WO2009030065A1 - 聚乙二醇修饰的干扰素α2a及其制备方法和应用 - Google Patents

聚乙二醇修饰的干扰素α2a及其制备方法和应用 Download PDF

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WO2009030065A1
WO2009030065A1 PCT/CN2007/002643 CN2007002643W WO2009030065A1 WO 2009030065 A1 WO2009030065 A1 WO 2009030065A1 CN 2007002643 W CN2007002643 W CN 2007002643W WO 2009030065 A1 WO2009030065 A1 WO 2009030065A1
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interferon
ifn
ypeg
peg
modified
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PCT/CN2007/002643
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French (fr)
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WO2009030065A8 (zh
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Weidong Zhou
Qingjiang Xiao
Li Sun
Tiebing Wang
Bin Liu
Song Lin
Min Liu
Fenghong Yin
Lu Zhuang
Lifang Lei
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Biosteed Gene Expression Tech. Co., Ltd.
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Priority to KR1020107007268A priority Critical patent/KR101483814B1/ko
Priority to DK07800860.4T priority patent/DK2196475T3/da
Priority to CN2007800505413A priority patent/CN101636411B/zh
Priority to CA2698396A priority patent/CA2698396C/en
Priority to PCT/CN2007/002643 priority patent/WO2009030065A1/zh
Priority to US12/676,511 priority patent/US8597634B2/en
Priority to PT07800860T priority patent/PT2196475E/pt
Priority to BRPI0721988-1A priority patent/BRPI0721988B1/pt
Application filed by Biosteed Gene Expression Tech. Co., Ltd. filed Critical Biosteed Gene Expression Tech. Co., Ltd.
Priority to ES07800860T priority patent/ES2386575T3/es
Priority to PL07800860T priority patent/PL2196475T3/pl
Priority to EP07800860A priority patent/EP2196475B1/en
Publication of WO2009030065A1 publication Critical patent/WO2009030065A1/zh
Publication of WO2009030065A8 publication Critical patent/WO2009030065A8/zh
Priority to ZA2010/01555A priority patent/ZA201001555B/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a Y-branched polyethylene glycol unit-site modified interferon ct2a and a preparation method thereof, and the use of the obtained single-site PEGylated interferon 012a in the pharmaceutical field. Background of the invention
  • Interferon is a type of small molecule protein or glycoprotein produced by eukaryotic cells against viral infection and other antigenic stimuli. It has broad-spectrum antiviral, anti-proliferative and immunomodulatory effects. Interferon has been widely used in the treatment of various diseases, such as viral infections such as hepatitis B, hepatitis C and HIV, and inflammatory response abnormalities such as multiple sclerosis, arthritis, asthma, gallbladder fibrosis and interstitial lung disease. Diseases, diseases such as myeloma, lymphoma, liver cancer, lung cancer, and hairy cell leukemia (Kenji Oritani, Paul W Kincade, et al.
  • diseases such as myeloma, lymphoma, liver cancer, lung cancer, and hairy cell leukemia (Kenji Oritani, Paul W Kincade, et al.
  • Type I interferon and limitin a comparison of structures , receptors, and functions. Cytokine and Growth Factor Reviews, 12, 337-348, 2001; Yu-Sen Wang? Stephen Youngster, et al. Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications. Advance Drug Delivery Reviews, 54, 547-570, 2002 ).
  • Interferons are classified into four classes based on chemical, immunological, and biological properties: interferons cc, beta, 7 and 8.
  • Interferon alpha is secreted by leukocytes.
  • Human interferon alpha is encoded by a multi-gene family of approximately 20 genes, and the amino acid sequence encoding the protein has up to about 90% homology (Henco K., Brosius F ⁇ , et al. J. Mol. Biol., 185 , 227-260, 1985).
  • Human interferon a2a is one of the subtypes of the human interferon alpha family ⁇ 2 subfamily and is a single-chain protein with various biological activities.
  • the single strand of the protein consists of 165 amino acid residues, as shown in SEQ ID ⁇ : 1, the N-terminal amino acid is Cys, and there is one free ⁇ - ⁇ 2; the amino acid sequence is 23, 31, 49, 70, 83 , 112, 121, 131, 133, 134 and 164 are Lys, each has 1 free ⁇ - ⁇ 2 ⁇
  • Polyethylene glycol is an inert, non-toxic, biodegradable organic polymer.
  • the PEG modification technique is the attachment of PEG to the active protein by covalent attachment.
  • PEGylation of protein drugs their traits are significantly improved, including prolonged drug half-life, decreased immunogenicity, improved safety, enhanced efficacy, reduced frequency of administration, improved drug solubility and water solubility, and resistance to protease digestion.
