CN101503457B - Column chromatography PEG and interferon analogue protein N-terminal site-directed coupling method and product thereof - Google Patents
Column chromatography PEG and interferon analogue protein N-terminal site-directed coupling method and product thereof Download PDFInfo
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- CN101503457B CN101503457B CN2009100137111A CN200910013711A CN101503457B CN 101503457 B CN101503457 B CN 101503457B CN 2009100137111 A CN2009100137111 A CN 2009100137111A CN 200910013711 A CN200910013711 A CN 200910013711A CN 101503457 B CN101503457 B CN 101503457B
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Abstract
The invention discloses a method of proteinic nitrogen fixed-point coupling of column chromatography PEG and interferon analog, which realizes the specific coupling of PEG-ALD and IFNs-A N-terminal methionine alfa amino by closing lysine epsilon amino on IFNs analogs, IFNs-A by ion exchange chromatography and obtains a single, fixed-point N-terminal coupling PEG-IFNs-A product by one-step purification at the same time. The invention also further discloses the application of PEGylation recombinant human interferon analog protein in medicines for curing virus B hepatitis or viral C hepatitis, multiple sclerosis, rheumatoid arthritis, malignancy, hairy cell leukemia and AIDS kaposi ' s sarcoma.
Description
Technical field
The present invention is under the jurisdiction of protein modification and water-soluble polymer coupling protein, polypeptide and two mutants thereof or analogue field in a broad sense; Statement is comparatively particularly; The present invention relates to a kind of column chromatography that utilizes and realize polyoxyethylene glycol and interferon analogue (Interferons Analogs; IFNs-A) protein-n distal point specificity link coupled method; And utilize one type of Pegylation recombinant human interferon alpha 2 analogue proteinate that this method obtains and said this type tool is pharmaceutically long-acting, efficient, the recombinant protein compounds of reduced immunogenicity characteristic is as prolonged action preparation class production of medicine and application.
Background technology
Interferon, rabbit (Interferon; IFN) be the general designation of a cytokine family; The Interferon, rabbit of mammal can be divided into few types such as α, β, γ, ω; Antiviral, antitumor and immunoregulation effect with wide spectrum; Recombinant human interferon alpha 2 (Recombiant Human Interferon wherein; RhIFN) (Interferon alfacon-1, IFN-con) all approved listings are used to treat B-mode or hepatitis C, multiple sclerosis, rheumatism or rheumatoid arthritis, malignant tumour (Fei Hejiejinshi disease, melanoma, mammary cancer etc.), hair white blood disease (HairyCell Leukemia) and AIDS card Podbielniak knurl etc. the consensusIFN of α-1b, α-2a, α-2b, β-1b, γ and α type; Calendar year 2001 and 2002, U.S. FDA ratified Pegylation (Polyethylene Glycol again respectively; PEG) two kinds of prolonged action preparation listings of Interferon Alpha-2b (PEG-Intron) and Intederon Alpha-2a (Pegasys); Compare with common Interferon, rabbit; Can realize 1～2 week injection administering mode once, obtain better accessibility, curative effect and security, become the drug of first choice or a line medicine of treatment chronic viral hepatitis and rheumatic arthritis at present; But the coupling of PEG and IFN is not dot characteristics (site-specific Pegylation) in PEG-Intron and the Pegasys molecule, and this has brought problem and trouble for the quality controllability of product, and the external activity of molecule is significantly reduced; PEG-Intron is that the PEG coupling of 12kDa gets by molecular weight; Its prolongs not enough plasma clearance transformation period, and Pegasys is that the PEG coupling of 40kDa get by molecular weight, and distribution is too small in its body; Make the recurrence rate of virus after the drug withdrawal higher, so prolonged action preparation exists further optimization and improved needs.
Obviously adopt some specificity PEG coupling mode, can realize the homogeneity of PEG-IFN molecule on molecular weight and molecular structure simultaneously, also can improve the external activity of PEG-IFN molecule.Existing multiple coupling strategy is realized the some specificity coupling of PEG-IFN; Comprising: 1) USP 5; 985,263 have announced the method and the product thereof of a kind of PEG relative specificity coupling Histidine (histidine), PEG succinic diamide carbonic ether (succinimidyl carbonate PEG; SC-PEG) but relative specificity and 34 hyte propylhomoserin (His34) couplings form PEG-His34-IFN alpha, the higher biological activity of conjugates tool in this site; 2) WO2006/004959 has described the method and the product thereof of 86 free halfcystines (Cys86) specificity coupling PEG maleinamide (PEG maleimide) in a kind of IFN of utilization alpha-1b aminoacid sequence; 3) tyrosine (Tyr) that discloses the 85th of a kind of point mutation IFN alfa or 86 among the WO2008/074230 is Cys; Utilize a kind of PEGization reagent of sulfydryl reaction again; Like maleimide-PEG, vinyl sulfone(Remzaol-PEG, iodo-acetamide-PEG and positive pyridine disulphide-PEG etc.; Realize some specificity PEG and IFN α-2a, IFN α-2b, the coupling of IFN-con etc.
Another kind of important some specificity coupling strategy is the coupling of protein-n terminal (N-is terminal) some specificity.(1) such as Wetzel R proposed to realize the amino highly selective coupling of the terminal alfa of polypeptide N-through control pH value in nineteen ninety; (3) such as Chang TK find that in 1994 subtilase enzymes (subtiligase) has the effect of enzymatic PEG selectivity link coupled; GaertnerHF etc. (2) have described various PEG reactive groups that can selectivity coupling protein N-terminal amino group in 1996, and on interleukin-8, G-CSF equimolecular, use; Nearer also have the scholar to adopt recombinant DNA technology (4), through expressing the terminal coupling target protein of PEG point specificity N-in trans-glutaminases (transglutaminase) the mediation microbe body.
The control pH value of passing through that proposes in nineteen nineties such as above-mentioned Wetzel R realizes on the amino highly selective link coupled basis of the terminal alfa of polypeptide N-; The Kinstler OB of U.S. Amgen company and Gabrial etc. have described in further detail through reducing method and the product thereof that the pH value can make N-terminal amino groups such as monomethyl polyoxyethylene glycol aldehyde radical (PEG-ALD) specificity coupling rhG-CSF and Interferon alfacon-1; See USP 5,824 for details, 784/USP 5; 985; 265/USP 7,090, patents such as 835 B2 and US2006/0233746 A1 and CN 1896103A; Kinstler etc. realize that through reducing the pH value PEG-ALD selectivity coupling protein N-is terminal; When the pH value is under the condition of acid (like pH 4.5), and the amino linked reaction probability with PEG-ALD of the terminal alfa of N-is amino far above the epsilon on the Methionin on rhG-CSF or the IFN-con molecule; So when linked reaction continues the long period, most PEG-ALD will occur and be coupled on rhG-CSF or the terminal alfa amino of IFN-con molecule N-, form PEG-rhG-CSF or PEG-IFN-con compound single, fixed point; Therefore, form condition single, the terminal highly selective of albumen N-and comprise low pH value and long reaction times, need relatively low PEG-ALD input amount in addition.And the pH value improves the coupling that can quicken PEG-ALD and protein molecular amino, just can lose PEG-ALD this moment to the terminal selectivity of albumen N-, forms and modifies the proteinic mixtures of PEG-more.In fact, U.S. Pat P 4,002,531 and the Shaw etc. that equal 1977 at Royer GI are at the U.S. Pat P4 of nineteen ninety, this are also all had in 904,584 and address.
Fee CJ (USP 2006/0128945 A1) has announced that a kind of gel chromatography that utilizes carries out the technology that protein PEG modifies; Propose because protein and the speed difference of PEGization protein in gel chromatography moving phase; Use chromatography column to carry out the PEGization linked reaction and possibly more be prone to produce single coupled product; But the method for Fee CJ (6) does not relate to the PEGization linked reaction that needs add catalyzer, the terminal specificity coupling technology of nonprotein N-yet.The method of utilizing a kind of solid phase PEGization technology point specificity modified recombinant human interferon alfa-2a (IFN a-2a) has been described in (5) such as nearest Lee BK; And obtained and IFNa-2a albumen N-end link coupled PEG-IFN; But this method is the coupled product that has just obtained the terminal highly selective of albumen N-through quite similar catalytic reaction conditions such as use and Kinstler OB; Therefore, in essence, pH value but not chromatography media or chromatography condition through the control reaction system make PEG-ALD highly selective coupling protein N-end; And because the restriction in reaction times reduces the linked reaction rate, more free IFN a-2a appears.
Just delivered crystal structure analysis as far back as (7) such as nineteen eighty-two Miller about recombinant human interferon alpha 2 alfa, after researched and analysed human interferon alfa and beta structure and function (8) (9) again, can learn that according to above-mentioned document the receptor binding domain of IFN alfa mainly comprises AB loop (Arg22, Leu26; Phe27, Leu30, Lys31, Arg33; His34), helixB (Ser68), helix C (Thr79, Lys83; Tyr85, and Tyr89), helix D (Arg120, Lys121; Gln124, Lys131, Glu132) with helix E (Arg144, Glu146).And point mutation Arg22, Leu26, Phe27, Leu30, Lys31; Arg33, His34, Phe36, Arg120; Lys121, Gln124, Tyr122, Tyr129; Lys131, Glu132, Arg144 or Glu146 obtain IFN alfa two mutants (IFN alfa mutants, IFN α m) biological activity and will significantly descend more than 5 times.Interferon, rabbit alfa has two disulfide linkage, respectively between Cys 1-Cys 98 and Cys 29-Cys 138; In PEG-Intron (the PEG coupling compound of one type of PEG-IFN alfa-2b and molecular weight 12kDa) molecule, be merely 11% (10) of common IFN alfa-2b in its activity of PEG-IFN alfa-2b of Cys 1 (N-is terminal) coupling PEG; And according to analyzing in the coupling site; In one type of PEG-IFN alfa-2a of Pegasys (the PEG coupling compound of one type of PEG-IFN alfa-2a and molecular weight 40kDa) molecule not with Cys 1 (N-is terminal) link coupled PEG-IFN alfa-2a; Terminal and the non-natural IFN alfa ideal coupling site (11) of N-is described, and the also non-suitable coupling site in Lys or His site.
Flexibly connect chain (Flexible linker) (GlyGlyGlyGlySer) n be used for two proteic fusions of different activities and be connected existing history (12) more than 20 years, and clinical application does not see that it is prone to by enzymolysis or tool immunogenicity.
In sum, external PEG albumen N-distal point specificity linked reaction has multiple, and its main policies is through improving the PEG activating group the amino selectivity of the terminal alfa of N-to be realized.Yet; Through retrieval; Through ion exchange chromatography with IFNs analogue (IFNs Analogs; IFNs-A) Methionin epsilon on amino " sealing " and realize the specificity coupling that PEG-ALD is amino with IFNs-AN-terminal methionine alfa, and realize that simultaneously single step purification obtains single, as to fix a point N-end coupling PEG-IFNs-A product research document and patent and also do not appear in the newspapers so far.
Reference:
〔1〕Wetzel?R，Halualani?R，Stults?JT，Quan?C.A?general?method?for?highly?selectivecross-linking?of?unprotected?polypeptides?via?pH-controlled?modification?ofN-terminal?a-amino?groups.Bioconjugate?Chemistry?1990；1：114-122。
〔2〕Gaertner?HF?and?Offord?RE.Site-specific?attachment?of?functionalizedpoly(ethylene?glycol)to?the?amino?terminus?proteins.Bioconjugate?Chemistry1996；7：38-44。
〔3〕Chang?TK，Jackson?DY，Burnier?JP，Wells?JA.Subtiligase：a?tool?for?semisynthesisof?proteins.Proc?National?Acad?Science?1994；91：12544-12548。
〔4〕Sato?H，Yamamoto?K，Hayashi?E，Takahara?Y.Transglutaminase-mediated?dual?andsite-specific?incorporation?of?poly(ethylene?glycol)derivatives?into?chimericinterleukin-2.Biocon?jugate?Chem?2000；11：502-509。
〔5〕Lee?BK，Kwon?JS，Kim?HJ，Yamamoto?S?and?Lee?EK.Solid-phase?PEGylation?ofrecombinant?interferon?a-2a?for?site-specific?modification：processperformance，characterization，and?in?vitro?bioactivity.Bioconjugate?Chem.2007；18：1728-1734。
〔6〕Fee?CJ。Size-exclusion?reaction?chromatography(SERC)：A?New?Technique?forprotein?PEGylation.Biotechnology?and?bioengineering?2003；82(2)：200-206。
〔7〕Miller?DL，Kung?H?&?Petska?S.Crystallization?of?recombinant?human?leukocyteinterferon?A.Science?1982；215：689-690。
〔8〕Radhakrishnan?R，et?al.Zinc?mediated?dimer?of?human?interferon-a2b?revealedby?X-ray?crystallography.Structure?1996；4(12)：1453-1463。
〔9〕Karpusas?M，et?al.The?crystal?structure?of?human?interferon?b?at?2.2-Aresolution.Proc.Natl.Acad.Sci.USA?1997；94：11813-11818。
〔10〕Wang?YS，Youngster?S，Grace?M，Bausch?J，Bordens?R?and?Wyss?DF.Structural?andbiological?characterization?of?pegylated?recombiant?interferon?alpha-2b?and?itstherapeutic?implications.Adv.Drug?Del?Rev?2002；54：547-570。
〔11〕Foser?S，et?al.Isolation，structural?characterization，and?antiviral?activitvof?positional?isomers?of?monopegylated?interferonα-2b(PEGASYS).Protein?Expression&?purification?2003；30：78-87。
〔12〕Hoston?JS，et?al.Protein?engineering?of?antibody?binding?sites：recovery?ofspecific?activity?in?an?anti-digoxin?single-chain?Fv?analogue?produced?inEscherichia?coli.Proc.Natl.Acad.Sci.USA?1988；85：5879-5883。
Summary of the invention
Deficiency to prior art; The problem that the present invention will solve provides a kind of column chromatography that utilizes and realizes polyoxyethylene glycol and interferon analogue (Interferons Analogs; IFNs-A) protein-n distal point specificity link coupled method, and the one type of Pegylation recombinant human interferon alpha 2 analogue proteinate that utilizes this method to obtain.The PEGization interferon analogue albumen coupling compound tool that the inventive method obtains is the characteristic of long-acting, the efficient and reduced immunogenicity of tool pharmaceutically, and is significant in preparing prolonged action preparation class medicine.
