or prevent some competitions.Some protein belongs to thermo-sensitivity protein, and modification reaction need carry out at low temperatures.In this case, the reaction times is wanted proper extension.Reaction medium can change proteinic conformation or capping position, thereby influences modification reaction.Experimental result by based on the orthogonal experiment design can easily obtain the modification reaction condition that suits.By control, just can obtain required be connected with single, two, the protein couplings of three or more modifier to one or more operation factors.
Disclosed polyethyleneglycol modified protein forms the method for albumen-polyethylene glycol conjugation thing and there are some problems in the conjugate that is produced by described method.Comprising, form the chemical modification method that conjugate adopted and may make protein denaturation or complete deactivation; In addition, because the characteristic of linked reaction itself tends to make the pH value of reaction system to reduce, be unfavorable for the quality control of coupled product.The reduction of using previous disclosed method to control this pH value may cause wider fluctuation of pH and the bioactive loss of protein; In addition, the previous disclosed protein-separation of polyethylene glycol conjugation thing, purification process always can not reach good effect, particularly under the situation of mass preparation; At last, hydrolytic rupture may take place in the chemical bond instability between polyoxyethylene glycol and the protein.If such cracking takes place after the administration, polyoxyethylene glycol will not reach expected effect to proteinic modification
The present invention by select for use chemical property good polyethyleneglycol modified dose and fully controlled modification reaction condition prepare high-quality protein-polyethylene glycol conjugation thing, and come the heterogeneity of classification, purifying polyoxyethylene glycol-protein conjugate by chromatography separating method.
1)Ame?M.Ryan，Timothy?G.Terrell“Biotechnology?and?its?products”in?(Eds)Handbookof?Toxicologic?pathology，Second?Edition，Volume?1，Acadamic?Press，New?York，2002
2)Walsh?G.Biopharmaceutical?Benchmarks.Nat.Biotechnol.，2000，18：831-833.
3)EF.Davis，a.m.Kazo，M.L.Nucci，A.Abuchowski，Reduction?of?immunogenicity?andextension?of?circulating?half-life?of?peptide?and?protein，in：V.H.L?Lee(Ed.)，Peptide?andProtein?Drug?Delivery，Marcel?Dekker，New?york，1990
4)J.M.Harris，S.Zalipsky(Eds)，Poly(ethylene?glycol)Chemistry?and?Biological?Applications，ACS?Symposium?Series?NO.680，American?Chemical?Society，Washington，DC，1997
5)Veronese?F?M，Harris?J?M.Introduction?and?Overview?of?Peptide?and?Protein?PegylationAdvanced?Drug?Delivery?Reviews，2002，54：453-456
6)Zalipsky，Functionalized?poly(ethylene?glycol)for?preparation?of?biologically?relevantconjugates，Bioconjugate?Chem，6；150-165，1995
7).Zalipsky，Chemistry?of?polyethylene?glycol?conjugates?with?biologically?active?molecules，Advanced?Drug?deliveryreviews，16；157-180，1995
8)M.J.Roberts，M.D.Bentley，J.M.Harris，Chemistry?for?peptide?and?protein?PEGylation，54；459-476，2002
Summary of the invention
The invention provides recombinant human Interferon alfacon-1-polyethylene glycol conjugation thing as shown below with physiologically active:
Wherein, R
1Be low alkyl group, for example methyl, ethyl, propyl group etc.; R
2For-O-or-NH-; R
3Be N-R
4, S, O; R
4Be low alkyl group or amino acid; M and n are the integer more than or equal to 1.
Definition
Used following definition in the context of the present invention:
Term " recombinant human Interferon alfacon-1 " is meant by gene recombination technology synthetic I type Interferon, rabbit, have 166 amino-acid residues, wherein about 30% locational amino acid is identical with IFN-β, amino acid more than 60% is identical with FN-γ, has only 20 the locational amino acid and the IFN-α of normal use clinically
2aThe amino acid difference of corresponding position.
Term " conjugate " is meant by one or more polypeptide, is generally an independent polypeptide and one or more non-peptide molecule, polymer molecule for example, the molecule of covalently bound formation.Term " covalently bound " is meant that polypeptide fraction and non-polypeptide fraction form covalent linkage by the chemical reaction between and both are linked together.