  • Inada et al Inada et al. J. Bioact. and Compatible Polymers, 5, 343, 1990
  • Delgado et al Delgado et al. Critical Reviews in Therapeutic Drug Carrier Systems, 9, 249, 1992
  • Katre et al Kertre Advanced Drug Delivery Systems, 10, 91, 1993
  • Davis et al. U.S. Patent No. 4,179,337).
  • U.S. Patent No. 4,179,337 discloses that when PEG is combined with an enzyme and insulin, the immunogenicity of the protein decreases while the activity of the protein decreases significantly. This effect is in G-CSF
  • PEG IFN-a2b PEG-Intron
  • PEG IFN-a2a PEG-Intron
  • PEG IFN-a2a PEG-Intron
  • a polyethylene glycol molecule modifies a protein by attachment to the N-terminal a-amino group of the protein molecule or the ⁇ -amino group of the lysine inside the molecule.
  • Polyethylene glycols that have been used to modify proteins generally come in three forms: a linear molecular form ( ⁇ 0593868), a branched molecular form of a U-shaped branch ( ⁇ 0809996 ), and a branched molecular form of a ⁇ branch ( CN1243779C ), but The preparation of branched PEG-modified interferon ot2a of the scorpion branch and the separation of PEG-modified interferon ct2 a at different amino sites have not been reported.
  • PEG capable of binding to a protein drug usually needs to be derivatized so that one or both terminal groups at both ends of the PEG are chemically activated to have an appropriate functional group, and the functional group is active against at least one of the drugs to be bound, and can form with it.
  • a stable covalent bond For example, PEG may be attached to the peptide chain of a protein £ Lys residue -> 3 ⁇ 4 2, coupled to a protein or peptide chain N-terminal amino acid residue of the ⁇ - ⁇ 2.
  • PEG-NHS is bound to ⁇ - ⁇ 2 of ⁇ - ⁇ 2 or Lys of the N-terminal amino acid of IFN- ⁇ by nucleophilic substitution.
  • the PEG-NHS described in this patent is a U-branched PEG derivative (PEG 2 -NHS) with the following molecular formula:
  • R and R are each independently a low molecular weight alkyl group; n and II' are between 600 and 1500; and the PEG has an average molecular weight of between 26 KD and 66 D.
  • the molecular formula of the PEG 2 -NHS modified product of IFN- ⁇ is as follows:
  • One or more PEG 2 -NHS molecules bind to a-H 2 of the N-terminal amino acid of IFN-a or ⁇ - ⁇ 2 of Lys, and the resulting product is non-PEGylated, single-site PEGylated and multi-site PEG Chemical
  • a mixture of IFN- ⁇ ; a single-site PEGylated IFN- ⁇ can be separated from the reaction product by known purification means. Since IFN-a has one N-terminal amino acid and multiple Lys, ie, multiple PEG 2 -NHS reaction sites, the isolated single-site PEGylated IFN-a is a plurality of single-site pegylated isomers. mixture.
  • European Patent EP 0593868 uses a linear linear PEG derivative to modify interferon.
  • the molecular formula of the modified product is as follows -
  • R is a low molecular weight alkyl group
  • R 2 , R 3 and R 4 are H or a low molecular weight sulfhydryl group
  • m is between 1 and the number of interferon potential PEG modification sites o M
  • W is O or NH
  • X is between 1 and 1000, y and z are between 0 and 1000, + + ⁇ is between 3 and 1000
  • R 2 , R 3 and at least one of them is a low molecular weight sulfhydryl group. Yu-Sen Wang et al. (Yu-Sen Wang et al, Advanced Drug Delivery Reviews, 54: 547-570, 2002.
  • Yu-Sen Wang et al, Biochemistry, 39, 10634-10640, 2000. reported a 12KD linear single armor.
  • the oxy PEG-modified rIFN-a2b (Peg-Intron) was confirmed by HPLC-IE separation analysis to be a mixture of 14 or more single-site modified isomers.
  • the linear PEG formula used by Yu-Sen Wang et al. is as follows:
  • the average molecular weight of the PEG is 12KD ( Inventive Overview
  • the PEG derivative used in the present invention is a Y-branch, which is a novel branched PEG derivative, and its structure is different from U-branched PEG, and the biggest difference between them is: 2 of the Y-type PEG derivatives used in the present invention
  • the PEG branching chains are linked together by N atoms, while the EP0809996 U-type PEG derivatives are linked together by C atoms.