Summary of the invention (Summary of The Invention)
The inventive method is with IFNs analogue (IFNs Analogs through ion exchange chromatography; IFNs-A) Methionin epsilon on amino " sealing " and realize the specificity coupling that PEG-ALD is amino with IFNs-A N-terminal methionine alfa, and realize that simultaneously single step purification obtains single, the N-end coupling PEG-IFNs-A product of fixing a point.
Above-mentioned IFNs-A is two mutants (the IFNs Mutants that will flexibly connect chain (GlyGlyGlyGlySer) n and IFNs or its IFNs through integration technology; IFNs-M) the terminal fusion rotein that obtains that merges of N-: (GlyGlyGlyGlySer) n-N-IFNs and (GlyGlyGlyGly Ser) n-N-IFNs-M; Be IFNs analogue (IFNs-A); Such IFNs-A finds unexpectedly; Can significantly improve the terminal linked reaction rate of macromole PEG and IFNs albumen N-, also can significantly reduce of the influence of coupling Cys1 site simultaneously for the IFNs protein biological activity.Therefore, the present invention intends with special, controllable mode directly influences proteic pharmacy of IFNs and pharmacological property, and exploitation compares with Pegasys with PEG-Intron, possesses long-acting, efficiently and the new prolonged action preparation of reduced immunogenicity characteristic simultaneously.
The invention discloses PEG and interferon analogue (Interferons Analogs; IFNs-A) albumen N-distal point specificity link coupled method promptly realizes PEG and IFNs-A albumen N-distal point specificity coupling simultaneously through ion exchange chromatography and realizes purifying single, fixed point PEG-IFNs-A compound synchronously.
Selection condition to PEG in the wherein said coupling method is:
1) can with the various activatory monomethyl of protein amino link coupled PEG (mPEG) molecule; Comprising mPEG-ALD, PEG-succinimide α methylbutyrate (mPEG-Succinimidyl α-methylbutanoate; MPEG-SMB), mPEG-succinimide propionic ester (mPEG-Succinimidyl Propionate; Various mPEG-N-HOSu NHS Acibenzolars (mPEG-N-Hydroxy succinimide active esters, mPEG-NHS ester) such as mPEG-SPA);
2) molecular weight of said mPEG is greater than 25kDa, preferred 26～40kDa, most preferably 30kDa;
3) said mPEG molecule can be strand, two strands or multichain, preferred strand;
Wherein: the preferred mPEG-ALD of said mPEG molecule; Its molecular formula is mPEG-(CH
2) r-CHO, wherein r is 1～3, the rightter scope of r is 2～3, i.e. mPEG-propionic aldehyde and mPEG-butyraldehyde.
Selection condition to IFNs-A in the said coupling method is:
1) said IFNs-A (IFNs analogue) merges for the N-that will flexibly connect chain (GlyGlyGlyGlySer) t and IFNs through integration technology is terminal; And the fusion rotein that obtains: (GlyGlyGlyGlySer) t-N-IFNs; Be IFNs analogue (IFNs-A), wherein t is 1～10, preferred 1～3; Unique characteristic of IFNs-A is that external activity is more than or equal to the former proteoplast of IFNs outer active 70%;
Wherein said IFNs is recombinant human interferon alpha 2 alfa or beta; Preferred recombinant human interferon alpha 2 alfa-2b (IFN-α 2b), recombinant human interferon alpha 2 alfa-2a (IFN-α 2a) or recombinant human interferon alpha 1 b eta-1b (IFN-β 1b), its aminoacid sequence should meet the aminoacid sequence formula shown in SEQ ID NO.1 or the SEQ ID NO.2;
2) can be divided into recombinant human interferon alpha 2 alfa analogue be IFNs-α-A to said IFNs-A, also can be recombinant human interferon alpha 1 b eta analogue IFNs-β-A;
3) said IFNs-A also can be (GlyGlyGlyGlySer) t and various IFNs two mutants (IFNsMutants; IFNs-M) merge the fusion rotein that is obtained; The characteristic of IFNs-M is that external activity is not less than 70% of the former proteic external activity of IFNs, and reduced immunogenicity or non-immunogenicity;
4) preferentially to adopt the procaryotic cell expression system be escherichia coli high-level expression to said IFNs-A; Still comprise synthetic and various other prokaryotic cell prokaryocytes, eukaryotic cell, insect, plant or animal expression system.
Selection condition to chromatography media, post and condition in the said coupling method is:
1) chromatography media can be selected various IXs, hydrophobicity, gel and chromatography media such as affine for use, and preferred cationic exchanger medium wherein is like MacroCap SP, Sepharose Fast Flow etc.;
2) chromatography column post bed height is preferably recommended the high upper limit of post;
3) the proteic applied sample amount of IFNs-A is the 10-70% that medium is surveyed maximum carrying capacity; Preferred 30-50%;
4) with under the lower condition that goes up an appearance flow velocity (less than 3ml/min), the long pH value that goes up an appearance time (3～5 hours), higher reactivity, last kind of activatory mPEG is to obtain PEGization reaction conditions preferably;
5) applied sample amount of activatory mPEG is 5～50mol: 1mol by the reacting weight ratio of activatory mPEG and IFNs-A, and preferred 5～20mol: 1mol is beneficial to the generation of mono-modified PEG-IFNs-A;
6) with 0.01M～1M sodium chloride salt solution gradient wash-out,, as shown in Figure 1 in order to effective separation of each reaction product.
With aforesaid method and selected condition thereof and the mono-modified PEG-IFNs-A elution peak sample that obtains through LC-MS chromatogram trypsin digestion peptide figure analysis; Important and find out of a clear sky: the coupling site of activatory PEG is that the N-of IFNs-A is terminal;, fixed point specificity coupled product, i.e. IFNsA-N-30, IFNsA-N-40 and IFNsA-N-20 (see embodiment 3) single like 30kDa PEG-ALD, 40kDa PEG-ALD or 20kDa PEG-ALD and IFNs-A albumen N-end; And the purity of mono-modified PEG-IFNsA elution peak sample is measured its purity of confirmation greater than 95% through efficient gel chromatogram (SEC) and performance liquid chromatography (HPLC), and is like Fig. 3, shown in Figure 4.
Adopt same chromatography and PEGization condition; The result who compares IFNs-α and IFNs-α-A chromatography column PEGization; Be surprised to find that greatly: the yield of the PEG-IFNs-α-A of the terminal pointed decoration of fusion rotein IFNs-N-that α-A obtains is significantly greater than not merging the PEG-IFNs-α that flexibly connects chain; And the external activity of PEG-IFNs-α-A is also apparently higher than PEG-IFNs-α (seeing table 1 and table 4 for details); Explain that fusion flexibly connects chain (GlyGlyGlyGlySer) t and can obviously improve the terminal site-directed coupling rate of N-,, also make PEGization that the influence of the external activity of IFNs-α-A is significantly reduced (seeing table 4 for details) simultaneously because of the avtive spot of coupling site away from IFNs-α.
The present invention also confirms (as shown in table 3); Only the pH value with above-mentioned reaction system is lowered to 4.0; Use mPEG-ALD can make mono-modified PEG-IFNs-α-A elution peak area account for total elution peak area and significantly be reduced to 50%; Unreacted IFNs-α-A elution peak accounts for total elution peak area can significantly be increased and reach 50%, modifies PEG-IFNs-α-A elution peak more and disappears; The sample of mono-modified PEG-IFNs-α-A elution peak confirms that through LC-MS trypsin digestion peptide figure analysis the coupling site of PEG-ALD is still terminal for the N-of IFNs-α-A; Prompting is under above-mentioned ion exchange chromatography condition, and the acid ph value condition is not the terminal principal element of PEG-ALD selectivity coupling IFNs-α-A N-.
The present invention also finds; Select for use can with PEG-NHS ester (the polyoxyethylene glycol N-hydroxy-succinamide Acibenzolar) modifier of protein propylhomoserin reaction; Adopt above-mentioned cation-exchange chromatography; The pH value of reaction system is 6.0～7.0, can obtain peak area greater than 70% PEG and single, the site-directed coupling product (as shown in Figure 5) of N-end of IFNs-α-A; Same reaction conditions adopts gel chromatography, also can obtain peak area greater than 70% PEG and single, the site-directed coupling product (as shown in Figure 6) of N-end of IFNs-α-A.
In liquid phase; If adopt and above-mentioned chromatography column PEGization reacting phase reactant ratio, condition and the time length together; The linked reaction product of mPEG-ALD and mPEG-SPA and IFNs-α-A; Measure through efficient gel and to show, 23%～48% the PEG-IFNs-α-A of only having an appointment usually is mono-modified PEG-IFNs-α-A, and all the other 37%～64% be many modification PEG-IFNs-α-A compound; 13～14% unreacted IFNs-α-A, obviously different with PEGization linked reaction proportion of products on the chromatography column (like Fig. 7, shown in 8).
Adopt strategy and the method identical with IFNs-α-A, analyze IFNs-β and can obtain the conclusion similar with IFNs-α-A (as shown in table 1) with above-mentioned IFNs-α with IFNs-β-A with above-mentioned IFNs-α.
The present invention also finds and confirms: under certain condition; Cation exchange medium absorption IFNs-A albumen; And can " seal " the Methionin epsilon amino that IFNs-A is positioned at molecular surface; Thereby make the alfa of PEG-ALD or PEG-NHS specificity coupling IFNs-A molecule N-terminal methionine amino; Form single, the terminal fixed point of N-PEG-IFNs-A, like IFNsA-N-30 etc. (30kDa PEG-ALD and IFNs-A albumen N-end are single, fixed point specificity coupled product), this PEG-ALD selects the terminal specificity of albumen N-and the reduction of unprovoked pH value or be due to the acidity; And terminal fusion of N-flexibly connects chain (GlyGlyGlyGlySer) t and can obviously improve the terminal site-directed coupling rate of N-; Meanwhile, the purity of collected single, the terminal fixed point of N-PEG-IFNs-A compound is greater than (like Fig. 2, shown in Figure 3) more than 95%; Explain that the reaction of chromatography column PEGization can realize single, the site-directed coupling of specificity IFNs-A N-end synchronously, and the purifying of PEG-IFNs-A coupling compound.
Further experiment is more wondrous; In chromatography (comprising IX and gel chromatography) or traditional liquid-phase reaction system; The linked reaction of interferon fusion protein IFNs-A and PEG-ALD or PEG-NHS obtains the N-end yield of PEG-IFNs-A single, fixed point and all is significantly higher than common interferon protein IFNs, the reaction tendency basically identical of IFN α and IFN β and PEG.
Another important discovery is; The external biological activity of PEG-IFNs-A is significantly higher than the PEG-IFNs with molecular weight; Explain that terminal flexible polypeptied chain (GlyGlyGlyGlySer) t that merges of IFNs N-also can make the avtive spot of the coupling site of PEG away from IFNs, thereby obviously reduced the PEG molecule the active influence of IFNs (seeing table 4 for details); In addition; Even the external activity of PEG-IFN α 2b-A-N30 is significantly higher than IFN α-2b-A and IFN α-2b; This result is unexpected, because it has been generally acknowledged that the coupling meeting of macromole PEG significantly reduces the activity of IFNs-A or IFNs; Obviously, this discovery has potential and important clinical application value; Along with increase PEG-IFNs-N10～40 of coupling PEG molecular weight or the external activity of PEG-IFNs-A-N10～40 reduce one by one; But at PEG-IFNs-N40 or PEG-IFNs-A-N40 place; The reduction of external activity is particularly remarkable, significantly active " flex point " that reduces occur; And the diPEG-IFNs-N40 of two 20kDa PEG of coupling or its external activity of diPEG-IFNs-A-N40 significantly are lower than PEG-IFNs-N40 or PEG-IFNs-A-N40 with equimolecular quantity; This points out simultaneously, double-stranded PEG or the coupling of dibit point to the influence of external activity greater than strand PEG or single site; Therefore, the present invention confirms, the N-terminal amino group of first-selected strand PEG-ALD coupling IFNs-A.
Comprehensive various physico-chemical properties, biological activity, medicine generation and immunogenicity testing data and the result who is obtained; The present invention confirms first and confirms: as long-acting, efficient and reduced immunogenicity preparation; PEG-IFN α 2bA-N30 or PEG-IFN β 1bA-N30 have more advantage than PEG-IFN α 2bA-N10, PEG-IFN α 2bA-N20, PEG-IFN α 2bA-N40, PEG-IFN β 1bA-N10, PEG-IFN β 1bA-N20 or PEG-IFN β 1b A-N40, should be most preferably.Therefore, the first-selected 30kDa PEG-ALD of Pegylation recombinant human interferon alpha 2 analogue albumen of the present invention and IFNs-A N-end is single, the polyethyleneglycol derivative of site-directed coupling, i.e. PEG-IFNsA-N30.
Detailed Description Of The Invention (Detailed Description of The Invention)
The method of column chromatography PEG according to the invention and interferon analogue protein-n terminal site-directed coupling, step comprises:
Select cation exchange medium, be loaded in the chromatography chromatographic column, the post bed height is the conventional upper limit of recommending height, and is connected in the LC system; 20mM sodium-acetate buffer and mass percent that use is equivalent to the pH 3.5～6.0 of 5～6 times of column volumes are the abundant balance chromatographic column of 0.005% polysorbate80; Use fresh feed pump,, IFNs-A stoste added in the chromatography column with the flow velocity of 8～10ml/min, applied sample amount be medium survey maximum carrying capacity 10%～70%; Again under the flow velocity of 13～15ml/min,, remove not material with chromatographic media generation electrostatic adhesion with the 20mM sodium-acetate buffer flushing pillar of the pH 3.5～6.0 of 5～6 times of column volumes; Reacting weight ratio in mPEG: IFNs-A is the ratio of 5～50mol: 1mol, and adding molecular weight ranges with appearance on the flow velocity of 0.5～3ml/min is the mPEG of 26～40kDa, and the mPEG otal investment X of said different molecular weight by formula (1) calculates:
(1) X＝5～50×m×MW
PEG/MW
IFNs-A
In the formula: X is the mPEG otal investment of different molecular weight, the IFNs-A protein content of m for adding, and MWPEG and MWIFNs-A are respectively the molecular weight of PEG and IFNs-A;
Calculate the add-on of reductive agent sodium cyanoborohydride again by following formula (2):
(2) NaBH3CN add-on=10 * MW
Reductive agent* X/MW
PEG
In the formula: the MW reductive agent is the molecular weight of reductive agent sodium cyanoborohydride, and X is the mPEG otal investment of different molecular weight, and MWPEG is the molecular weight of mPEG;
Went up appearance 240 ± 5 minutes continuously; The pH of reaction system is 3.5～6.0; Again with the flow velocity of 13～15ml/min,, remove not material with chromatographic media generation electrostatic adhesion with the 20mM sodium-acetate buffer flushing pillar of the pH 3.5～6.0 of 5～6 times of column volumes; Use the 20mM sodium-acetate buffer of pH 3.5～6.0 and the mixed solution of 0.01M～1M sodium-chlor subsequently; With type of elution 0～8%, 8%～18% and 18%～100% wash-out, continue 150 ± 2 minutes, obtain two elution peaks; Be P1 and P2; Wherein the peak area of P1 accounts for 72 ± 1% of total peak area, is mono-modified PEG-IFNs-A, i.e. single, the fixed point specificity coupling compound of polyoxyethylene glycol and IFNs-A albumen N-end;
Wherein:
Above-mentioned IFNs-A is the terminal fusion rotein that obtains that merges of the N-that will flexibly connect chain (GlyGlyGlyGlySer) t and IFNs through integration technology: (GlyGlyGlyGlySer) t-N-IFNs; Wherein t is 1～10; IFNs is recombinant human interferon alpha 2 alfa or beta; Its aminoacid sequence should meet the aminoacid sequence formula shown in SEQ ID NO.1 (wherein 23 are Arg, are Interferon alfa-2b, are Interferon alfa-2a as if 23 for Lys) or the SEQ ID NO.2;
Above-mentioned mPEG refer to can with the various activatory monomethyl of protein amino link coupled PEG molecule, be PEG-succinimide α methylbutyrate in mPEG-ALD or the mPEG-N-HOSu NHS Acibenzolar or mPEG-succinimide propionic ester.