The term " polymer " molecule " be by the covalently bound macromole that forms of a plurality of monomer molecules, can be human or animal's serum albumin or immunoglobulin (Ig), also can be the macromolecular material of synthetic, for example polyoxyethylene glycol.
Term " but the reactive group on the peptide molecule " be meant the peptide molecule surface various can with the chemical group of non-polypeptide fraction generation chemical reaction, for example sulfydryl of the α of lysine residue and ε amino, N-terminal amino, halfcystine, the imidazolyl of Histidine etc.
Term " body in function half life " is meant that still there are the time of 50% biologic activity in polypeptide in vivo or conjugate.The another kind of method of determining function in body half life is definite " serum half life ", promptly 50% polypeptide or conjugate before being eliminated in blood circulation the time." serum half life " and other term synonym, for example " blood plasma half life ", " circulation half life ", " blood clearance ", " plasma clearance ", " removing half life " or the like.
Be meant the mensuration of corresponding half life under comparable conditions of conjugate about " body in function half life " or " serum half life " used term " increase ", with respect to reference molecule, for example the half life of not modified recombinant human interferon alpha 2, increase on statistical significance.For example, corresponding half life, may increase about 25% or 50% or 100% or the like at least.
Term " kidney removing " is meant that kidney passes through the filtration of renal glomerulus, any removing that the secretion of uriniferous tubules or the drainage of uriniferous tubules are carried out.Kidney is removed the physical features that depends on polypeptide or conjugate molecule, for example molecular size (diameter), symmetry, rigidity and electric charge.
Prepare recombinant human Interferon alfacon-1 of the present invention-selected polymkeric substance of polyethylene glycol conjugation thing
With peptide molecule link coupled polymer molecule can be any suitable polymer molecule, for example homopolymer or heteropolymer, usually and the molecular weight of polypeptide link coupled polymkeric substance 300 to 100,000Da, 500-20 for example, 000Da, preferred 1000-15,000Da, more preferably 2000-12,000Da.
The example of suitable polymer molecule comprises various polyalkylene oxides (PAO), polyalkylene glycol (PAG) for example, polyoxyethylene glycol (PEG) and glycol polypropylene (PPG), polyvinyl alcohol (PVA) etc.Usually the polymkeric substance of selecting for use is the water-soluble polymer of biocompatible, nontoxic, no antigen, non-immunogenicity.
Polyoxyethylene glycol (PEG) is preferred polymer molecule, and mono methoxy polyethylene glycol (mPEG) is preferred polymer molecule.Because the hydroxyl of mPEG one end is replaced by methoxyl group, thus can avoid and cause crosslinked between the peptide molecule during polypeptide linked reaction.Selected polyoxyethylene glycol can be a polyoxyethylene glycol linear or that have branch or the capable structure of star.
In order to realize the covalently bound of polymer molecule and peptide molecule, the terminal hydroxyl of the peg molecule that provides must change the reactive group of certain form earlier into, is beneficial to the linked reaction of polymer molecule and peptide molecule.Suitable activated polyethylene glycol molecule can be the arylation polyethylene glycol reagents, mPEG-dichlorotriazine (mPEG-dichlorotriazine) and 2 for example, 4-two (mono methoxy polyethylene glycol)-6-chloro-sym-triazine (2,4-bis-(methoxypolyethylene glycol)-6-chloro-s-triazine); Acylations polyethylene glycol reagents, for example SucCINFnimidyl SucCINFnate-mPEG, SucCINFnimidylCarboxymethylate-mPEG, SucCINFnimidyl Amino aCINFd-mPEG, p-Nitro phenylCarbonate-mPEG; Alkylation polyethylene glycol reagents, for example Tresylate-mPEG, mPEG-acetaldehyde, FMP-mPEG.Suitable activated polyethylene glycol molecule can also be polyoxyethylene glycol branch shape or star, for example mPEG2-NHS, mPEG2-ALD, mPEG2-MAL, multi-armsmPEG.In order to obtain the coupled product that specificity is modified, preferred mPEG-acetaldehyde, mPEG-MAL etc.In order to obtain maximum molecular weight with minimum decorating site, preferred mPEG2-NHS, mPEG2-ALD, mPEG2-MAL, multi-arms mPEG etc.