  • P nP b is the same or different polyethylene glycol; j is an integer from 1 to 12; is a substituted or unsubstituted alkyl, substituted aryl, arylalkyl or heteroalkyl group of U, C 1-12 ⁇ ⁇ 2 is independently a linking group, wherein (CH 2 ) n , X 2 is a group selected from the group consisting of: (CH 2 ) n, (CH 2 ) n OCO, (C3 ⁇ 4) n HCO, (CH 2 ) n CO, and n is an integer from 1 to 10; F is a terminal group selected from the group consisting of hydroxyl, carboxyl, ester, acid chloride, hydrazide, maleimide, pyridine
  • the Y-type PEG derivative molecule is represented by the formula: Wherein, R and R 'are each independently - alkyl, preferably methyl; m and m' characterization of the degree of polymerization, is any integer; m + m, preferably from 600 to 1500; is 11, d_ 12 substituted or unsubstituted An alkyl group, a substituted aryl group, an arylalkyl group, or a heterofluorenyl group; j is an integer from 1 to 12; F is a terminal group selected from the group consisting of a hydroxyl group, a carboxyl group, an ester group, an acid chloride, a hydrazide, and a male
  • the imide, pyridine disulfide can form a covalent bond with the therapeutic drug or the amino, hydroxyl or sulfhydryl groups on the substrate.
  • the polyethylene glycol has a total average molecular weight of from about
  • R and R' are each independently CVC 4 alkyl, preferably methyl; m and m' characterize the degree of polymerization, any integer; m+m, preferably 600 to 1500; j is an integer from 1 to 12; Total average molecule The amount is about 40,000 Daltons.
  • the present inventors modified interferon c 2 a (IFN-a2a) using a branched-chain PEG (YPEG) derivative of Y-branches, and isolated YPEG-IFN-a2a modified by Q Sepharose FF ion exchange chromatography. Further, the isolated single-site modified YPEG-IFN-a2a was resolved by SP Sepharose FF chromatography to further obtain the ⁇ -amino group-based linkage of IFN-oc2a corresponding to lysine at position 134 of SEQ ID NO: 1. YPEG-IFN-a2a of YPEG is called YPEG-IFN-a2a (134).
  • the in vitro specific activity of YPEG-IFN-a2a (134) was determined to be significantly higher than that of YPEG-IFN-a2a linked to YPEG at other amino acid positions, and the serum half-life was significantly longer than that of unmodified IFN-a2a.
  • the present invention provides a single-site modified pegylated interferon a2a having the following structure:
  • ⁇ P b is the same or different polyethylene glycol; j is an integer from 1 to 12; is H, substituted or unsubstituted alkyl, substituted aryl, aryl fluorenyl, or hetero fluorenyl ⁇ ⁇ 2 are each independently a linking group, wherein (C3 ⁇ 4) n , X 2 is a group selected from the group consisting of: (CH 2 ) n , (CH 2 ) n OCO, (CH 2 n HCO, (C3 ⁇ 4) n CO, and n is an integer from 1 to 10.
  • the pegylated interferon c 2a of the invention has the formula:
  • R and R' are each independently - fluorenyl, preferably methyl; j is an integer from 1 to 12; m and m' are characterized by degree of polymerization, any integer, which may be the same or different integers; m + m' is preferred 600 to 1500.
  • the Y-branched PEG is linked to the interferon oi2 a molecule by unit point binding.
  • the molecular weight of YPEG-IFN-a 2a of formula II depends primarily on the degree of polymerization m and m'.
  • m+m' is preferably from 600 to 1500, and the average molecular weight of the corresponding YPEG is from about 26,000 Daltons to about 66,000 Daltons; m+m' is preferably from 795 to 1030, and the average molecular weight of the corresponding YPEG is from 35,000 Daltons to 45,000 Daltons. ; m+m' is particularly preferably 885 to 1030, corresponding to the average molecular weight of YPEG 39000 Daltons to 45,000 Daltons; m+m' is most preferably 910, corresponding to an average molecular weight of Y80 of 40,000 Daltons.
  • the ratio of m and m' may range from 0.5 to 1.5, preferably from 0.8 to 1.2.
  • the amide bond of polyethylene glycol to interferon a2a in the pegylated interferon a2a of the invention is located in interferon a2a corresponding to 23, 31, 49 of SEQ ID NO: 1, 70, 83, 112, 121, 131, 133, 134 or 164 lysine side chain ⁇ amino or a terminal amino acid a amino group.
  • the amide linkage of polyethylene glycol to interferon a2a in the pegylated interferon a2a of the invention is predominantly located in the interferon c 2a corresponding to 134 of the SEQ ID NO: 1
  • the side chain ⁇ amino group of the acid is predominantly located in the interferon c 2a corresponding to 134 of the SEQ ID NO: 1
  • the interferon a2a of the invention may be interferon a2a obtained from a natural source or obtained by recombinant biotechnology.