Further, the method for above-mentioned column chromatography PEG and interferon analogue protein-n terminal site-directed coupling preferred embodiment is:
Preferred MacroCap SP of said cation exchange medium or Sepharose Fast Flow.
Said IFNs-A is the terminal fusion rotein that obtains that merges of the N-that will flexibly connect chain (GlyGlyGlyGlySer) t and IFNs through integration technology: (GlyGlyGlyGlySer) t-N-IFNs; Wherein t is 1～3, and IFNs is recombinant human interferon alpha 2 alfa-2b (IFN-α 2b), recombinant human interferon alpha 2 alfa-2a (IFN-α 2a) or recombinant human interferon alpha 1 b eta-1b (IFN-β 1b);
Said IFNs-A preferably adopts the escherichia coli expression preparation in the procaryotic cell expression system, still comprises synthetic and various other prokaryotic cell prokaryocytes, eukaryotic cell, insect, plant or animal expression system.
Aminoacid sequence by the Interferon beta 1b of escherichia coli expression is compared with the natural Interferon beta of people 1b aminoacid sequence; The N-end has increased Met (methionine(Met)) and Cys17Ser (17 Cys become Ser); And people Interferonbeta 1a is identical with the natural Interferon beta of people 1b aminoacid sequence by the gp of expressing cho cell.
Unique characteristic of said IFNs-A is that external activity is more than or equal to the former proteoplast of IFNs outer active 70%.
The applied sample amount of said IFNs-A is preferably the 30-50% that medium is surveyed maximum carrying capacity.
The preferred 30kDa of the molecular weight of said mPEG; The preferred strand of mPEG molecule; The applied sample amount of said mPEG is preferably the ratio of 5～20mol: 1mol in the reacting weight ratio of mPEG: IFNs-A.
The preferred mPEG-ALD of said mPEG; Its molecular formula is mPEG-(CH
2) r-CHO, wherein r selects 2～3, i.e. mPEG-propionic aldehyde or mPEG-butyraldehyde;
Wherein the molecular structural formula of mPEG-propionic aldehyde is as follows;
The molecular formula of mPEG-butyraldehyde
The pH of the reaction system of said column chromatography IFNs-A nitrogen-terminal fixed-point coupling mPEG is preferably 5.0～6.0.
One type of Pegylation recombinant human interferon alpha 2 analogue albumen that the method for column chromatography PEG according to the invention and interferon analogue protein-n terminal site-directed coupling makes is formed greater than the PEG molecule site-directed coupling of 26kDa by the N-terminal amino group of nonglycosylated IFNs-A and single, strand, molecular weight; It is characterized in that: said Pegylation recombinant human interferon alpha 2 analogue protein molecular formula is: CH
3-(CH
2CH
2-O) n-(CH
2) r-NH-(GlyGlyGlyGlySer) t-IFNs, n is 570～2200 in the formula, and r is 0～4, and t is 1～10; IFNs is recombinant human interferon alpha 2 alfa or beta; Its aminoacid sequence should meet SEQID NO.1, and (wherein 23 are Arg; Be Interferon alfa-2b, if 23 are Interferonalfa-2a for Lys) or SEQ ID NO.2 shown in the aminoacid sequence formula; Said Pegylation recombinant human interferon alpha 2 analogue albumen also should possess feature simultaneously:
1) IFNs-A and mPEG link coupled molecule ratio are 1: 1, and the coupling site is an IFNs-A molecule N-terminal amino group;
2) in specific activity is measured; Measure the protein concentration of PEG-IFNs-A to be measured with the Lowry method; WISH cell/Viola crystallina method is surveyed external activity, calculates every milligram of proteic activity and promptly gets specific activity, and the specific activity of PEG-IFNs-A should be more than or equal to 7.0X10E7IU/mg albumen;
3) be that the plasma clearance long half time of mono-modified product of 12kDa PEG and IFN a-2b is more than 50% the plasma half-life of PEG-IFNs-A than PEG-Intron;
4) in immunogenicity determining, PEG-IFNs-A should be lower than 50% in the 12nd all IFN antibody positive rate.
Wherein: the rightter scope of n is 570～1000 in the above-mentioned Pegylation recombinant human interferon alpha 2 analogue protein molecular structural formula, and the n optimum value is 700 ± 100; R is 1～3, and the rightter scope of r is 2～3; T is 1～3; Above-mentioned IFNs is recombinant human interferon alpha 2 alfa-2b (IFN-α 2b), recombinant human interferon alpha 2 alfa-2a (IFN-α 2a) or recombinant human interferon alpha 1 b eta-1b (IFN-β 1b);
One type of Pegylation recombinant human interferon alpha 2 analogue albumen according to the invention possesses the homogeneity on molecular weight and the molecular structure simultaneously, is the terminal site-directed coupling product of strand, single macromole PEG and IFNs-A N-; Wherein more preferably coupled product is PEG-IFNsA-N30, PEG-IFNsA-N10, PEG-IFNsA-N20 and PEG-IFNsA-N40.
First-selected molecular weight 30kDa PEG-ALD of one type of Pegylation recombinant human interferon alpha 2 analogue albumen according to the invention and the terminal site-directed coupling product of IFNs-A N-; Be the PEG-IFNsA-N30 molecule, concrete preferred PEG-IFN α 2bA-N30 or PEG-IFN β 1b A-N30.
One type of Pegylation recombinant human interferon alpha 2 analogue albumen according to the invention obviously is superior to common IFNs-A on thermostability and resistance to enzymolysis property stability, on the cell in vitro activity more than or equal to 7.0X10E7IU/mg albumen; When using in vivo, compare with said PEG-Intron, have obviously prolong plasma half-life, activity in vivo obviously increases with long-acting preparation medicines such as immunogenicity reduction for kinetics and pharmacodynamic profile; Compare with said Pegasys, have obviously characteristics such as raising and the interior significantly increase that distributes of body of external activity.Based on this; One type of Pegylation recombinant human interferon alpha 2 analogue albumen according to the invention; Like PEG-IFNsA-N30 (the terminal site-directed coupling product of 30kDa PEG-ALD and IFNs-A albumen N-), be the prolonged action preparation that possesses long-acting, efficient and reduced immunogenicity characteristic simultaneously.
Pegylation recombinant human interferon alpha 2 analogue albumen according to the invention is treated the application in B-mode or hepatitis C, multiple sclerosis, rheumatism or rheumatoid arthritis, malignant tumour, hair white blood disease and the AIDS card Podbielniak tumor medicine in preparation.
Further provide; Be used to treat parenteral formulations B-mode or hepatitis C, multiple sclerosis, rheumatism or rheumatoid arthritis, malignant tumour, hair white blood disease and AIDS card Podbielniak knurl etc., wherein contain the Pegylation recombinant human interferon alpha 2 analogue albumen according to the invention and the pharmaceutically acceptable carrier of treating significant quantity; Further, said preparation contains PEG-IFN α 2bA-N30 according to the invention or the PEG-IFN β 1b A-N30 and the pharmaceutically acceptable carrier of treating significant quantity.
By the molecular parenteral prolonged action preparation of PEG-IFNs-A of treatment significant quantity, can realize 1-4 week injection once; Different according to applicating finger syndrome and therapeutic purpose, the dosage that PEG-IFNs-A uses in 0.1ug～300ug/kg body weight/1 day～4 weeks, the advantageous applications dosage range in 0.3ug-100ug/kg body weight/1 day～4 weeks.
Adopt the expression of procaryotic cell expression system (intestinal bacteria) to prepare IFNs and IFNs-A:
IFNs and IFNs-A gene clone with efficiently express
IFNs and IFNs-A gene clone
The invention discloses the method for the intestinal bacteria recombinant bacterial strain that makes up efficiently expressing recombinant human Interferon, rabbit and analogue (IFNs and IFNs-A) thereof, form by following step;
Synthesizing of recombinant human interferon alpha 2 (IFNs) gene with known IFNs (IFN-α 2b; IFN-α 2a; IFN-β 1b) the Argine Monohydrochloride sequence is the basis, and the IFNs base sequence is designed again, codon all is changed to the codon of intestinal bacteria hobby; Consider that simultaneously DNA secondary structure and GC content makes suitable modification, make whole gene not contain rare codon.With gene difference called after IFN-α 2b, IFN-α 2a, IFN-β 1b, and be structured on the pMD18-T carrier, further carrier is transformed in the Top10 bacterial strain and preserves.
The structure of IFNs recombinant expression is a template with synthetic IFNs gene, through design primer PCR amplification in vitro, is cloned on the colibacillus expression plasmid, obtains the IFNs recombinant expression.According to amended IFNs gene order, the design primer is:
IFN-α_F：5’-ATTAAT
CATATG?TGCGATCTGC?CGCAGACCCA-3
IFN-α_R：5’-ACTTGC
CTCGAGTCATTCTTTGCTACGCAG-3
IFN-β1b_F：5’-ATTAAT
CATATGTCTTACAACCTGCTGGGT-3
IFN-β1b_R：5’-CGC
CTCGAG?TTA?TTA?GTT?ACG?CAG?GTA?ACC?CGT-3’
With the synthetic gene is template, utilizes PCR (polymerase chain reaction) amplification in vitro IFNs gene.Gene is cut through Nde I and Xho I enzyme, is cloned into coli expression carrier, the exactness of checking plasmid, recombinant plasmid called after vector-IFNs.
The structure of IFNs-A recombinant expression is a template with synthetic IFNs gene; Through design primer PCR amplification in vitro; And on primer, add (GlyGlyGlyGlySer) n, product cloning to colibacillus expression plasmid, is obtained IFN α-2b recombinant expression.
IFN-α-A_F:5 '-ATTAAT
CATATG(GGTGGAGGCGGTTCA) n TGCGATCTGCCGCAGACCCA-3 (wherein n is 1～10, preferred 1～3)
IFN-α-A_R：5’-ACTTGC
CTCGAGTCATTCTTTGCTACGCAG-3
IFN-β 1b-A_F:5 '-TAAT
CATATG(GGTGGAGGCGGTTCA) n TCTTACAACCTGCTGGGT-3 (wherein n is 1～10, preferred 1～3)
IFN-β1b-A_R：5’-CGC?
CTCGAG?TTA?TTA?GTT?ACG?CAG?GTA?ACC?CGT-3’
With the synthetic gene is template, utilizes PCR amplification in vitro IFNs-A gene.Gene is cut through Nde I and Xho I enzyme, is cloned into coli expression carrier, and the exactness of checking plasmid, recombinant plasmid called after vector-IFNs-A.
Efficiently express IFNs and IFNs-A
Be transformed into e. coli bl21 (DE3) pLysS with extracting recombinant plasmid vector-IFNs or the DH5 α bacterial strain of vector-IFNs-A after activation.The screening efficient expression strain.
Recombinant expression vector-IFNs or vector-IFNs-A are transformed into e. coli bl21 (DE3) pLysS.Positive colony in the picking LB flat board after the screening; Under aseptic condition, use inoculation articulating 1～2 to encircle and be added with in the fermention medium of penbritin that final concentration is 50～150 mcg/ml in 5ml; Under 30～40 ℃ of conditions; 150～300 rev/mins of shaking table shaking culture 8～16 hours make seed liquor; Transfer in the 300ml triangular flask that the 50ml substratum is housed with 1%～5% inoculum size.37 ℃ of thermal agitations are cultivated 3h, make bacterium be in logarithmic growth mid-term, OD
600Value is about 0.8.From 50ml logarithmic phase bacterium liquid, get 1mL and do contrast, in all the other bacterium liquid, add 1mol/L IPTG solution, making its ultimate density is 1mmol/L.Bacterium liquid continues to cultivate 1～4h after adding IPTG, the centrifugal 10min of 12000rpm, get supernatant and deposition preserve respectively for use, last centrifugal results bacterium, the proteic expression of testing goal is chosen the highest bacterial strain of expression amount and is preserved.
In aforesaid method, escherichia coli vector preferably has the carrier of strong promoter, like one of T7 or T5, and preferably has Nde I, and the colibacillus expression plasmid of Nco I restriction enzyme site is like one of pET22b, pET15b, pET28a; One of the above-mentioned preferred Top10 of host bacterium that is used for plasmid amplification, DH5 α, JM109; One of the above-mentioned preferred BL21 of host bacterium that is used to express, BL21 (DE3) pLysS.
The preparation of IFNs or IFNs-A
Application of I FNs or the strain of IFNs-A colibacillus engineering obtain highly purified IFNs albumen through technologies (seeing embodiment 1 for details) such as high density fermentation cultivation, fermentation aftertreatment, the washing of albumen inclusion body, renaturation and separation and purification.