The method for preparing recombinant human Interferon alfacon-1 of the present invention-polyethylene glycol conjugation thing
The coupling of polypeptide and activated polyglycol molecule can be by any suitable method, and for example liquid phase is freely modified and solid phase is assisted modification etc.To those skilled in the art, it is very easy adopting suitable modification reaction according to the activated polyethylene glycol reagent of selecting for use.But the coupling of polyoxyethylene glycol and polypeptide can occur on any reactive group, but also can specificity occurs on certain or some reactive groups.
In order to prepare best conjugate, at the quantity of link coupled PEG molecule, the molecular weight of each molecule and shape (for example, these molecules are linearity or branch), factors such as the coupling position of PEG and polypeptide can be modified PEG and be designed.Selection to the molecular weight of the polyoxyethylene glycol that uses will be considered the effect that expectation reaches.If for example the link coupled main purpose is the conjugate (for example reducing the kidney clearance rate) that obtains high molecular and larger molecular weight, can select so and one or two high-molecular weight peg molecule coupling.But if exist a plurality of immune sites to cover on the peptide molecule, the low molecular poly molecule that can use sufficient amount is to cover the immune site on all or most of polypeptide surface effectively.
Usually, but the coupling of polyoxyethylene glycol and peptide molecule be make reactive group as much as possible and peg molecule the reaction condition under carry out.This can reach by the mode of the suitable mole number that surpasses polypeptide of the mole number that makes polyoxyethylene glycol.Usually, the mol ratio of activated polyethylene glycol and polypeptide can be 1000: 1～1: 1, for example, about 100: 1, preferably approximately 50: 1～1: 1, more preferably about 10: 1～2: 1.
The present invention optimizes the linked reaction of polyoxyethylene glycol and polypeptide.Be appreciated that most modification reaction is to be undertaken by the nucleophillic attack that carries out at the nucleophilic group on the peptide molecule.Therefore, most modification reaction is to carry out under alkaline a little environment, for example pH7.0～10.0.And in the process of reaction, constantly produce acidic substance, make the pH value of reaction system reduce.And the fluctuation of pH value of reaction system is unfavorable for keeping the repeatability of modification reaction, thereby is unfavorable for the quality control of conjugate.Different with previously disclosed method, for example suppress the reduction of pH value of solution value by the method that in the modification system, adds alkaline matter, the present invention passes through alkaline matter, borax for example, be compressed into fine and close chip solid by a certain percentage with polyoxyethylene glycol, and in reaction buffer, slowly discharge activated polyethylene glycol reagent gradually in the dissolved process and alkaline matter realizes that the linked reaction of activated polyethylene glycol reagent and polypeptide suppresses the reduction of pH value of buffer solution simultaneously by it.Use method of the present invention can keep the repeatability of modification reaction, thereby help the quality control of conjugate.
When linked reaction proceeds to the degree of expection, can be by in reaction system, adding excessive primary amine, Methionin for example, method stop linked reaction, also can be by the method for conditioned reaction system pH, for example the pH value of solution value is adjusted to below 4.0 with acid, stops linked reaction.
The recombinant human Interferon alfacon-1 of the present invention-separation of polyethylene glycol conjugation thing, purification process
The linked reaction of polyoxyethylene glycol and polypeptide is except the coupled product that can produce expectation, also can produce some undesirable by products, for example the polyoxyethylene glycol of the free small molecules that breaks away from from activated polyethylene glycol reagent, hydrolytic inactivation, without modified polypeptides molecule etc.Before using polypeptide-polyethylene glycol conjugation thing, these materials must be removed.In addition, linked reaction produces various multi-form conjugates usually, conjugate of two peg molecules of coupling etc. on the conjugate of a peg molecule of coupling on peptide molecule, the peptide molecule for example, before using these conjugates, preferably they are separated into single composition.Another aspect of the present invention is about the chromatographic separation of polypeptide-polyethylene glycol conjugation thing, purification process.With previously disclosedly separate, purification process is different, for example, removes the polyoxyethylene glycol of small molecules free group and hydrolytic inactivation by the method for dialysis or ultrafiltration.By gel filtration chromatography or ultrafiltration conjugate and unreacted polypeptide are separated.But there are some problems in these methods, for example often small molecules and unnecessary peg molecule can not be removed fully by the method for dialysis or ultrafiltration, and the treatment capacity of gel filtration chromatography are limited, and this may limit preparative scale amplification.The present invention removes byproduct of reaction and separates different coupled products by ion-exchange chromatography, for example, utilize the desalination chromatographic column soon the small molecules group that dissociates to be removed fully, the buffer system with product is replaced into the required damping fluid of next step chromatographic separation simultaneously; Utilize the cation-exchange chromatography post to separate with coupled product with without the modified polypeptides molecule easily; Utilize anion-exchange column can be easily unnecessary peg molecule to be removed, and classification, the multi-form conjugate of purifying.This method has fast, efficient, protein-active is not had the advantage of injury, and can be easy to carry out more massive separation preparation.