  • the interferon (x2a is human interferon ot2a (WFN-a2a) having the sequence of SEQ ID ⁇ : 1 extracted from a natural source or obtained by recombinant biotechnology.
  • the human interference The cc2a is a recombinant human interferon o 2a (rhIFN-a2a). rhIFN-a2a may be artificially synthesized, or may be expressed in a prokaryotic system such as E.col. E.col or a eukaryotic yeast system such as Pichia.
  • Pastoris can also be expressed by other insect cell systems or mammalian cell systems such as CHO.
  • Methods of preparing natural or recombinant IFN- a 2a and methods for detecting the activity of IFN-a2a and its YPEG modified products are prior art in the art.
  • the YPEG-IFN-a 2a of the present invention has the same clinical use as IFN-a2a, and is suitable for the treatment of tumors and antiviral infections, such as hepatitis, hairy cell leukemia, cell-mediated lymphocyte lysis, Kapasi's sarcoma and the like.
  • tumors and antiviral infections such as hepatitis, hairy cell leukemia, cell-mediated lymphocyte lysis, Kapasi's sarcoma and the like.
  • YPEG-IFN- ⁇ 2a of the present invention has significant improvements in stability, solubility, serum half-life and clinical efficacy relative to IFN-ot2a.
  • the YPEG-IFN-a 2a of the present invention can be administered to a patient in the form of a composition comprising a pharmaceutically effective amount of YPEG-IFN-a 2a and a pharmaceutically acceptable carrier or agent.
  • Shape agent a pharmaceutically effective amount of a pegylated interferon a2a of the invention and a pharmaceutically acceptable carrier or excipient.
  • the composition comprises mannitol, an amino acid, sodium chloride and sodium acetate, wherein the amino acids are preferably aspartic acid, asparagine and glycine.
  • the present invention provides a pegylated interferon a2a of the present invention or a composition comprising the pegylated interferon oa of the present invention in the preparation of a medicament for treating a disease requiring treatment with interferon (x2a) Application in medicine.
  • the disease requiring treatment with interferon a2a is selected from a viral infection such as hepatitis B, hepatitis C, hepatitis D, genital warts, etc., tumors such as hairy cell leukemia, chronic myeloid cells Leukemia, low-grade non-Hodgkin's leukemia, cell-mediated lymphocyte lysis, Kapasi's sarcoma, multiple myeloma, Malignant melanoma, cutaneous T-cell lymphoma, laryngeal papillomatosis, recurrent or metastatic renal cell carcinoma, inflammatory allergic diseases such as multiple sclerosis, arthritis, asthma, gallbladder fibrosis and interstitial lung disease Thrombosis associated with myeloproliferative diseases.
  • a viral infection such as hepatitis B, hepatitis C, hepatitis D, genital warts, etc.
  • tumors such as hairy cell leukemia, chronic
  • the YPEG-activated derivative such as polyethylene glycol succinimidyl ester (YPEG-NHS)
  • YPEG-NHS polyethylene glycol succinimidyl ester
  • the PEG moiety is covalently bound to the amino group (-NH 2 ) of the protein, including the a-amino group at the N-terminus of the protein and the ⁇ -amino group of the lysine residue.
  • the reaction equation for the reaction of IFN-a2a with YPEG to form YPEG-IFN-a2a is as follows:
  • the reaction conditions are mild, the pH range is 4.5-9.5, the temperature is 0-25 ° C, and stirring or other means of mixing is required. Specific conditions are described, for example, in the examples.
  • YPEG of various molecular weights can be linked to IFN-a2a by this method, and the reaction products include single site (YPEG-IFN-a2a), two sites (YPEG 2 -IFN-2a) and multiple sites (YPEG n -IFN -a2a)
  • the product is modified, and the product is mainly substituted by a single point by controlling the reaction conditions.
  • cation exchange chromatography, anion exchange chromatography and exclusion chromatography were used to separate the single-site modified YPEG-IFN-a2a from various YPEG-modified IFN-a2a mixtures, and the single-site modified YPEG- IFN-(x2a is further resolved to obtain YPEG-IFN-a2a which is linked to YPEG at different sites.
  • Common methods such as cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography and exclusion chromatography, etc. Know the method for the property analysis, such as mass spectrometry, polyacrylamide gel electrophoresis, high performance liquid chromatography exclusion chromatography, etc.
  • the in vitro biological activity of various modified products is detected according to the interferon. Knowing the activity detection method, such as cytopathic inhibition method.