From liquid nitrogen container, take out IFNs or IFNs-A bacterial classification, 37 ℃ of water-bath speed are melted; In Strict aseptic operation down, IFNs or IFNs-A colibacillus engineering are inoculated in LB/Amp, put 37 ℃ of incubators and cultivated 12-15 hour, choose single bacterium colony, transfer in containing the LB/Amp culture tube, 37 ℃ of 250rpm shaking culture 12-14 hour; Get 10ml fermented liquid switching shake-flask culture (37 ℃, the 250rpm vibration) 8-15 hour in the 1000mlLB/Amp fermented liquid, when bacterial concentration reaches OD
6001.5～2.5 o'clock; Be inoculated in 15 liters of fermentor tanks and cultivate, establishing fermentation parameter is 37 ℃ of temperature, pH7.0, dissolved oxygen 35% and mixing speed and dissolved oxygen interlock.Work as OD
600Value reaches at 20 o'clock, is the mid-term of engineering bacteria logarithmic growth, adds IPTG and induces, and continues to stop after 2 hours fermentation.Cooling and fermentation liquid makes it be cooled to 4-8 ℃.Adopt freezing low speed centrifuge centrifugal (4000rpm, 30 minutes) to collect bacterium.
In the engineering bacteria bacterial sediment of collecting, add the broken damping fluid (50mM Tris, 1mM EDTA, pH7.5 damping fluid) of engineering bacteria.Broken thalline under high pressure homogenizer 10000psi pressure.Liquid obtains inclusion body (IB) bullion through high speed freezing centrifuge 11000rpm rotating speed centrifugal (4 ℃, 20 minutes) behind the broken bacterium.Use inclusion body washings (50mM Tris, 1mM EDTA, pH7.5; 2.5% Triton * 100; 0.15M NaCl) repetitive scrubbing is three times, is about to inclusion body washings and inclusion body through high-speed liquid stirrer thorough mixing, collects inclusion body in 50-55 minute at 4 ℃ of 10000g high speed centrifugations again;, accomplish inclusion body washing and purifying.The inclusion body of purifying is dissolved in (50mM Tris, 1mM EDTA, 8M Guanidinium hydrochloride in the solubilization of inclusion bodies sex change liquid in the ratio of 10 gram weight in wet bases than 500 milliliters; 1mM dimercapto tetrahydroxybutane), put 4 ℃, 12 hours; Obtain solubilization of inclusion bodies sex change liquid, again through 4 ℃ of 10000g high speed centrifugations 40-45 minute (Sorvall RC5B, 11; 000rpm), collect supernatant.
By 1: 100 volume ratio solubilization of inclusion bodies sex change liquid is slowly added 20mM sodium-acetate buffer (pH 6.0); 150mM sodium-chlor; 3M urea, 0.005% gathers in sharp alcohol ester 80 (polysorbate 80) the renaturation solution in mountain, and 4 ℃ left standstill 24 hours; Behind 0.45 μ m aperture membrane filtration, carry out protein and purify.
IFNs or IFNs-A protein renaturation liquid are through ion exchange chromatography, and level pad is 20mM sodium-acetate buffer (pH4.5), 150mM sodium-chlor, 0.005% polysorbate80; Went up appearance in 60ml/ minute, with 0～1M sodium-chlor gradient elution, separating resulting is as shown in Figure 3, collects A280 albumen elution peak liquid.With above-mentioned level pad balanced gel chromatography column (chromatography column BPG100; Chromatography media Superdex 75 prep grade); Press appearance on 30ml/ minute the flow velocity, go up appearance and continue 5 minutes (the about 150ml of applied sample amount) at every turn, last kind after 120 minutes; Collect the A280 albumen absorption peak that occurs, be IFNs or IFNs-A stoste.
With PEG-propionic aldehyde and the terminal specificity site-directed coupling method of IFNs-A albumen N-is preference, describes PEGization IFNs-A albumen and preparation technology thereof
Select MacroCap SP or Sepharose FF cation exchange medium; Load the chromatography chromatographic column; The post bed height reaches the upper limit of recommending height; And be connected to AKTA Explorer 100 LC systems (AmershamBioscience, Sweden) on, use 20mM sodium-acetate buffer pH 3.5～6.0 (buffer A) and the abundant balance chromatographic column of 0.005% polysorbate80 be equivalent to 5 times of column volumes; Use fresh feed pump,, IFNs-A stoste added in the chromatographic column with the flow velocity of 10ml/min, applied sample amount be medium survey maximum carrying capacity 10%～70%, more fitting scope is 30-50%; Again under the flow velocity of 15ml/min,, remove not material with chromatographic media generation electrostatic adhesion with the buffer A flushing pillar of about 5 times of column volumes; With the flow velocity of 0.5～3ml/min, add PEG propionic aldehyde (PEG-propionaldehyde) and the 2.2mM～22mM NaBH of 0.22mM～2.2mM 30kDa
3CN solution 250ml～1500ml went up appearance about 240 minutes continuously; The pH of reaction system is 3.5～6.0, more fits to be pH 5.0～6.0; Again with the flow velocity of 15ml/min,, remove not material with chromatographic media generation electrostatic adhesion with the buffer A flushing pillar of about 5 times of column volumes; Use buffer B (being buffer A+1mol sodium-chlor) subsequently,, continue about 150 minutes, can record ion exchange chromatography spectrogram as shown in Figure 1 with type of elution 0～8%, 8%～18% and 18%～100% wash-out.Fig. 1 shows, can obtain-2 two elution peaks in peak-1 and peak altogether, i.e. P1 and P2, and wherein the peak area of P1 accounts for 72% of total peak area; P1 is mono-modified PEG-IFN α 2b-A through reduced form SDS-PAGE, efficient gel chromatogram (SEC) and performance liquid chromatography (HPLC) purity check; Purity greater than 95% (like Fig. 2; Shown in 3,4), this product is through LC-MS trypsin digestion peptide figure analysis; Confirm it is the PEG-ALD and the terminal site-directed coupling of IFNs-A N-of 30kDa molecular weight, coupled product is PEG-IFN α 2bA-N30.
Can get PEG-IFNsA-N10, PEG-IFNsA-N20 and PEG-IFNsA-N40 etc. with method.
Similarly, be example with the terminal specificity site-directed coupling method of PEG-NHS (polyoxyethylene glycol N-hydroxy-succinamide) and IFNs-A albumen N-, PEGization IFNs-A albumen and preparation technology thereof are described:
Select MacroCap SP or Sepharose FF cation exchange medium; Load the chromatography chromatographic column; The post bed height reaches the upper limit of recommending height; And be connected to AKTA Explorer 100 LC systems (AmershamBioscience, Sweden) on, use 20mM sodium-acetate buffer pH 3.5～6.0 (buffer A) and the abundant balance chromatographic column of 0.005% polysorbate80 be equivalent to 5 times of column volumes; Use fresh feed pump,, IFNs-A albumen stoste added in the chromatographic column with the flow velocity of 10ml/min, applied sample amount be medium survey maximum carrying capacity 10%～70%, more fitting scope is 30-50%; Again under the flow velocity of 15ml/min,, remove not material with chromatographic media generation electrostatic adhesion with the buffer A flushing pillar of about 5 times of column volumes; With the flow velocity of 0.5～3ml/min, add PEG-NHS (polyoxyethylene glycol N-hydroxy-succinamide) solution 250ml～1500ml of 0.22mM～2.2mM 30kDa, went up appearance about 240 minutes continuously; The pH of reaction system is 4.5～7.0, more fits to be pH 6.0～7.0; Again with the flow velocity of 15ml/min,, remove not material with chromatographic media generation electrostatic adhesion with the buffer A flushing pillar of about 5 times of column volumes; Use buffer B (being buffer A+1mol sodium-chlor) subsequently,, continue about 150 minutes, can record ion exchange chromatography spectrogram as shown in Figure 5 with type of elution 0～8%, 8%～18% and 18%～100% wash-out.Fig. 5 shows that the peak area of mono-modified PEG-IFNs-A accounts for 79% total peak area, confirms as 30kDa molecular weight PEG-NHS and single, the site-directed coupling product of the proteic N-end of IFNs-A through LC-MS trypsin digestion peptide figure analysis.
Can get PEG-IFNsA-N10, PEG-IFNsA-N20 and PEG-IFNsA-N40 etc. with method.
Similarly, be example with the terminal specificity site-directed coupling method of PEG-NHS on gel chromatography column (polyoxyethylene glycol N-hydroxy-succinamide) and IFNs-A albumen N-, PEGization IFNs-A albumen and preparation technology thereof are described:
Select Superdex 200 gel chromatography media; Load chromatography chromatographic column (2.6/60cm); And be connected to AKTAExplorer 100 (the Amersham Bioscience of LC system; Sweden) on, use 20mM sodium-acetate buffer pH3.5～6.0 (buffer A) and abundant balance chromatographic column of 0.005% polysorbate80; Use fresh feed pump, with the IFNs-A albumen stoste of 2～5mg/ml concentration, press appearance on 30ml/ minute the flow velocity, each applied sample amount is 1/10～20 column volume; With the flow velocity of 10～30ml/min, add PEG-NHS (polyoxyethylene glycol N-hydroxy-succinamide) solution 250ml～1500ml of 0.22mM～2.2mM 30kDa, went up appearance about 120 minutes continuously; The pH of reaction system is 3.5～6.0, more fits to be pH 5.0～6.0; Collect the A280 albumen absorption peak that occurs, can record gel displacement chromatography spectrogram as shown in Figure 6.Can be seen by Fig. 6: main peak is the coupled product PEG-IFNs-A (being called for short PEG-IFNsA-N30) of albumen N-end, the single modification of 30kDaPEG.The peak area of mono-modified PEG-IFNs-A accounts for 76% total peak area, confirms as 30kDa molecular weight PEG-NHS and single, the site-directed coupling product of the proteic N-end of IFNs-A through LC-MS trypsin digestion peptide figure analysis.
Can get PEG-IFNsA-N10, PEG-IFNsA-N20 and PEG-IFNsA-N40 etc. with method.
In liquid-phase reaction system, be example with PEG-propionic aldehyde and the terminal specificity site-directed coupling method of IFNs-A albumen N-, the amino specificity coupling of PEG-ALD and IFNs-A albumen is described:
Get above-mentioned IFNs-A albumen stoste and make protein concentration reach 2mg/ml through ultrafiltration and concentration, in 8～40mol: the 1mol ratio is the IFNs-A albumen stoste that the PEG propionic aldehyde (PEG-propionaldehyde) of 30kDa is dissolved in 2mg/ml with molecular weight; The ratio of fitting is that PEG-ALD and the IFNs-A stoste molar ratio of 30kDa is 8～20mol: 1mol; Reaction solution is 20mM～50mM sodium-acetate buffer, and pH 5.0～6.0; Adding concentration in 1: 1～5 (w/w) ratio again is the sodium cyanoborohydride (NaBH of 7.5mg/ml
3CN), reaction solution left standstill 4 hours under 4 ℃～25 ℃, added 1mM dilute hydrochloric acid solution (pH 3.5) termination reaction in 1: 100 (v/v) ratio, got PEG-IFNs-A.
With damping fluid 20mM～50mM sodium-acetate buffer; PH 3.5～9.0; 150mM～500mM sodium-chlor and 0.005% polysorbate80 solution equilibria Sepharose Fast Flow ion exchange column; Press 50ml/ minute flow velocity and go up appearance PEG-IFNs-A reaction solution continuously,, collect A280 albumen elution peak through 0～1M sodium chloride solution gradient elution; Obtain three elution peaks, be respectively the PEG-IFNs-A of modification, mono-modified PEG-IFNs-A and the elution peak of IFNs-A more.Through calculating, 48% the PEG-IFNs-A of only having an appointment is mono-modified PEG-IFNs-A, and 37% be many modification PEG-IFNs-A compounds in addition; Surplus 14% for unreacted IFNs-A, as shown in Figure 7.
In liquid-phase reaction system, describe with mPEG-SPA and the terminal specificity site-directed coupling of IFNs-A albumen N-:
Getting above-mentioned IFNs-A albumen stoste makes protein concentration reach 1～2mg/ml through ultrafiltration and concentration.
In 5～50mol: the 1mol ratio is the IFNs-A stoste that the mPEG-SPA (mPEG-succinimide propionic ester) of 30kDa is dissolved in 2mg/ml with molecular weight; Reaction solution is 20mM～50mM PBS damping fluid, and pH 6.0～9.0; Reaction solution left standstill 15～180 minutes under 4 ℃～25 ℃, got PEG-IFNs-A.
With damping fluid 20mM～50mM PBS damping fluid; PH 4.5～7.0 and 0.005% polysorbate80 solution; Balance Sepharose Fast Flow ion exchange column; Press 50ml/ minute flow velocity and go up appearance PEG-IFNs-A reaction solution continuously,, collect A280 albumen elution peak through 0～1M sodium chloride solution gradient elution; Obtain three elution peaks, be respectively the PEG-IFNs-A of modification, mono-modified PEG-IFNs-A and the elution peak of IFNs-A more.Through calculating, 23% the PEG-IFNs-A of only having an appointment is mono-modified PEG-IFNs-A, and 64% be many modification PEG-IFNs-A compounds in addition; Surplus 13% for unreacted IFNs-A, as shown in Figure 8.
The present invention discloses first in chromatography column and traditional liquid-phase reaction system, and the comparative data of the pegylation reaction product yield of interferon fusion protein and common interferon protein is as shown in table 1, and under identical reaction conditions, its reaction product is obviously different; These results show; In chromatography (comprising IX and gel chromatography) or traditional liquid-phase reaction system; The linked reaction of interferon fusion protein IFNs-A and PEG-ALD or PEG-NHS obtains the N-end yield of PEG-IFNs-A single, fixed point and all is significantly higher than common interferon protein IFNs; The reaction tendency basically identical of IFN α and IFN β and PEG, just the IFN α relative yield amplification that to obtain mono-modified N-end PEGization product be PEG-IFN α-A is bigger.
The mono-modified product comparison of table 1 chromatography and liquid phase, interferon fusion protein and interferon protein PEGization (X ± SD, n=8)
* more remarkable with the mono-modified product yield of the PEGization of common Interferon, rabbit than difference, P＜0.01 or P＜0.05
The physico-chemical property of PEG-IFNs and PEG-IFNs-A and biological activity
The invention discloses external physico-chemical property and the biologically active data of PEG-IFNsA-N30, PEG-IFNsA-N40, PEG-IFNsA-N20 and PEG-IFNsA-N10 etc., see embodiment 3 for details.
The present invention finds, molecular weight is thermostability and the resistance to enzymolysis stability etc. that significantly improved PEG-IFNs-A after macromole PEG and the coupling of IFNs-A N-end of 30kDa, and this is that this quasi-molecule has one of bioactive basis, inside and outside (as shown in table 2).