Recombinant human Interferon alfacon-1 of the present invention-polyethylene glycol conjugation thing can be analyzed, identify with methods such as SDS-PAGE or MALDI-TOF.The biologic activity of conjugate is measured by the method for Chinese biological goods rules, promptly passes through the antiviral activity of Wish cell (people's amnion passage cell)-VSV virus (folliculus stomatitis virus) various conjugates of systems measurement.
The purposes of recombinant human interferon alpha 2 of the present invention-polyethylene glycol conjugation thing
The purposes of polyethyleneglycol modified recombinant human Interferon alfacon-1 of the present invention is to be used to prepare the medicine that antiviral, cancer therapy drug and treatment immunologic hypofunction and Interferon, rabbit lack syndromes.
Polyethyleneglycol modified recombinant human Interferon alfacon-1 of the present invention can keep the antiviral activity of the natural Interferon, rabbit of people, it has better thermostability and resistance to enzymolysis stability than the natural Interferon, rabbit of people again simultaneously, its plasma clearance is lower, and the body-internal-circulation transformation period is longer.Therefore, as antiviral, anticancer medicine, it is more effective than natural Interferon, rabbit.
Description of drawings
The SDS-PAGE electrophoretogram of FIG.1 Pegylation Interferon alfacon-1, wherein first swimming lane is a molecular weight standard; Second swimming lane is an Interferon alfacon-1; The 3rd swimming lane is the product that the polyoxyethylene glycol succinimdyl carbonate is modified Interferon alfacon-1; The 4th swimming lane is the product that FMP-PEG modifies Interferon alfacon-1.
Specific implementation method
In the purpose of this embodiment of listing only is for the present invention is described better, and does not have the meaning of any restriction scope of the invention.To those skilled in the art, the possibility that method of the present invention, application are changed, revise, recombinated all is conspicuous, but anyly do not exceed scope and spirit of the present invention at change of the present invention, modification and reorganization, it will further obtain clear and definite in claims part of the present invention.
The preparation of mono methoxy polyethylene glycol succinimdyl carbonate (SC-PEG): take by weighing 5 gram mono methoxy polyethylene glycols 5000, and it is dissolved in 25 milliliters of anhydrous dioxane, heating is dissolved polymkeric substance fully; With 6 mmole N, N '-two succinimdyl carbonate is dissolved in 10 milliliters of anhydrous second eyeballs; 6 mmole 4-(dimethylamine) pyridines are dissolved in 10 milliliters of anhydrous second eyeballs; Under agitation condition, with N, the second eyeball solution of N '-two succinimdyl carbonate is added in the dioxane solution of mono methoxy polyethylene glycol slowly, then, adds the second eyeball solution that is dissolved with 4-(dimethylamine) pyridine more at leisure in above-mentioned solution; Reaction continues at least 6 hours; Reaction is precipitated out mono methoxy polyethylene glycol succinimdyl carbonate (SC-PEG) with ether after finishing.With ethyl acetate or the above-mentioned Acibenzolar of Virahol recrystallization; With the dry Acibenzolar of vacuum drier, and in 4 ℃ of following lucifuge exsiccant environment, preserve.