  • the activity detection method such as cytopathic inhibition method.
  • PEG unit point modification IFN different modification site isomers are not affected by the retention of IFN activity domain by PEG component.
  • the biological activities between the modified site isomers are quite different; in general, the in vitro biological activity of IFN is significantly decreased after PEG modification.
  • the isolates of the three peaks obtained in the ion exchange chromatography results were subjected to in vitro specific activity measurement, and the results showed that the third peak isolate (SP2) was separated from other peaks and PEGASYS (Basel Hoff, Switzerland).
  • Roche Co., Ltd. has a significantly higher specific activity and has a significantly longer serum pharmacological half-life than unmodified IFN-a2a.
  • the Y-branched PEG-linked peptide of SP2 is isolated and subjected to amino acid sequence determination by Edman degradation. The result shows that the main component of SP2 is YPEG-IFN-a2a (134).
  • the invention also provides a method of making and purifying YPEG-IFN-a2a, comprising:
  • R and R are each independently C r C 4 alkyl, preferably methyl; j is an integer from 1 to 12; m and m' are characterized by any degree of polymerization; m + m' is preferably from 600 to 1500;
  • step (b) capturing the reaction product obtained in the step (a) with an anion exchange resin, preferably Q Sepharose FF, and eluting with an anion gradient, preferably using a chloride ion gradient, to obtain a modified product;
  • an anion exchange resin preferably Q Sepharose FF
  • step (c) using a cation exchange resin, preferably SP Sepharose FF, subjecting the reaction product captured in step (b) to a cationic gradient elution, preferably using a sodium ion gradient, to collect each peak separately;
  • a cation exchange resin preferably SP Sepharose FF
  • Figure 1 SDS-PAGE results of three batches of IFN-a2a Y-PEG (40KD) modification reaction. The separation gel concentration was 12%, and Coomassie Brilliant Blue R-250 stained. Lanes 1-2: 20060804; Lanes 3-4: 20060807-1; Lanes 5-6: 20060807-2.
  • Figure 2 YPEG-IFN-a2a SP Sepharose FF purification resolution modification site isomer map.
  • Figure 3 YPEG-IFN-a2a SP Sepharose FF purified sample SDS-PAGE (12%) Electrophoresis silver staining results.
  • Lane 1 molecular weight reference; lanes 2, 4, 6, 8: blank; lanes 3, 5, 7, 9: correspond to 1-4 peaks of the elution profile, respectively.
  • Figure 4 Results of SDS-PAGE electrophoresis silver staining for apparent molecular weight of YPEG-IFN-a2a modified site isomer.
  • Lane 1 Molecular Weight Reference (GE Lifescience);
  • Lane 2 YPEG-IFN-a2a SP3, 0 ⁇ g ⁇ ,
  • Lane 3 YPEG-IFN-a2a SP2, 0A ⁇ g ⁇ ,
  • Lane 4 YPEG-IFN-a2a SP1, 0 g.
  • FIG. 5 YPEG-IFN-2a SP Sepharose FF purification split sample MALDI-TOF MS method molecular weight determination results.
  • YPEG-IFN-a2a SP1 corresponds to the lane 4 sample of Fig. 4
  • YPEG-IFN-a2a SP2 corresponds to the lane 3 sample of Fig. 4
  • YPEG-IFN-a2a SP3 corresponds to the lane 2 sample of Fig. 4.
  • Figure 6 YPEG-NHS (40 D) MALDI-TOF MS method for molecular weight determination.
  • Figure 7 Comparison of serum drug concentration and 2'-5,A concentration in cynomolgus monkey single sc 30 g.kg- 1 YPEG-rhIFN-a2a SP2
  • FIG. 8 YPEG-IFN-2a SP2 trypsin digestion Oh-like HPLC-RP C 18 trypsin peptide map detection results.
  • the retention time of YPEG-IFN- ct 2a SP2 was 62.105 min, the elution peak at 71.882 min was the background peak of the flux, and the elution peak at 2-3 min was the trypsin peak.
  • Figure 9 YPEG-IFN-a 2a SP2 trypsin digestion 48h sample HPLC-RP C 18 trypsin peptide map detection results. A solvent peak of 71.581 min was obtained, which corresponds to the 71.882 min solvent peak of the peptide map of the enzymatic Oh sample; no substrate protein peak (62.105 min) was detected between 59.5 min and 62.5 min, indicating that the sample was completely enzymatically digested.
  • FIG. 10 YPEG-IFN-a2a SP2 trypsin complete digestion sample YPEG modified peptide Sephacryl S-100 HR separation map. Detailed description of the invention
  • the final concentration of IFN-ct2a reaction was 4 mg/ml, and the molar ratio of IFN-oc2a to YPEG reaction was 1:2.