Table 2 PEG-IFN α 2b-A-N30 stoste physico-chemical property
The present invention is important and be surprised to find that (seeing for details shown in the table 4); The PEGization interferon fusion protein is that the external biological activity of PEG-IFNs-A is significantly higher than the PEG-IFNs with molecular weight; Explain that terminal flexible polypeptied chain (GlyGlyGlyGlySer) t that merges of IFNs N-can make the avtive spot of the coupling site of PEG away from IFNs, thereby obviously reduced the PEG molecule the active influence of IFNs; In addition, the external activity of PEG-IFN α 2b-A-N10 is significantly higher than IFN α-2b-A and IFN α-2b, and this result is unexpected; Because; It has been generally acknowledged that the macromolecular coupling of PEG meeting significantly reduces the activity of IFNs-A or IFNs, obviously, this discovery has potential and important clinical application value; The progressively echelon reduction of PEG-IFNs-N10～40 that the terminal coupling PEG of IFNs or IFNs-A protein-n propionic aldehyde is obtained or its external activity of PEG-IFNs-A-N10～40 along with the increase of coupling PEG molecular weight; But at PEG-IFNs-N40 or PEG-IFNs-A-N40 place; The reduction of external activity is particularly remarkable, significantly active " flex point " occur; In addition, the diPEG-IFNs-N40 of two 20kDa PEG of coupling or its external activity of diPEG-IFNs-A-N40 significantly are lower than PEG-IFNs-N40 or PEG-IFNs-A-N40 with equimolecular quantity.This points out simultaneously, double-stranded PEG (is duplex structure like 40kDa PEG-ALD) or the coupling of dibit point to the influence of external activity greater than singly connecting PEG (is single-stranded structure like 30kDa PEG-ALD) or single site.Therefore, the present invention confirms, the N-terminal amino group of first-selected strand PEG-ALD coupling IFNs-A.
The pharmacokinetics test of PEG-IFNs-A
The plasma clearance transformation period (t1/2) (seeing for details shown in the table 5) of healthier rat tail vein injection PEG-IFN α 2bA-N10, PEG-IFN α 2bA-N20, PEG-IFN α 2bA-N30 and PEG-IFN α 2bA-N40; Discovery is along with the increase of PEG molecular weight; The plasma clearance transformation period significant prolongation of PEG-IFN α 2bA-N10～40; But occur once more in the plasma half-life of PEG-IFN α 2bA-N40 " flex point "; Promptly the increasing degree in the plasma half-life that the increase of this PEG molecular weight brings obviously reduces, and increases to 20kDa with from 20kDa from 10kDa like the molecular weight of PEG to increase to 30kDa, and all increased about about 50% the plasma half-life of PEG-IFN α 2bA-N20 or PEG-IFN α 2bA-N30; And the molecular weight of PEG increases to 40kDa from 30kDa, and but only increased about 10% the plasma half-life of PEG-IFN α 2bA-N40; Compare with PEG-IFN α 2bA-N40; Though the plasma half-life of PEG-IFN α 2bA-N30 and the difference of maximum plasma concentration are not remarkable; But nearly four times of its volume of distribution apparently higher than PEG-IFN α 2bA-N40; This possibly obviously reduce relevantly with the subcutaneous absorption of PEG-IFN α 2bA-N40, also has potential clinical therapeutics meaning simultaneously.
The further drug metabolism unexpected discovery of test (shown in figure 18); Rat tail vein injection super large dosage PEG or PEG-IFNs-A; Adopt the ELISA method to measure the PEG content in the urine; Injection PEG20 and PEG-IFN α 2bA-N20 group can measure a considerable amount of PEG to be got rid of through urine, and injection PEG30, PEG40, PEG-IFN α 2bA-N30 and PEG-IFN α 2bA-N40 group can obviously reduce by detected PEG content.Such result has exceeded people's understanding and experimental result in the past; Because; It has been generally acknowledged that: GCW molecular filtration aperture is 60-70kDa or 3-5nm, and the PEG molecule has the hydrodynamic force volume much bigger than sphaeroprotein (hydrodynamic volume), and the molecular scale of the PEG of 20kDa molecular weight (molecular size) can reach 7nm; Much larger than aforementioned GCW molecular filtration aperture; Inject PEG20 in theory and PEG-IFN α 2bA-N20 should not remove from kidney, but adopt the content of PEG in the responsive anti-PEG antibody ELISA method monitoring urine, the PEG that but can detect a great deal of gets rid of from urine; This possibly be simultaneously the explanation that the above-mentioned plasma clearance transformation period " flex point " phenomenon occurs.Obviously, this discovery especially has meaning and value for the exploitation prolonged action preparation.
The immunogenicity test of PEG-IFNs-A
The invention also discloses the immunogenic testing data of above-mentioned PEG-IFNs and IFNs; On normal rat; Adopt the administering mode in 1 time/week, 12 weeks of continuous application, the result shows that the immunogenicity of PEG-IFN α 2bA-N20, PEG-IFN α 2bA-N30 and PEG-IFN α 2bA-N40 all is lower than IFN-α 2b-A; And the immunogenicity of PEG-IFN α 2bA-N30 and PEG-IFN α 2bA-N40 all is lower than PEG-IFN α 2bA-N20, and is as shown in table 6.
With above-mentioned same method PEG-IFN β 1bA-N10, PEG-IFN β 1bA-N20, PEG-IFN β 1bA-N30 and PEG-IFN β 1b A-N40 are made an experiment, the result shows that each item index of PEG-IFN β 1bA-N30 is more excellent.
Comprehensive various physico-chemical properties, biological activity, medicine generation and immunogenicity testing data and the result who is obtained; The present invention confirms first and confirms: as long-acting, efficient and reduced immunogenicity preparation; PEG-IFN α 2bA-N30 or PEG-IFN β 1bA-N30 have more advantage than PEG-IFN α 2bA-N10, PEG-IFN α 2bA-N20, PEG-IFN α 2bA-N40, PEG-IFN β 1bA-N10, PEG-IFN β 1bA-N20 or PEG-IFN β 1b A-N40, should be most preferably.Therefore, the first-selected 30kDa PEG-ALD of Pegylation recombinant human interferon alpha 2 analogue albumen of the present invention and IFNs-A N-end is single, the polyethyleneglycol derivative of site-directed coupling, i.e. PEG-IFNsA-N30.
IFNs among the present invention and IFNs-A also can have multiple source, except that the above-mentioned coli expression system of enumerating especially, still comprise synthetic and various other prokaryotic cell prokaryocytes, eukaryotic cell, insect, plant or animal expression system; IFNs and IFNs-A can be the albumen that whole aminoacid sequences is formed simultaneously; Also can be IFNs and the IFNs-A varient of forming through the IFNs and the IFNs-A sequence of sudden change; Or change the IFNs analogue that partial amino-acid series is formed, and be M1C for halfcystine (Cys) at the molecule N-terminal sudden change methionine(Met) (Met) of said IFNs-A; But it is characterized in that: external activity IFNs albumen former with it quite or be not less than that it is active 70%, and reduced immunogenicity or non-immunogenicity; Above-mentioned various IFNs albumen is all within scope involved in the present invention.
PEG-IFNs-A molecule involved in the present invention is the effective constituent of clinical medicinal prepns, also possibly comprise in the said preparation: thinner (diluents), stablizer (stabilizers), sanitas (preservatives), solvating agent (solubilizers), emulsifying agent (emulsifiers), adjuvant (adjuvants) and carrier (carriers) etc.For example: damping fluids such as 1) diluent: Tris-HCL, phosphoric acid, acetic acid; 2) pH value and ionic strength; 3) stain remover and solvating agent: polysorbate20, polysorbate80; 4) filling agent (bulking substances): lactose, N.F,USP MANNITOL.See Remington ' s Pharmaceutical Science for details, 18
ThEd (1990, Mack PublishingCo., Easton, Pa.18042) Pages 1435:1712.The effective dose of effective constituent is meant the efficacious therapy or the preventive dose of factors such as considering body weight, age.Its effective dosage ranges of PEG-IFNs-A involved in the present invention in 0.1ug-300ug/kg body weight/1 day～4 weeks, better suited effective dosage ranges in 0.3ug-100ug/kg body weight/1 day～4 weeks.
The clinical medicinal prepns of PEG-IFNs-A involved in the present invention is long-acting, efficient, reduced immunogenicity preparation, can reach the long-acting effect of every 1-4 week injection one pin usually; The application of the clinical medicinal prepns of PEG-IFNs-A designs also relevant with indication of using and patient's practical situation at interval.The clinical medicinal prepns of PEG-IFNs-A can be subcutaneous, muscle, vein and intraperitoneal injection; Also can be through suction, hypogloeeis or transdermal administration.
The present invention can be applicable to treat various serious virus infectiones and malignant tumour, and common indication comprises: treat B-mode or hepatitis C, multiple sclerosis, rheumatism or rheumatoid arthritis, malignant tumour (Fei Hejiejinshi disease, melanoma, mammary cancer etc.), hair white blood disease (Hairy Cell Leukemia) and AIDS card Podbielniak knurl etc.
Data provided by the present invention and method are equally applicable to other cytokines and antibody passage Fab '; ScFv and Fc Pegylation and exploitation long-acting, efficient, the reduced immunogenicity preparation are like recombinant methionyl human G-CSF, recombinant human erythropoietin, recombinant human tumor necrosis factor's receptor protein or the like.
Description of drawings
The terminal specificity site-directed coupling of Fig. 1 PEG-ALD and IFNs-A N-ion exchange chromatography spectrogram
Wherein: P1 is mono-modified PEG-IFN α 2b-A, and P2 is unreacted IFN α 2b-A.
Fig. 2 SDS-PAGE PG-N-30 molecular-weight determination (silver dyes)
The molecular weight that shows PEG-IFN α 2bA-N30 is 66.2kDa, is mono-modified PEG-IFN α 2bA-N30.
Fig. 3 efficient gel chromatogram (SEC) is measured PEG-IFNsA-N30 purity
Show the main peak area greater than 95%, be mono-modified PEG-IFN α 2bA-N30 through calibrating, the small peak before it is the PEG-IFN of modification α 2bA-N30 more.
Fig. 4 performance liquid chromatography (RT-HPLC) is measured PEG-IFNsA-N30 purity
Show the main peak area greater than more than 98%, be mono-modified PEG-IFN α 2bA-N30 through calibrating.
The terminal specificity site-directed coupling of Fig. 5 PEG-NHS and IFNs-A N-ion exchange chromatography spectrogram
Wherein: P1 be mono-modified PEG-IFNs-A account for total peak area 79%, P2 be unreacted IFNs-A account for total peak area 28%.
The terminal specificity site-directed coupling of Fig. 6 PEG-NHS and IFNs-A N-gel chromatography spectrogram
Wherein: peak 1 accounts for 10% of total peak area for the PEG-IFNs-A that modify more,, peak 2 accounts for 76% of total peak area for mono-modified PEG-IFNs-A, and peak 3 accounts for 14% of total peak area for unreacted IFNs-A.
Fig. 7 efficient gel stratographic analysis liquid phase P EG-ALD and IFNs-A linked reaction product
Wherein: peak-1 is the PEG-IFNs-A of modification more, accounts for 33.90% of total peak area, and peak-2 is two modification PEG-IFNs-A; Account for 3.14% of total peak area, peak-3 is mono-modified PEG-IFNs-A, accounts for 48.47% of total peak area; Peak-4 is unreacted IFNs-A, accounts for 14.49% of total peak area.
Fig. 8 efficient gel stratographic analysis liquid phase mPEG-SPA and IFNs-A linked reaction product
Wherein: peak-1 is the PEG-IFNs-A of modification more, accounts for 63.81% of total peak area, and peak-2 is mono-modified PEG-IFNs-A, accounts for 22.50% of total peak area, and peak-3 is unreacted IFNs-A, accounts for 13.69% of total peak area.
Fig. 9 IFNs and IFNs-A prokaryotic expression system plasmid construction figure.
The expression amount analysis of Figure 10 SDS-PAGE rhG-CSF.
Figure 11 reduced form SDS-PAGE measures the IFNs-A molecular weight
Wherein: IFN α 2a-A, the molecular weight of IFN α 2b-A and IFN β 1b-A is all at 18kDa.
Figure 12 performance liquid chromatography (HPLC) method is measured IFN α 2b-A stoste purity.
Figure 13 efficient gel chromatography (SEC) is measured the purity of IFN α 2b-A stoste.
The MALDI-TOF-MS molecular-weight determination result of Figure 14-1 PEG-ALD (30kDa).
Figure 14-2 embodiment 2 peaks 1 MALDI-TOF-MS molecular-weight determination result.
Figure 15-1 PEG-IFN α 2bA-N30 (1mg/mL/ props up) is after leaving standstill 1 month under 37 ℃ of conditions, through the efficient gel detected result
Wherein: peak 1 accounts for 0.5% for PEG-IFN α 2bA-N30 polymer, and peak 2 is a PEG-IFN α 2bA-N30 dimer 1.1%, and peak 3 accounts for 98.4% for mono-modified PEG-IFN α 2bA-N30.
Figure 15-2 IFN-α-2b-A (1mg/mL/ props up) is after leaving standstill for 1 week under 37 ℃ of conditions, through the efficient gel detected result
Wherein: peak 1 accounts for 38.4% for IFN-α-2b-A, and peak 2 accounts for 61.6% for IFN-α-2b-A aggregate.
The external enzymolysis response diagram of Figure 16 IFN-α 2b-A and PEG-IFN α 2bA-N30.
Figure 17 PEG-IFN α 2bA-N10～40 compound molecular weights and relation of plasma half-life.
Figure 18 ELISA method animals urine PEG assay result
PEG-IFNsA-N20 is PEG-IFN α 2bA-N20 among the figure.
* with PEG30, PEG40, PEG-IFN α 2bA-N30 compares significant difference (P＜0.05 or P＜0.01) with each class value of PEG-IFN α 2bA-N40.
Embodiment
Below the enforcement example help further to set forth the present invention, but that content involved in the present invention and scope are not limited only to these examples is said.