The preparation of FMP-mPEG: 250ml toluene and 50gmPEG5000 are added in the round-bottomed flask of 1000ml, on round-bottomed flask, connect water trap, reflux condensing tube and drying tower successively, heat toluene solution to 140 ℃ then.Kept reflux state two hours.Change reflux into vacuum distillation apparatus then, steam 200ml toluene.Add the 500ml ether again in round-bottomed flask, cooling naturally is precipitated out fully up to solid under stirring condition.Remove by filter ether then, and throw out is put in the vacuum drier remaining ether is removed.Obtain the anhydrous mPEG5000 of exsiccant.With the moisture content among the dry anhydrous mPEG5000 in back of Ka Shi moisture content tester mensuration, require moisture content to be lower than 50ppm then.Pour in the round-bottomed flask of 500ml with the dry 25ml acetonitrile of graduated cylinder weighing, 567mg FMP is dissolved in the acetonitrile.In flask, add the 10g molecular weight then and be 5000 mono methoxy polyethylene glycol (mPEG).In flask, drip triethylamine in the time of stirred solution lentamente to keep the pH value of solution value between 8～9.Reaction is 15 minutes under the condition of normal temperature and pressure.Reaction finishes the back adds capacity in acetonitrile solution ether, till not having white solid to generate.Filter the mixing solutions of ether and acetonitrile with funnel, and with 300ml ether washing precipitate.Then, the solid of white is dissolved in the 250ml Virahol again, and is heated to 55 ℃ of complete dissolved solidss.Naturally cooling is precipitated out fully up to solid under stirring condition.Remove by filter Virahol then, and throw out is put in the vacuum drier Virahol of remnants removed in dry 3 hours.Obtain fluffy white solid.
The recombinant human Interferon alfacon-1 is dissolved in the pH value is 7.0-9.0, concentration is in the boric acid-borate buffer solution or phosphate buffered saline buffer of 25mM-100mM.With activated polyglycol reagent, for example succinimdyl carbonate polyoxyethylene glycol (SC-PEG) and borax for example 1: 1～1: 10, mix and with powerful pressure both are pressed into the sheet of densification or the solid of other shape according to a certain percentage.In protein solution, add this fine and close flap then, and slowly stir protein solution and make its slow dissolving.Be reflected under 4-37 ℃ and carry out, the reaction times is 30-120 minute.Reaction finishes to come termination reaction by add excessive glycine in above-mentioned system.
Reaction mixture is carried out chromatographic separation.At first sloughing the free small molecules of reaction generation and proteic buffer system is replaced into the pH value with gel-filtration column is 4.0-5.0, and concentration is acetic acid-sodium-acetate buffer of 25mM-100mM.Then the protein ingredient of collecting is separated with the recombinant human interferon alpha 2 of cation-exchange chromatography with polyethyleneglycol modified recombinant human Interferon alfacon-1 and unmodified, chromatographic condition is: be 4.0-5.0 with pH, concentration is that acetic acid-sodium-acetate buffer of 25mM-100mM is an A moving phase, with pH is 4.0-5.0, and concentration is that to add 0.2M NaCl be B moving phase to acetic acid-sodium-acetate buffer of 25mM-100mM.Under this chromatographic condition recombinant human Interferon alfacon-1-polyethylene glycol conjugation thing not with chromatograph packing material generation electrostatic interaction, therefore pass in the peak at stratographic and can collect conjugate, and unreacted recombinant human Interferon alfacon-1 can be adsorbed on the chromatograph packing material, it can be eluted with B liquid, and collect again and utilize.Remove unnecessary peg molecule and classification, the multi-form recombinant human Interferon alfacon-1-polyethyleneglycol modified conjugate of purifying with anion-exchange chromatography at last, the recombinant human Interferon alfacon-1 of modified by polyethyleneglycol for example, the two recombinant human Interferon alfacon-1s of modifying of polyoxyethylene glycol and the polyoxyethylene glycol recombinant human Interferon alfacon-1s of modifying more.Chromatographic condition is: be 8.0-10.0 with pH, concentration is that the phosphate buffered saline buffer of 25mM-100mM is an A moving phase; With pH is 8.0-10.0, and concentration is that to add 1M NaCl be B moving phase to the phosphate buffered saline buffer of 25mM-100mM.Under this chromatographic condition unnecessary polyoxyethylene glycol not with chromatograph packing material generation electrostatic interaction, therefore pass in the peak at stratographic and can collect polyoxyethylene glycol, and various multi-form recombinant human Interferon alfacon-1s can be adsorbed on the chromatograph packing material, and can utilize the NaCl gradient in the B moving phase that its classification, purifying are obtained single component.
Embodiment 4
By Chinese biological goods rules (Ministry of Health of the People's Republic of China, one one of Chinese biological goods rules, nineteen ninety-five version, Beijing: the China Population Press, 1995:268-269) method is measured.