  • the mixture was stirred at 0-25 ° C for 2 h under stirring to form PEGylated IFN-a2a, and glacial acetic acid (Xifu Chemical Plant, Shantou City) was added to make the pH ⁇ 4.0 to terminate the reaction, and the sample was subjected to SDS-PAGE electrophoresis.
  • the reaction system was diluted 50 times with water, filtered at 0.2 ⁇ m, and placed at 4 ° C until use.
  • the target product was mainly PEGylated YPEG-IFN-oc2a with a yield of 20-40%.
  • the target product was mainly PEGylated YPEG-IFN-a2a with a yield of 35-50%.
  • Figure 1 shows the results of SDS-PAGE of three batches of YPEG 0KD) modified IFN-a2a. From the results of SDS-PAGE electrophoresis in Figure 1, it can be seen that under the conditions of this reaction, the PEG modification rate of rhIFN-a2a is between 35-50%, and the modification rate is stable; the modified product is mainly composed of single-point modification products. Site modification product.
  • YPEG-IFN-a2a Q Sepharose FF capture sample was adjusted to pH 5.0 with 20% acetic acid, diluted 15 times with 5 mM NaAc/HAc (pH 5.0) (Xifu Chemical Plant, Shantou City); 0.5 mg/ml load Sample SP Sepharose FF 100ml (GE Healthcare) (18mmx394mm), 5mM NaAc/HAc (pH 5.0) equilibrated 3CV, 0%-30% 5mM NaAc/HAc-70mM NaCl (pH5.0) gradient elution 2.5CV, 30 %-100% of 5111] ⁇ NaAc/HAc-70 mM NaCK pH 5.0) was eluted with a gradient of 50 CV.
  • SP Sepharose FF 100 ml was split into 4 eluting peaks, and samples were collected by peaks, sampled separately for SDS-PAGE electrophoresis, and stained with silver staining.
  • SP Sepharose FF split 1 peak is mainly YPEG multi-site modification product (YPEG n -IFN-a2a);
  • SP Sepharose FF split 2 peak is based on single-point modification product (YPEG-IFN-a2a) , contains a part of multi-site modification products; SP Sepharose FF split 3 peaks, 4 peaks are single point modification products.
  • SP Sepharose FF resolved 2-4 peaks as single-point modification product isomers of different YPEG modification sites, designated as YPEG-IFN-a2a SP1, YPEG-IFN-a2a SP2 and YPEG-IFN-a2a SP3, respectively.
  • the split map and electrophoretic silver staining results are shown in Figures 2 and 3, respectively.
  • Each sample of YPEG-IFN-a2a SP1-3 was separately added with sodium chloride, sodium acetate, mannitol, aspartic acid, 0.22 ⁇ filter, and placed at 4 ° C for use.
  • the modified site isomer of YPEG-IFN-a2a was assayed for protein concentration by Kjeldahl method.
  • the apparent molecular weight of the modified site isomer of YPEG-IFN-a2a was determined by SDS-PAGE electrophoresis. The experimental procedure was carried out as reported by Laemmli (Nature 227:680, 1970) with a gel concentration of 7.5% and silver staining. The apparent molecular weight of the modified site isomer of YPEG-IFN-a2a is basically the same, and the size is about 120KD (Fig. 4).
  • the molecular weight of the modified site isomer of YPEG-rHuIFN-o ⁇ a was determined by the German BRU ER autoflex TOF/TOF mass spectrometer and the MALDI-TOF MS method.
  • the substrate was treated with sinapinic acid (Sinapinic acid, SA, C n H 12 0 5 , MW 224.22; batch number 2006 236870 002, BRUKER, Germany), and the protein molecular weight standard was analyzed by BRUKER's Protein Calibration Standard II (Part No. 207234).
  • the software is flexAnalysis Ver.3.0.54.0.
  • the MS of the modified site isomer of YPEG-IFN-a2a has a uniform molecular weight of about 59,000 Daltons (Fig. 5).
  • the purity of the modified site isomer of YPEG-IFN-a2a was determined by HPLC-SE method.
  • the HPLC analytical column was TSK G4000 SW XL (7.8mmx300mm) (TOSOH), the sample loading volume was 20 ⁇ 1, about 10 ⁇ ⁇ protein, the mobile phase was 0.1M PBNa-O.lM NaCl (pH7), and the elution flow rate was 0.8ml/min.
  • the detection wavelength was 280 nm.
  • the detection result of YPEG-IFN-a2a SP2 is a single main peak with a purity greater than 99%.