1-1: express IFNs and IFNs-A
Make up prokaryotic expression system pET22b-IFNs and pET22b-IFNs-A and efficiently express IFNs and IFNs-A; Bacterial strain uses therefor is bacillus coli DH 5 alpha (E.coli DH5 α LacZ Δ M15 hsdR recA), e. coli bl21 (DE3) (E.coliBL21 (DE3)) and e. coli bl21 (DE3)/pLysS (E.coli BL21 (DE3)/pLys); Used plasmid is pET22b; Above bacterial strain and plasmid are all available from Novagen company.Used molecular cloning comprises with enzyme and reagent: restriction enzyme Nde I, Xho I; Pfu archaeal dna polymerase (available from the rich inferior biological reagent in Shanghai company), Agarose Gel DNAPurification Kit; The T4 ligase enzyme, DNA Fragment Purification Kit (available from OMEGA Ltd.), nucleic acid molecular weight standard 1Kb Marker (available from BioLab) and molecular weight of albumen standard (14.4-97.OkDa) (available from the biochemical institute in Shanghai).
Synthetic human interferon (IFNs) gene
IFNs is recombinant human interferon alpha 2 alfa and beta; Preferred recombinant human interferon alpha 2 alfa-2b (IFN-α 2b), recombinant human interferon alpha 2 alfa-2a (IFN-α 2a) and recombinant human interferon alpha 1 b eta-1b (IFN-β 1b), its aminoacid sequence should meet the aminoacid sequence formula shown in SEQ IDNO.1 and the SEQ ID NO.2.Base sequence designs again, and their codon all is changed to the codon of intestinal bacteria hobby, considers that simultaneously DNA secondary structure and GC content makes suitable modification, makes whole gene not contain rare codon.(wherein the 67-69 position is Interferon alfa-2b for AGA for password replacement presequence such as SEQ ID NO.3; If the 67-69 position is Interferon alfa-2a for aag) and SEQ ID NO.4 shown in; Replacement back synthetic encoding sequence such as SEQ ID NO.5 and SEQ ID NO.6 (wherein the 67-69 position is Interferonalfa-2b for AGC, if the 67-69 position is Interferon alfa-2a for aag).Product is totally 165 amino acid, and proteic primary structure does not have other variations.Being recombinant expressed product can Duo a Met than natural product in that N-is terminal.Synthetic is gene constructed on the pMD18-T carrier, and carrier is transformed in the Top10 bacterial strain and preserves.
The clone of IFNs gene
According to amended IFNs gene order, the design primer:
IFN-α_F：5’-ATTAAT
CATATG?TGCGATCTGC?CGCAGACCCA-3
IFN-α_R：5’-ACTTGC
CTCGAGTCATTCTTTGCTACGCAG-3
IFN-β1b_F：5’-ATTAAT
CATATGTCTTACAACCTGCTGGGT-3
IFN-β1b_R：5’-CGC?
CTCGAG?TTA?TTA?GTT?ACG?CAG?GTA?ACC?CGT-3’
With pMD18-T/IFNs is template, utilizes PCR (polymerase chain reaction) amplification in vitro IFNs sequence.
The PCR reaction system consists of: (primer concentration is 20 μ mol/L) 10 * damping fluid, 5 μ l, 25mmol/L MgCl
24 μ l, 10mmol/L, four kinds of dNTP mixed solution l μ l; Each 1 μ l of upstream and downstream primer; TaqDNA polysaccharase 0.5 μ l; Template DNA 1 μ l adds water and mends to 50 μ l.If PCR reaction conditions: 97 ℃ of preparatory sex change 10 minutes, 94 ℃ of sex change 60 seconds, 56 ℃ of annealing 60 seconds, 72 ℃ were extended 60 seconds, and 30 circulations were extended 4 ℃ of preservations 10 minutes for back 72 ℃.The segmental size of electrophoresis detection.Agarose concentration is 0.8%.
The clone of IFNs-A gene
According to amended IFNs gene order, the design primer, and on primer, add (GlyGlyGlyGlySer) n:
IFN-α-A_F:5 '-ATTAAT
CATATG(GGTGGAGGCGGTTCA) n TGCGATCTGCCGCAGACCCA-3 (wherein n is 1～5, preferred 1～3)
IFN-α-A_R：5’-ACTTGC
CTCGAGTCATTCTTTGCTACGCAG-3
IFN-β 1b-A_F:5 '-TAAT
CATATG(GGTGGAGGCGGTTCA) n TCTTACAACCTGCTGGGT-3 (wherein n is 1～5, preferred 1～3)
IFN-β1b-A_R：5’-CGC?
CTCGAG?TTA?TTA?GTT?ACG?CAG?GTA?ACC?CGT-3’
With pMD18-T/IFNs is template, utilizes PCR (polymerase chain reaction) amplification in vitro IFNs sequence.The PCR reaction system is the same.
PCR is obtained segment for structure IFNs and IFNs-A recombinant expression and the pET22b plasmid utilizes Nde I, XhoI endonuclease to carry out double digestion digestion.Utilize the PCR product to reclaim test kit (available from OMEGA company) then and reclaim the fragment that purifying enzyme is cut.Utilize T4 ligase enzyme (buying the company in MBI) to connect, the reaction compositional system is:
IFN α-2b gene fragment 6 μ l, pET22b segment 2 μ l connect liquid damping fluid (10X) 1 μ l, T4 ligase enzyme 1 μ l, ligation is carried out under 22 ℃, and the reaction times is 4 hours, and reaction system is 10 μ l.Environmental induction type expression plasmid pET22b-IFNs and pET22b-IFNs-A obtain recombinating after connecting.
The checking amplification of IFNs and IFNs-A recombinant expression
To contain recombinant plasmid pET22b-IFNs or pET22b-IFNs-A and transform the entering bacillus coli DH 5 alpha.Single bacterium colony picking is changed in the LB liquid nutrient medium of the penbritin that contains 100 mcg/ml, 37 ℃, 225 rev/mins of following overnight cultures.Utilize plasmid kit to extract recombinant plasmid pET22b-IFNs and pET22b-IFNs-A, the size of checking plasmid.And with NdeI, the analysis of Xho I endonuclease double digestion, screening contains recombinant plasmid transformed of IFNs or IFNs-A gene fragment.The recombinant plasmid collection of illustrative plates is seen Fig. 9.
IFNs and IFNs-A gene are at expression in escherichia coli
The recombinant plasmid pET22b-IFNs or the pET22b-IFNs-A that will pass through checking are transformed into e. coli bl21 (DE3) pLysS.Positive colony in the picking LB flat board after the screening; Under aseptic condition, use inoculation articulating 1～2 to encircle and be added with in the fermention medium of penbritin that final concentration is 50～150 mcg/ml in 5ml; Under 30～40 ℃ of conditions; 150～300 rev/mins of shaking table shaking culture 8～16 hours make seed liquor; Transfer in the 300ml triangular flask that the 50ml substratum is housed with 1%～5% inoculum size.37 ℃ of thermal agitations are cultivated 3h, make bacterium be in logarithmic growth mid-term, OD
600Value is about 0.8.From 50ml logarithmic phase bacterium liquid, get 1mL and do contrast, in all the other bacterium liquid, add 1mol/L IPTG solution, making its ultimate density is 1mmol/L.Bacterium liquid continues to cultivate 1～4h after adding IPTG, the centrifugal 10min of 12000rpm, get supernatant and deposition preserve respectively for use, last centrifugal results bacterium, the proteic expression of testing goal.
Target protein detects
Get 1.5mL inductive culture, centrifugal 30 seconds of 12000rpm abandons supernatant; Deposition is washed once with PBS, adds 50 μ L sterilization distilled water and 50 μ L, 2 * SDS sample loading buffer, and resuspended deposition was boiled 5-10 minute, the centrifugal 10min of 10000rpm, and it is subsequent use to get supernatant; Carry out 8V/cm voltage stabilizing electrophoresis at concentrated glue; After the bromjophenol blue indicator gets into separation gel, change 15V/cm voltage stabilizing electrophoresis to bromjophenol blue band into and migrate to 1cm from the gel bottom; Take out gel and, change over to subsequently in the destainer more than 4 hours with the dyeing of Coomassie brilliant blue staining fluid, it is clear to background to decolour.SDS-PAGE result sees Figure 10, and the product expression amount accounts for about 25% of bacterial protein.
1-2: preparation IFNs and IFNs-A
From liquid nitrogen container, take out IFNs or IFNs-A bacterial classification, 37 ℃ of water-bath speed are melted; Under the Strict aseptic operation; IFNs or IFNs-A colibacillus engineering are inoculated in LB/Amp (prescription consists of and contains 5 milligrams of Tryptones 1 gram, yeast extract 0.5 gram, sodium-chlor 0.5 gram, 4 milligrams in sodium hydroxide, agar powder 1.5 grams and penbritins among the 100ml); Putting 37 ℃ of incubators (SANYO MCO-17AIC) cultivated 12-15 hour; Choose single bacterium colony; Switching in 12ml LB/Amp (prescription consists of and contains 5 milligrams of Tryptones 1 gram, yeast extract 0.5 gram, sodium-chlor 0.5 gram, 4 milligrams in sodium hydroxide and penbritins among the 100ml) culture tube, 37 ℃ of 250rpm shaking culture (HWY 111 constant temperature shaking tables) 12 hours; Get 10ml fermented liquid switching shake-flask culture (37 ℃, the 250rpm vibration) 8-12 hour in 1000ml LB/Amp fermented liquid, when bacterial concentration reaches OD
6001.5～2.5 o'clock; Be inoculated in 15 liters of fermentor tanks (B.Braun BIOSTAT C) and cultivate, every liter of fermented liquid contains Tryptones 12 grams, yeast extract 24 grams, potassium primary phosphate 3.8 grams, potassium hydrogenphosphate 12.5 grams, sal epsom 0.5 gram, glycerine 6.3 grams, 0.9 liter of pure water, glucose 20 grams and penbritin 0.1 gram; If fermentation parameter is 37 ℃ of temperature, pH7.0, dissolved oxygen 35% and mixing speed and dissolved oxygen interlock.Work as OD
600Value reaches at 20 o'clock, is the mid-term of engineering bacteria logarithmic growth, adds IPTG and induces, and continues to stop after 2 hours fermentation.Cooling and fermentation liquid makes it be cooled to 4-8 ℃.Adopt the freezing low speed centrifuge of Sorvall considerable low-temperature (Sorvall RC3B) centrifugal (4000rpm, 30 minutes) to collect bacterium.
In the engineering bacteria bacterial sediment of collecting, add the broken damping fluid (50mM Tris, 1mM EDTA, pH7.5 damping fluid) of engineering bacteria.Broken thalline under APV high pressure homogenizer (APV 2000) 10000psi pressure.Liquid obtains inclusion body (IB) bullion through high speed freezing centrifuge (Sorvall RC5B) SLA-3000 rotary head 11000rpm rotating speed centrifugal (4 ℃, 20 minutes) behind the broken bacterium.Use inclusion body washings (50mM Tris, 1mM EDTA, pH7.5; 2.5% Triton * 100,0.15M NaCl) repetitive scrubbing is three times, is about to inclusion body washings and inclusion body through high-speed liquid stirrer thorough mixing; Again through low temperature (4 ℃) high speed centrifugation (Sorvall RC5B; 11000rpm, 50 minutes) collect inclusion body, accomplish inclusion body washing and purifying.The inclusion body of purifying is dissolved in (50mM Tris, 1mMEDTA, 8M Guanidinium hydrochloride in the solubilization of inclusion bodies sex change liquid in the ratio of 10 gram weight in wet bases than 500 milliliters; 1mM dimercapto tetrahydroxybutane), put 4 ℃, 12 hours; Obtain solubilization of inclusion bodies sex change liquid, again through 4 ℃ of 10000g high speed centrifugations 40 minutes (Sorvall RC5B, 11; 000rpm), collect supernatant.
By 1: 100 volume ratio solubilization of inclusion bodies sex change liquid is slowly added 20mM sodium-acetate buffer (pH 6.0); 150mM sodium-chlor; 3M urea, 0.005% gathers in sharp alcohol ester 80 (polysorbate 80) the renaturation solution in mountain, and 4 ℃ left standstill 24 hours; Behind 0.45 μ m aperture membrane filtration, carry out protein and purify.
IFNs or IFNs-A protein renaturation liquid are through ion exchange chromatography (tomography devices AKTA purifier; Chromatography column INdEX200/500, chromatography media Sepharose big bead), level pad is 20mM sodium-acetate buffer (pH 4.5); 150mM sodium-chlor, 0.005% polysorbate80; Went up appearance in 60ml/ minute, and, collected A280 albumen elution peak liquid with 0～1M sodium-chlor gradient elution.With above-mentioned level pad balanced gel chromatography column (chromatography column BPG100; Chromatography media Superdex75 prep grade); Press appearance on 30ml/ minute the flow velocity, go up appearance and continue 5 minutes (the about 150ml of applied sample amount) at every turn, last kind after 120 minutes; Collect the A280 albumen absorption peak that occurs, be IFNs or IFNs-A stoste.
Reduced form SDS-PAGE silver dyes measures IFNs-A molecular weight (like Figure 11), and performance liquid chromatography (HPLC) method and efficient gel chromatography (SEC) mensuration IFN α 2b-A stoste, result such as Figure 12 and shown in Figure 13, and method is identical with embodiment 3 said methods.The result shows that the molecular weight of IFNs-A is about 18kDa; And IFN α 2b-A stoste HPLC and SEC purity are all greater than 98%.
The terminal site-directed coupling system PEG-IFNs-A of embodiment 2 PEG-ALD and IFNs-A N-
Load MacroCap SP strong cation exchange chromatographic column (2.6cmX20cm); And be connected to AKTA Explorer 100 (the Amersham Bioscience of LC system; Sweden) on, use 20mM sodium-acetate buffer pH 5.0 (buffer A) and the abundant balance chromatographic column of 0.005% polysorbate80 500ml; Use fresh feed pump,, IFNs-A stoste 0.5mg/ml 1500ml is added in the chromatographic column with the flow velocity of 10ml/min; Again under the flow velocity of 15ml/min,, remove not material with chromatographic media generation electrostatic adhesion with about 500ml buffer A flushing pillar; With the flow velocity of 3ml/min, add PEG propionic aldehyde (PEG-propionaldehyde) and the 0.1mg/ml NaBH of 5mg/ml30kDa
3CN solution 1500ml went up appearance about 240 minutes continuously; The pH of reaction system is 5.0; Again with the flow velocity of 15ml/min,, remove not material with chromatographic media generation electrostatic adhesion with about 500ml buffer A flushing pillar; Use buffer B (being buffer A+1mol sodium-chlor) subsequently,, continue about 150 minutes, can record ion exchange chromatography spectrogram as shown in Figure 1 with type of elution 0～8%, 8%～18% and 18%～100% wash-out.