Antiviral activity with Wish cell (people's amnion passage cell)-VSV virus (folliculus stomatitis virus) various conjugates of systems measurement.Adopt CPE (cell cause a disease effect) to suppress the inhibition micromethod for the basis, examining the dilution inverse that the high dilution of product still can protect half cell (50%) to avoid virus attack with every milliliter of Interferon, rabbit is Interferon, rabbit unit.Represent with international unit (IU), and proofread and correct with national IFN α standard substance.
Embodiment 5
Thermostability: get rhCINF, mono-mPEG-rhCINF, each 1 mmole of di-mPEG-rhCINF, poly-mPEG-rhCINF, place respectively under 4 ℃, 37 ℃, the 56 ℃ conditions, timing sampling is measured the antiviral activity of Interferon, rabbit.The result shows: rhCINF is at 56 ℃ of following 4-6 hours, loss of activity 73%; Mono-mPEG-rhCINF loss of activity 52%; Di-mPEG-rhCINF loss of activity 37%; Poly-mPEG-rhCINF loss of activity 22%.RhCINF is at 37 ℃ of following 10-12 hours, loss of activity 51%; Mono-mPEG-rhCINF loss of activity 33%; Di-mPEG-rhCINF loss of activity 17%; Poly-mPEG-rhCINF loss of activity 6%.RhCINF is in 4 ℃ of following 1 week-2 weeks, loss of activity 45%; Mono-mPEG-rhCINF loss of activity 11%; Di-mPEG-rhCINF loss of activity 4%; Poly-mPEG-rhCINF loss of activity 0%.The aforementioned stable test is in liquid state, does not have to carry out under any stablizer and the protectant condition, can think tentatively that therefore recombinant human Interferon alfacon-1 and polyethylene glycol conjugation can increase its thermostability afterwards.
Resistance to enzymolysis stability: get 2.5 milliliters of 0.1% trypsin solutions (pH8.0), each adds in 0.3 milliliter of rhCINF, mono-mPEG-rhCINF, di-mPEG-rhCINF, the poly-mPEG-rhCINF sample, 25 ℃ of insulations are measured antiviral activity at 0,10,30,50,70,90 minute time sampling respectively.The result shows that rhCINF activity within 5～7 minutes is reduced to 50%, 30 minute original activity rapidly and almost completely loses; Mono-mPEG-rhCINF activity within 10～13 minutes is reduced to 50%, 50 minute original activity and almost completely loses; Di-mPEG-rhCINF activity within 50～54 minutes is reduced to 50%, 90 minute original activity and almost completely loses; Poly-mPEG-rhCINF is active within 82～85 minutes to be reduced to original 50%.
Claims (11)
1. recombinant human Interferon alfacon-1-polyethylene glycol conjugation thing, its structure is as shown below:
Wherein, R
1Be low alkyl group, for example methyl, ethyl, propyl group etc.; R
2For-O-or-NH-; R
3Be N-R
4, S, O; R
4Be low alkyl group or amino acid; M and n are the integer more than or equal to 1.CINF is an Interferon alfacon-1, it is characterized in that in the amino acid of primary structure that 20% is identical with INF-β, and 78% is identical with INF-γ; And compare it with existing any natural interferon active high 6～11 times.
2. recombinant human Interferon alfacon-1-polyethylene glycol conjugation thing is characterized in that comprising the polypeptide with recombinant human Interferon alfacon-1 physiologically active, but and has the reactive group coupling mutually of a non-polypeptide fraction and polypeptide fraction at least.
3. a method for preparing claim 1,2 described recombinant human Interferon alfacon-1-polyethylene glycol conjugation things is characterized in that linked reaction can be the auxiliary coupling of free coupling of liquid phase or solid phase.
4. preparation method according to claim 3 is characterized in that polyoxyethylene glycol and alkaline matter are compacted into the fine and close sheet or the solid of other shapes.
5. according to the preparation method of claim 4, it is characterized in that realizing the reduction of linked reaction and inhibited reaction system pH by the slow dissolving of compact solid in reaction buffer.
6. a claim 1, the 2 described recombinant human Interferon alfacon-1-separation of polyethylene glycol conjugation thing, purification process is characterized in that removing byproduct of reaction and classification, purifying conjugate by chromatographic process.
7. according to separation, the purification process of claim 6, it is characterized in that removing the free small molecules that reaction produces by the method for desalination chromatogram or dialysis.