  • the endotoxin content of YPEG-IFN-a2a was determined by the sputum reagent method ("People's Republic of China Pharmacopoeia" 2005 edition three appendix XC), and the test results of each sample were ⁇ 5.0 EU/mg.
  • IFN Intra-viral effect
  • 2'-5'AS 2'5'-oligoadenylate synthetase
  • 125 1 the activity of 2'-5'AS in vivo was measured to reflect the pharmacodynamics of IFN.
  • 2'-5'AS is capable of catalyzing the production of 2'-5 by ATP in the presence of poly(I)poly(C) agar, A (2'-5'AS activity produced by 2,-5'A (2'5 '- oligoadenosine concentration) expressed.
  • the substrate ATP is catalyzed to generate 2'-5,A.
  • the binding rate of 125 1 labeled 2'-5'A was initially added, and a standard curve was prepared using a four-parameter logistic regression to calculate the concentration of 2,-5,A induced by 2,-5,AS in the unknown sample.
  • Table 1 and Figure 7 are the 2'-5, A method of cynomolgus monkeys (15 cynomolgus monkeys, 7 males and 8 males). Laboratory Animal Center of the Academy of Medical Sciences (Certificate No. SCXK- (Jun) 2002-001), weighing 2.5 ⁇ 3.7kg. Cage feeding, fed with standard monkey feed, free water) single subcutaneous injection (sx) 30 ⁇ -1 YPEG-IFN-a 2a SP2 serum 2,-5,A concentration, as can be seen from Figure 5 The activity of 2,-5,AS in serum was significantly increased after administration, and the peak time of 2,-5,A in serum was delayed compared with the peak time of YPEG-IFN-ct2a SP2. The average peak time is 24 ⁇ 18.33 h, and the peak concentration is KS ltSSjg Pmol-dl o
  • the HRP substrate solution was added to each well to produce a color that was proportional to the amount of IFN-a2a or YPEG-IFN-cc2a SP2 bound in the first step.
  • the reaction was terminated and the color intensity was measured.
  • the higher the absorbance OD value the higher the concentration of IFN-a2a or YPEG-IFN-a2a SP2 in the sample.
  • the standard curve of the product is used to determine the serum drug concentration of each blood sample.
  • Cytopathic inhibit the biological activity in vitro method of detecting each modification site YPEG-IFN-cc2 a isomers According to the method described in the Pharmacopoeia of the People's Republic of China 2005 Edition, Appendix XC, Interferon Potency Method, interferon can protect human amniotic cells (WISH) from vesicular stomatitis virus (VSV) destruction.
  • WISH human amniotic cells
  • VSV vesicular stomatitis virus
  • the viable WISH cells were stained with crystal violet, the absorbance was measured at 570 nm, and the protective effect curve of interferon on WISH cells was plotted to determine the in vitro biological activity of interferon.
  • the in vitro biological activity test results of each sample are shown in Table 2, and each sample was simultaneously tested for 3 parallel samples.
  • the SP2 sample in each of the modified site isomers of the single-point modification product had the highest in vitro specific activity, 1-2 times higher than SP1 and SP3, than the unreplicated sample and PEGASYS (Basel Hoff, Switzerland) Made by Roche Co., Ltd., Shanghai Roche Co., Ltd., product batch number B1016, sub-package lot number SH0020) is also 1-2 times higher.
  • YPEG-IFN-2a SP2 modification site analysis YPEG-IFN-a2a SP2 was replaced with 50 mM H 4 HC0 3 (pH 8.0 ) by a 5K ultrafilter (Millipore, polyethersulfone), and the protein concentration was determined to be 4.02 mg/ml by UV scanning.
  • TPCK TrypsinC Promega is formulated into 0.5 ⁇ ⁇ / ⁇ 1 using the solution supplied with the product. Add according to Table 3:
  • the reaction system was heated at 37 ° C for 48 h, and the reaction was terminated by adding 1.52 ml of 20% acetic acid.
  • the detection instrument is Waters HPLC system, host model 600, 2487 dual wavelength detector, data processing software Empower 2.
  • the HPLC analytical column was Jupiter C 18 , particle size 5 ⁇ , pore size 30 ⁇ , O4.6 ⁇ l50 mm, produced by Phenomenex, USA.
  • the flow phase A was 0.1% TFA/H 2 O
  • the flow phase B was 0.1% TFA/90% ACN/H 2 O; the flow rate was measured at 1 ml/min, and the detection wavelength was 214 nm.
  • the elution gradient is shown in Table 4, and the results are shown in Figures 8-9.