Fig. 1 shows, can obtain peak 1 and 2 two elution peaks in peak altogether, i.e. P1 and P2, and wherein the peak area of P1 accounts for 72% of total peak area; P2 accounts for 28% of total peak area.P1 is mono-modified PEG-IFN α 2b-A through reduced form SDS-PAGE, efficient gel chromatogram (SEC) and performance liquid chromatography (HPLC) purity check, and purity is greater than 95% (like Fig. 2, shown in 3,4).This product confirms that through LC-MS trypsin digestion peptide figure analysis the coupling site of 30kDa molecular weight PEG-ALD is the N-end of IFNa2b-A, i.e. PEG-IFNa2bA-N30.
Same quadrat method, the terminal site-directed coupling of PEG-ALD and IFNs N-can be made PEG-IFNs.
In the terminal site-directed coupling method of above-mentioned chromatography column PEG albumen N-; If only change the pH value of reaction system; As under pH4.0, pH5.0 and pH6.0, (seeing table 3 for details); The composition of elution peak and ratio will change, but mono-modified PEG-IFN α 2b-A identifies PEG-ALD and the terminal site-directed coupling of IFN α 2b-A N-, i.e. the PEG-IFN α 2bA-N30 that is still the 30kDa molecular weight through LC-MS trypsin digestion peptide figure analysis.Point out this PEG-ALD that the terminal specificity of albumen N-is selected and the reduction or acidity of unprovoked pH value due to.
The change of table 3 pH value of reaction system is to the influence of the terminal site-directed coupling of chromatography column PEG albumen N-
Physico-chemical property and the external activity of embodiment 3 PEG-IFN α-A
1) MALDI-TOF-MS molecular-weight determination
In ground substance assistant laser parsing-ionization time flight mass spectrometer Bruker Daltonics Autoflex 3 (BrukerDaltonics Billerica companies; The U.S.) on; Carry out the cleaning of MALDI target in operation sequence and dry; Adopt Zip TipC4 (Millipore company, the U.S.) rifle head desalination, concentrate testing sample; Draw 10ul testing sample and standard substance point target; If the instrumentation procedure condition is: postponing extraction time (Delayed extraction) 190ns, detecting pattern and be Linearion mode/positive ion mode, laser frequency (Laser frequency) 50Hz, acceleration voltage (AceceleratingVoltage) 20KV, figure stack (sum shots) 400 times, correction mode is External calibration; Through the correction of standard model, carry out sample determination, and processing can obtain MALDI-TOF-MS molecular-weight determination collection of illustrative plates to data; Shown in Figure 14-1 and Figure 14-2.Figure 14-1 is the MALDI-TOF-MS molecular-weight determination result of used modifier PEG-ALD (30kDa) among the embodiment 2, is 32439.3 dalton, and the result shows that molecular weight and its tagged molecule amount of MALDI-TOF-MS method mensuration PEG-ALD (30kDa) is approaching; Figure 14-2 is depicted as the institute peak that obtains 1 mensuration result in the foregoing description 2, and the molecular weight of PEG-IFN α 2bA-N30 is 50664.679 dalton, is illustrated as mono-modified PEG-IFNaA.
2) performance liquid chromatography (High Performance Liquid Chromatography, HPLC) measure by the albumen degree
On Agilent 1100 chromatographic instruments (production of U.S. Agilent company); Select carbon 4 Vydac C4 albumen chromatographic columns (company) for use, with A phase trifluoroacetic acid aqueous solution (get 1.0ml trifluoroacetic acid add water cause 1000ml) and B mutually trifluoroacetic acid acetonitrile solution (get 1.0ml trifluoroacetic acid add trifluoroacetic acid aqueous solution cause 1000ml) be moving phase; At room temperature, establishing flow velocity is that 1ml/min, pressure are 76bar, carries out gradient elution (0～70%B phase); Appearance volume 20ul on the testing sample, applied sample amount is not less than 30ug, detects in wavelength 280nm.Calculate each peak area by area normalization method.Like Fig. 4, shown in Figure 12.
3) the efficient gel chromatogram (Size Exclusion Chromatography, SEC) measure by purity of protein
On Agilent 1100 chromatographic instruments (production of U.S. Agilent company), select gel filtration chromatography post Superdex for use
TM200, HR10/30 (GE Healthcare Biosciences company), moving phase is the sodium phosphate buffer (containing the sodium sulfate of 0.1M) of 50mM, pH6.7; If flow velocity is 0.5ml/min, pressure is 16bar, temperature: be 25 ℃; Detect wavelength 280nm, the wide 4nm of ripple; Reference wavelength 360nm, the wide 100nm of ripple; The testing sample applied sample amount is not less than 20ug; Adopt area normalization method to calculate each peak area.Like Fig. 3, Fig. 7, Fig. 8, Figure 13, Figure 15-1 is shown in the 15-2.
4) IFNs external activity
Adopt external WISH cell/Viola crystallina method to measure and compared the external activity of IFNs and various PEG-IFNs-A, the result is as shown in table 4.
WISH cell/Viola crystallina method: detailed method is seen three appendix X of Pharmacopoeia of People's Republic of China version in 2005 C.WISH cell and vesicular stomatitis virus (VSV) are available from Nat'l Pharmaceutical & Biological Products Control Institute; Under 37 ℃, 5% carbon dioxide conditions, make WISH cell adherent growth in the RPMI that contains 10% NBCS 1640 substratum (containing penicillium mould 10E5IU/L and Streptomycin sulphate 10E5IU/L), went down to posterity 2～3 times weekly by 1: 4; Discard nutrient solution, with PBS buffer solution for cleaning cell 2 times, collecting cell is made into the cell suspension that every 1ml contains 2.5 * 10E5～3.5 * 10E5 cell with above-mentioned substratum again, continues to cultivate 4～6 hours; Every hole adds cell suspension 100 μ l in being added with 96 porocyte nutrient solution plates of standard solution and need testing solution, cultivates 18-24 hour in 37 ℃, 5% carbonic acid gas; Discard the supernatant in the culture hole, every hole adds 100 μ l and attacks malicious nutrient solution (containing VSV 100CCID50 in RPMI 1640 substratum of 3% NBCS), continues to cultivate 24 hours; 50% pathology point of microscopy validation criteria article solution discards the supernatant in the culture hole behind 1IU/ml, every hole adds violet staining liquid 50 μ l, and (Viola crystallina 50mg is dissolved in the 20ml absolute ethyl alcohol; Add water to 100ml); Put room temperature after following 30 minutes, carefully wash away dye liquor with flowing water, and blot residual moisture; Every hole adds destainer 100 μ l (absolute ethyl alcohol 50ml, acetate 0.1ml add water to 100ml), and room temperature was placed 3～5 minutes; Behind the mixing, (Bio-Tek company USA), is a reference wavelength with 630nm, measures absorbance in wavelength 570nm place to put into ELIASA.
Table 4 IFNs active determination in vitro result
*PEG-IFNs-A is PEGization interferon fusion (Gly Gly Gly Gly Ser) 3 albumen;
△PEG-IFNs-A compares significant difference, P＜0.01 with the PEG-IFNs of coupling same molecular amount PEG;
The variation tendency of PEG-IFN α 2a-N10～40 external activities is similar with PEG-IFN α 2b-N10～40, concrete data not shown.
From table 4, can find out: the external biological activity of PEG-IFNs-A is significantly higher than the PEG-IFNs with molecular weight, explains that the terminal flexible polypeptide (Gly Gly Gly Gly Ser) 3 that merges of IFNs N-can significantly improve the influence of PEG molecule to the IFNs molecular activity; PEG-IFN α 2b-A-N30 is evident as height than the external activity of IFN α-2b-A and IFN α-2b; PEG-IFNs-N10～40 that the terminal coupling PEG of IFNs or IFNs-A protein-n propionic aldehyde is obtained or its external activity of PEG-IFNs-A-N10～40 are along with the increase of coupling PEG molecular weight; External activity progressively echelon reduces; But at PEG-IFNs-N40 or PEG-IFNs-A-N40 place; The reduction of external activity is particularly remarkable, significantly active " flex point " occur; The diPEG-IFNs-N40 of two 20kDaPEG of coupling or its external activity of diPEG-IFNs-A-N40 significantly are lower than PEG-IFNs-N40 or PEG-IFNs-A-N40 with equimolecular quantity.
5) thermostability of PEG-IFN α 2bA-N30 preparation
In the solution of 10mM sodium-acetate buffer (pH 4.5), 0.005% polysorbate80,5% sorbyl alcohol; Protein concentration is 1mg/ml PEG-IFN α 2bA-N30; Kept 1 month down at 37 ℃; Do not find protein aggregation or degraded (shown in Figure 15-1) through SEC mensuration, 1 week promptly occurs a small amount of protein aggregation, be soluble poly collective (shown in Figure 15-2) and preserve under this condition of IFN-α 2b-A; Explain that PEGization IFN-α 2b-A has obviously increased proteic thermostability.
6) the external resistance to enzymolysis stability of PEG-IFN α 2bA-N30
With ultrapure water dialysis testing sample 24 hours, freeze-drying (freeze drier, Alphal-2; Matin Christ USA) behind the testing sample, causes 1.5mg/ml with the dissolving of 1% ammonium bicarbonate aqueous solution; Add the trypsin TPCK treated TRYPSIN that TPCK handled by 1: 25 (w/w), Sigma, USA) and testing sample; PEG-IFN α 2bA-N30 and IFN-α 2b-A are hatched under 37 ℃ of constant temperature jointly, and sampling in 15,30,60,120 and 240 minutes, the SDS-PAGE method was surveyed former proteolysis per-cent respectively; The result is shown in figure 16, and the result shows that PEG-IFN α 2bA-N30 enzymolysis is after 4 hours; Still have 50% not by enzymolysis, and IFN-α 2b-A at 1 hour promptly by enzymolysis about 50%.The resistance to enzymolysis stability that PEG-IFN α 2bA-N30 is described is higher than common IFN-α 2b-A far away.
The test of embodiment 4 PEG-IFNs-A pharmacokineticss
4-1: select the healthy SD rat to carry out the pharmacokinetics test, through IFNs, PEG-Intron (the happy ability of wearing, Shanghai Schering Plough company), PEGASYS (Pai Luoxin, Shanghai company of Roche Group) and the various PEG-IFNs-A of tail vein injection 100ug/kg; 0,15min, 30min, 1; 3,5,8,24; Got blood in 48,72,96,120 hours; Each gets every kind of compound the blood time point and gets three animals simultaneously, and ELISA method (U.S. R&D company, the packing of brilliant U.S. company) is measured Plasma Concentration, and every kind of testing sample all should be gone the vitro test of susceptibility, particularity and linearity range before animal experiment; And make corresponding standard curve, and measure the result and obtain the plasma clearance elimination half life values through calculating, as shown in table 5.
Table 5 rat is injected the pharmacokinetic parameter of various PEG-IFNs-A
Route is a route of administration, and Tmax (h) is a peak time, and t1/2 (h) is the plasma clearance transformation period, and Cmax (ng/ml) is a peak concentration, and CL (ml/h/kg) is a clearance rate, and Vss (ml/kg) is a volume of distribution.
Plasma clearance transformation period (the t of healthier rat tail vein injection PEG-IFN α 2bA-N10, PEG-IFN α 2bA-N20, PEG-IFN α 2bA-N30 and PEG-IFN α 2bA-N40
1/2); Discovery is along with the increase of PEG molecular weight; The plasma clearance transformation period significant prolongation of PEG-IFN α 2bA, but occur " flex point " (seeing Figure 17) in plasma half-life of PEG-IFN α 2bA-N40, promptly the increasing degree in the plasma half-life that the increase of this PEG molecular weight brings obviously reduces; Increase to 20kDa with from 20kDa from 10kDa like the molecular weight of PEG and to increase to 30kDa; All increase the plasma half-life of PEG-IFN α 2bA-N20 or PEG-IFN α 2bA-N30 approximately about 50%, and the molecular weight of PEG increases to 40kDa from 30kDa, has but only increased about 10% the plasma half-life of PEG-IFN α 2bA-N40; But the volume of distribution (Vss) of PEG-IFN α 2bA-N40 obviously reduces nearly four times (as shown in table 5); Compare with PEG-IFN α 2bA-N30 simultaneously, the plasma half-life of subcutaneous injection (sc) PEG-IFN α 2bA-N40 and maximum plasma concentration increase few, and this maybe be more relevant with the subcutaneous absorption of PEG-IFN α 2bA-N40.
4-2: select 36 of healthy Wistar rats, be divided into 6 groups at random, the tail vein is slowly injected PEG20 respectively; PEG30, PEG40, PEG-IFN α 2bA-N20, PEG-IFN α 2bA-N30 and PEG-IFN α 2bA-N40 4; 000ug/kg, every animals urine ,-20 ℃ of preservations collected at a distance from 6 hours; Use anti-PEG ELISA test kit (Epitomics company, the U.S.) to measure the PEG content in the urine, the result is shown in Figure 180.Greater than 1ng/ml, within 500ng/ml～1ng/ml, PEG concentration logarithm is linearly relevant with the light absorption value logarithm to the detection sensitivity of above-mentioned various PEGs and PG-Ns for anti-PEG ELISA reagent.
The results suggest of Figure 18, more than the molecular weight of PEG is greater than 30kDa after, PEGs and PEG-IFNs-A significantly reduce from the permeability of GCW, so " flex point " of pharmacokinetics plasma clearance transformation period parameter appears in PEG-IFN α 2bA-N30.
The immunogenicity test of embodiment 5 PEG-rhG-CSF
On the healthy SD rat, test-compound (every group of 15 rats) by subcutaneous injection weekly once, the mode of administration of each 50 μ g/kg, successive administration, observed for 12 weeks, in the 1st week and the anti-PEG-IFNs antibody of the 8th, 10,12 weeks blood drawing mensuration; Employing indirect elisa method mensuration PEG-IFNs antibody (U.S. R&D company, soon PEG-IFNs (antigen) becomes 10 μ g/ml concentration with the Na2CO3-NaHCO3 solution dilution of pH9.6, encapsulate on 96 hole enzyme plates, every hole 100 μ l, 4 ℃ are spent the night.Behind 37 ℃ of sealings of 2%BSA confining liquid 2h, each hole adds the to be checked serum sample of 100 μ l with the sample diluting liquid dilution, establishes the positive and negative control hole simultaneously, hatches 1h for 37 ℃.Every hole, washing back adds the goat-anti rat IgG (1: 6 ten thousand) of 100 μ l horseradish peroxidase-labeled, hatches 1h again for 37 ℃.Each hole, washing back adds 100 μ l enzyme reaction substrate TMB, 37 ℃ of reaction 20min.With 50 μ l stop buffer termination reactions, on enzyme mark microplate reader, read the OD value at each 450nm place, hole.The judgement of antibody positive rate: with 2.1 times of threshold values as generation antibody of the measured OD value of the vehicle control treated animal serum specimen same period, the OD value that serum specimen records after all administrations is judged to be the positive more than or equal to threshold value person.The result shows (as shown in table 6): the immunogenicity of PEG-IFN α 2bA-N20, PEG-IFN α 2bA-N30 and PEG-IFN α 2bA-N40 all is lower than IFN-α 2b-A, and the immunogenicity of PEG-IFN α 2bA-N30 and PEG-IFN α 2bA-N40 all is lower than PEG-IFN α 2bA-N20.