8. according to separation, the purification process of claim 6, it is characterized in that removing unreacted protein, and collect and utilize by the cation-exchange chromatography process.
9. according to separation, the purification process of claim 6, it is characterized in that removing unnecessary peg molecule by the anion-exchange chromatography process, and classification, the various recombinant human Interferon alfacon-1 of purifying-polyethylene glycol conjugation thing.
10. purification of recombinant human Interferon alfacon-1-polyethylene glycol conjugation thing that method according to claim 6 obtains, it is characterized in that to keep the antiviral activity of the natural Interferon, rabbit of people, simultaneously has better thermostability and resistance to enzymolysis stability than natural Interferon, rabbit again, plasma clearance is lower, and the body-internal-circulation transformation period is longer.
11. according to claim 1,2 described recombinant human Interferon alfacon-1-polyethylene glycol conjugation things, it is characterized in that to prepare together with other drug and become pharmaceutical composition, can be separately as medicinal application, and can with the various viral infections of above-mentioned medicine composite for curing, cancer, immunologic hypofunction and Interferon, rabbit lack syndromes etc. the method for disease.
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| CN101768205A (en) * | 2010-03-01 | 2010-07-07 | 安徽安科生物工程（集团）股份有限公司 | Method for recovering interferon from polyethylene glycol modified interferon reaction liquid |
| CN101724142B (en) * | 2008-10-22 | 2011-12-14 | 中国科学院过程工程研究所 | Poly alkyl ether polymer using thiosulfonate as terminal group and synthesis and application thereof |
| CN101002944B (en) * | 2006-01-17 | 2012-07-25 | 中国科学院过程工程研究所 | Conjugate of branched chair polymacrogol-interferon, and its preparing method |
| CN106632588A (en) * | 2015-08-27 | 2017-05-10 | 江苏众红生物工程创药研究院有限公司 | A purifying process for polyethylene glycol modified protein |
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| CN101002944B (en) * | 2006-01-17 | 2012-07-25 | 中国科学院过程工程研究所 | Conjugate of branched chair polymacrogol-interferon, and its preparing method |
| CN101724142B (en) * | 2008-10-22 | 2011-12-14 | 中国科学院过程工程研究所 | Poly alkyl ether polymer using thiosulfonate as terminal group and synthesis and application thereof |
| CN101768205A (en) * | 2010-03-01 | 2010-07-07 | 安徽安科生物工程（集团）股份有限公司 | Method for recovering interferon from polyethylene glycol modified interferon reaction liquid |
| CN106632588A (en) * | 2015-08-27 | 2017-05-10 | 江苏众红生物工程创药研究院有限公司 | A purifying process for polyethylene glycol modified protein |
| CN112442180A (en) * | 2019-09-04 | 2021-03-05 | 北京化工大学 | Amphiphilic polymer for promoting stem cell interface adhesion growth and preparation method and application thereof |
| CN110591077A (en) * | 2019-10-17 | 2019-12-20 | 江南大学 | Method for preparing tyrosine oligopeptide and grafted monomethoxy polyethylene glycol through enzyme catalysis |
| CN110669810A (en) * | 2019-10-17 | 2020-01-10 | 江南大学 | Method for preparing lysine oligopeptide through enzyme catalysis and modifying lysine oligopeptide by monomethoxypolyethylene glycol |
| CN110563939A (en) * | 2019-10-17 | 2019-12-13 | 江南大学 | Method for preparing phenylalanine oligopeptide-monomethoxypolyethylene glycol copolymer by enzyme catalysis |
| CN110591077B (en) * | 2019-10-17 | 2022-01-07 | 江南大学 | Method for preparing tyrosine oligopeptide and grafted monomethoxy polyethylene glycol through enzyme catalysis |
| CN110669810B (en) * | 2019-10-17 | 2023-08-08 | 江南大学 | Method for preparing lysine oligopeptide and modifying monomethoxy polyethylene glycol thereof by enzyme catalysis |
| CN111100894A (en) * | 2019-12-31 | 2020-05-05 | 江苏苏南药业实业有限公司 | Method for carrying out fixed-point polyethylene glycol long-acting modification on interferon IFN α -2b |
| CN112251484A (en) * | 2020-10-26 | 2021-01-22 | 江南大学 | Method for preparing glycine oligopeptide-polyethylene glycol copolymer under catalysis of lipase |
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