The immunogenicity of the various PEG-IFNs-A of table 6 rat continuous application relatively
Annotate: * and the remarkable P of IFN α 2b-A facial difference＜0.05; △ compares significant difference P＜0.05 with PEG-IFN α 2bA-N20.
Sequence table
< 110>Tianjin Paige Biotechnology Co., Ltd.
< 120>column chromatography PEG and interferon analogue protein-n terminal site-directed coupling method and product thereof
<141>2008-12-28
<160>6
<210>1
<211>165
<212>PRT
< 213>human interferon (Interferon)
< 221>human interferon alfa aminoacid sequence
<222>(1)…(165)
<400>1
Met(Gly?Gly?Gly?Gly?Ser)t
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met?Leu
5 10 15
Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp?Arg?His
20 25 30
Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln?Lys?Ala?Glu
35 40 45 50
Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe?Asn?Leu?Phe?Ser
55 60 65
Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu?Leu?Asp?Lys?Phe?Tyr
70 75 80 85
Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Gly
90 95 100
Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val
105 110 115
Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser
120 125 130 135
Pro?Cys?Ala?Trp?Glu?Val?Val?Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu
140 145 150
Ser?Thr?Asn?Leu?Gln?Glu?Ser?Leu?Arg?Ser?Lys?Glu?＊＊＊
155 160 165
<210>2
<211>166
<212>PRT
< 213>human interferon (Interferon)
< 221>human interferon alpha 1 b eta aminoacid sequence
<222>(1)…(166)
<400>2
Met(Gly?Gly?Gly?Gly?Ser)t
Met?Ser?Tyr?Asn?Leu?Leu?Gly?Phe?Leu?Gln?Arg?Ser?Ser?Asn?Phe?Gln
5 10 15
Ser?Gln?Lys?Leu?Leu?Trp?Gln?Leu?Asn?Gly?Arg?Leu?Glu?Tyr?Cys?Leu
20 25 30
Lys?Asp?Arg?Met?Asn?Phe?Asp?Ile?Pro?Glu?Glu?Ile?Lys?Gln?Leu?Gln
35 40 45
Gln?Phe?Gln?Lys?Glu?Asp?Ala?Ala?Leu?Thr?Ile?Tyr?Glu?Met?Leu?Gln
50 55 60
Asn?Ile?Phe?Ala?Ile?Phe?Arg?Gln?Asp?Ser?Ser?Ser?Thr?Gly?Trp?Asn
65 70 75 80
Glu?Thr?Ile?Val?Glu?Asn?Leu?Leu?Ala?Asn?Val?Tyr?His?Gln?Ile?Asn
85 90 95
His?Leu?Lys?Thr?Val?Leu?Glu?Glu?Lys?Leu?Glu?Lys?Glu?Asp?Phe?Thr
100 105 110
Arg?Gly?Lys?Leu?Met?Ser?Ser?Leu?His?Leu?Lys?Arg?Tyr?Tyr?Gly?Arg
115 120 125
Ile?Leu?His?Tyr?Leu?Lys?Ala?Lys?Glu?Tyr?Ser?His?Cys?Ala?Trp?Thr
130 135 140
Ile?Val?Arg?Val?Glu?Ile?Leu?Arg?Asn?Phe?Tyr?Phe?Ile?Asn?Arg?Leu
145 150 155 160
Thr?Gly?Tyr?Leu?Arg?Asn?＊＊＊
165
<210>3
<211>498
<212>DNA
< 213>human interferon (Interferon)
< 221>natural coding human Interferon, rabbit (IFN α) gene
<222>(1)…(498)
<400>3
tgtgatctgc?ctcaaaccca?cagcctgggt?agcaggagga?ccttgatgct?cctggcacag?60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag?120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc?180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc?240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata?300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg?360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg?420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt?480
ttaagaagta?aggaatga
<210>4
<211>498
<212>DNA
< 213>human interferon (Interferon)
< 221>natural coding human interferon alpha-1 b eta-1b (IFN-β 1b) gene
<222>(1)…(498)
<400>4
agctacaact?tgcttggatt?cctacaaaga?agcagcaatt?ttcagtgtca?gaagctcctg?60
tggcaattga?atgggaggct?tgaatactgc?ctcaaggaca?ggatgaactt?tgacatccct?120
gaggagatta?agcagctgca?gcagttccag?aaggaggacg?ccgcattgac?catctatgag?180
atgctccaga?acatctttgc?tattttcaga?caagattcat?ctagcactgg?ctggaatgag?240
actattgttg?agaacctcct?ggctaatgtc?tatcatcaga?taaaccatct?gaagacagtc?300
ctggaagaaa?aactggagaa?agaagatttc?accaggggaa?aactcatgag?cagtctgcac?360
ctgaaaagat?attatgggag?gattctgcat?tacctgaagg?ccaaggagta?cagtcactgt?420
gcctggacca?tagtcagagt?ggaaatccta?aggaactttt?acttcattaa?cagacttaca?480
ggttacctcc?gaaactga
<210>5
<211>498
<212>DNA
< 213>human interferon (Interferon)
< 221>synthetic human interferon alpha-2 b gene again
<222>(1)…(498)
<400>5
tgcgatctgc?cgcagaccca?tagcctgggc?agccgtcgta?ccctgatgct?gctggcgcag?60
atgcgtcgta?ttagcctgtt?tagctgcctg?aaagatcgtc?atgattttgg?ctttccgcag?120
gaagaatttg?gcaaccagtt?tcagaaagcg?gaaaccattc?cggtgctgca?tgaaatgatt?180
cagcagattt?ttaacctgtt?tagcaccaaa?gatagcagcg?cggcgtggga?tgaaaccctg?240
ctggataaat?tttataccga?actgtatcag?cagctgaacg?atctggaagc?gtgcgtgatt?300
cagggcgtgg?gcgtgaccga?aaccccgctg?atgaaagaag?atagcattct?ggcggtgcgt?360
aaatattttc?agcgtattac?cctgtatctg?aaagaaaaaa?aatatagccc?gtgcgcgtgg?420
gaagtggtgc?gtgcggaaat?tatgcgtagc?tttagcctga?gcaccaacct?gcaggaaagc?480
ctgcgtagca?aagaatga
<210>6
<211>498
<212>DNA
< 213>human interferon (Interferon)
< 221>synthetic human interferon beta 1b (IFN-β 1b) gene again
<222>(1)…(498)
<400>6
tcttacaacc?tgctgggttt?cctgcagcgt?tcttctaact?tccagtctca?gaaactgctg 60
tggcagctga?acggtcgtct?ggaatactgc?ctgaaagacc?gtatgaactt?cgacatcccg 120
gaagaaatca?aacagctgca?gcagttccag?aaagaagacg?ctgctctgac?catctacgaa 180
atgctgcaga?acatcttcgc?tatcttccgt?caggattctt?cttctacggg?ctggaacgaa 240
acgatcgttg?aaaacctgct?ggctaacgtt?taccatcaga?tcaaccatct?gaaaacggtt 300
ctggaagaaa?aactggaaaa?agaagatttc?acccgtggta?aactgatgtc?ttctctgcac 360
ctgaaacgtt?actacggtcg?tatcctgcat?tacctgaaag?ctaaagaata?ctctcactgc 420
gcttggacca?tcgttcgtgt?tgaaatcctg?cgtaacttct?acttcatcaa?ccgtctgacg 480
ggttacctgc?gtaactaa
Claims (3)
1. the method for column chromatography PEG and interferon analogue protein-n terminal site-directed coupling, step comprises:
Select cation exchange medium, be loaded in the chromatography chromatographic column, the post bed height is the conventional upper limit of recommending height, and is connected in the LC system; 20mM sodium-acetate buffer and mass percent that use is equivalent to the pH 3.5～6.0 of 5～6 times of column volumes are the abundant balance chromatographic column of 0.005% polysorbate80; Use fresh feed pump,, IFNs-A stoste added in the chromatography column with the flow velocity of 8～10ml/min, applied sample amount be medium survey maximum carrying capacity 30%～50%; Again under the flow velocity of 13～15ml/min,, remove not material with chromatographic media generation electrostatic adhesion with the 20mM sodium-acetate buffer flushing pillar of the pH 3.5～6.0 of 5～6 times of column volumes; Reacting weight ratio in mPEG:IFNs-A is the ratio of 5～20mol: 1mol, and with the mPEG and the sodium cyanoborohydride solution of appearance adding different molecular weight on the flow velocity of 0.5～3ml/min, the mPEG otal investment X of said different molecular weight by formula (1) calculates:
(1)X＝5～20×m×MWPEG/MWIFNs-A
In the formula: X is the mPEG otal investment of different molecular weight, the IFNs-A protein content of m for adding, and MWPEG and MWIFNs-A are respectively the molecular weight of mPEG and IFNs-A;
Calculate the add-on of reductive agent sodium cyanoborohydride again by following formula (2):
(2) NaBH3CN add-on=10 * MW reductive agent * X/MWPEG
In the formula: the MW reductive agent is the molecular weight of reductive agent sodium cyanoborohydride, and X is the mPEG otal investment of different molecular weight, and MWPEG is the molecular weight of mPEG;
Went up appearance 240 ± 5 minutes continuously; The pH of reaction system is 3.5～6.0; Again with the flow velocity of 13～15ml/min,, remove not material with chromatographic media generation electrostatic adhesion with the 20mM sodium-acetate buffer flushing pillar of the pH 3.5～6.0 of 5～6 times of column volumes; Use the 20mM sodium-acetate buffer of pH 3.5～6.0 and the mixed solution of 0.01M～1M sodium-chlor subsequently; With type of elution 0～8%, 8%～18% and 18%～100% wash-out, continue 150 ± 2 minutes, obtain two elution peaks; Be P1 and P2; Wherein the peak area of P1 accounts for 72 ± 1% of total peak area, is mono-modified PEG-IFNs-A, i.e. single, the fixed point specificity coupling compound of polyoxyethylene glycol and IFNs-A albumen N-end;
Wherein:
Above-mentioned IFNs-A is by escherichia coli expression, preparation; The fusion rotein that said IFNs-A obtains for the terminal fusion of the N-that will flexibly connect chain (GlyGlyGlyGlySer) t and IFNs through integration technology: i.e. (GlyGlyGlyGlySer) t-N-IFNs; Wherein t is 3; IFNs is recombinant human interferon alpha 2 alfa-2b or beta-1b, and its aminoacid sequence is the aminoacid sequence shown in SEQ IDNO.1 or the SEQ ID NO.2; The external activity of said IFNs-A is more than or equal to the former proteoplast of IFNs outer active 70%;
Above-mentioned mPEG is mPEG-ALD; Said cation exchange medium is MacroCap SP or Sepharose Fast Flow; Said mono-modified PEG-IFNs-A is PEG-IFNsA-N10, PEG-IFNsA-N20, PEG-IFNsA-N30 and PEG-IFNsA-N40.
2. the Pegylation recombinant human interferon alpha 2 analogue albumen that makes of the method for said column chromatography PEG of claim 1 and interferon analogue protein-n terminal site-directed coupling is formed by N-terminal amino group and the mPEG molecule site-directed coupling single, strand, that molecular weight is 26～40kDa of nonglycosylated IFNs-A; Said mPEG molecule is mPEG-ALD, and its molecular formula is mPEG-(CH2) r-CHO, and wherein r is 2～3; Said IFNs is recombinant human interferon alpha 2 alfa-2b, and its aminoacid sequence is the aminoacid sequence shown in the SEQ ID NO.1; Pegylation recombinant human interferon alpha 2 analogue albumen also possesses feature simultaneously:
1) IFNs-A and mPEG link coupled molecule ratio are 1: 1, and the coupling site is an IFNs-A molecule N-terminal amino group;
2) in specific activity is measured; Measure the protein concentration of PEG-IFN-A to be measured with the Lowry method; WISH cell/Viola crystallina method is surveyed external activity, calculates every milligram of proteic activity and promptly gets specific activity, and the specific activity of PEG-IFNs-A is more than or equal to 7.0X10E7IU/mg albumen;
3) be that the plasma clearance long half time of mono-modified product of 12kDa PEG and IFN a-2b is more than 50% the plasma half-life of PEG-IFNs-A than PEG-Intron;
4) in immunogenicity determining, PEG-IFNs-A is lower than 50% in the 12nd all IFN antibody positive rate;
Described Pegylation recombinant human interferon alpha 2 analogue protein molecular is PEG-IFN2bA-N30.
3. be used to treat the parenteral formulation of B-mode or hepatitis C, wherein contain the described Pegylation recombinant human interferon alpha 2 of the claim 2 of treating significant quantity analogue albumen and pharmaceutically acceptable carrier.
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| CN103467591B (en) * | 2013-09-03 | 2015-09-09 | 长春海伯尔生物技术有限责任公司 | A kind of coupling method for Peg-IFN alpha-2b |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1289528C (en) * | 2004-10-29 | 2006-12-13 | 上海生物制品研究所 | Recombined human interferon-alpha 1b compound and process for preparation |
| CN1966527A (en) * | 2005-11-16 | 2007-05-23 | 中国科学院过程工程研究所 | Polyethylene glycol-interferon coupler and its preparation method |
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| CN1966527A (en) * | 2005-11-16 | 2007-05-23 | 中国科学院过程工程研究所 | Polyethylene glycol-interferon coupler and its preparation method |
Non-Patent Citations (2)
| Title |
|---|
| 吴影新等.干扰素α-2b的聚乙二醇修饰.《生物工程学报》.2008,第24卷(第9期),640-644. * |
| 张霖琳等.不同分子量聚乙二醇单修饰重组人干扰素α-2a.《过程工程学报》.2006,第6卷(第4期),619-622. * |
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