  • the first elution peak sample (sample number: YPEG-IFN-a2a S100-l, Fig. 10) was collected, and the solvent system was replaced with 5 mM PBNa (pH 7) by 5K ultrafiltration, and vacuum-dried and dried.
  • the N-terminal amino acid sequence was determined by Edman degradation method.
  • the N-terminal 7 amino acid sequence of the sample was XYSPXAW (Table 5), where X refers to the cysteine (Cys) of the ⁇ -amino acid which cannot be detected by Edman degradation method.
  • Alpha amino Acids and other modified amino acids Based on the alignment with the sequence shown in SEQ ID ⁇ : 1, it was confirmed that YPEG-IFN-2a SP2 is mainly a YPEG-modified product of Lysl34.
  • X refers to the amino acid cysteine (Cys), non-a amino acids and other modified amino acids that cannot be detected by Edman's degradation method.

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Description

聚乙二醇修饰的干扰素 a2a及其制备方法和应用 发明领域
本发明涉及 Y型分支的聚乙二醇单位点修饰的干扰素 ct2a及其制备 方法， 以及获得的单位点聚乙二醇化千扰素 012a在制药领域中的应用。 发明背景
干扰素 (interferon, IFN)是真核细胞针对病毒感染和其它抗原刺激而 产生的一类小分子蛋白质或糖蛋白， 具有广谱抗病毒、 抗增殖和免疫调 节作用。 干扰素已被广泛应用于多种疾病的治疗， 如乙型肝炎、 丙型肝 炎和 HIV等病毒性感染， 多发性硬化、 关节炎、 哮喘、 胆囊纤维化和间 质性肺病等炎症反应异常性疾病， 骨髓瘤、 淋巴瘤、 肝癌、 肺癌和毛状 细胞白血病等肿瘤等疾病 （Kenji Oritani, Paul W Kincade, et al. Type I interferon and limitin: a comparison of structures , receptors, and functions. Cytokine and Growth Factor Reviews, 12， 337-348, 2001； Yu-Sen Wang? Stephen Youngster, et al. Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications. Advance Drug Delivery Reviews, 54, 547-570, 2002)。
根据化学、 免疫学和生物学性质的不同， 干扰素被分为 4类： 干扰 素 cc、 β、 7和8。 干扰素 α由白细胞分泌产生。 人干扰素 α由约 20个基 因组成的多基因家族编码， 编码蛋白质的氨基酸序列具有高达约 90%的 同源性（Henco K.， Brosius F丄， et al. J.Mol.Biol., 185, 227-260, 1985 )。人 干扰素 a2a是人干扰素 α家族 α2亚族的亚型之一， 是一种具有多种生物 学活性的单链蛋白。 该蛋白单链由 165个氨基酸残基组成， 如 SEQ ID ΝΟ:1所示， N-端氨基酸为 Cys，有 1个游离 α-ΝΗ2;氨基酸序列之第 23、 31、 49、 70、 83、 112、 121、 131、 133、 134和 164位为 Lys， 均有 1个 游离 ε- Η2ο
干扰素用于临床治疗时， 通常为胃肠外给药。 由于体内药代半衰期 短 （2-4h)、 免疫原性强， 导致给药间隔短、 给药频度高， 并且由于抗体 的生成使疗效显著降低， 因此目前还难以达到理想的临床疗效。 近年来 发展的聚乙二醇 （PEG) 修饰技术为克服上述问题提供了一种可能的选 择。
聚乙二醇是一种惰性、 无毒、 可生物降解的有机多聚物， 在生物技 术和制药领域有重要用途。 PEG修饰技术是通过共价结合将 PEG连接到 活性蛋白上。 蛋白质药物经 PEG化后， 其性状有显著改善， 具体包括药 代半衰期延长、 免疫原性降低、 安全性提高、 疗效增强、 给药频度降低、 药物可溶性和水溶性提高、 蛋白酶酶解抗性增强、 方便药物的控释等。 具体可见 Inada等 ( Inada et al. J.Bioact.and Compatible Polymers， 5， 343， 1990)、 Delgado等 (Delgado et al. Critical Reviews in Therapeutic Drug Carrier Systems, 9， 249, 1992)、 Katre等 (Katre. Advanced Drug Delivery Systems, 10， 91， 1993 )和 Davis等 （美国专利 UP 4179337) 的报道。
美国专利第 4179337号披露， PEG与酶和胰岛素结合后， 蛋白质的 免疫原性降低的同时蛋白质的活性明显下降。 这种效应在 G-CSF
(Satake-Ishikawa et al. Cell Structure and Function, 17， 157-160, 1992)、 IL-2( Katre et al. Proc.Natl.Acad.Sci.USA, 84， 1487， 1987)>TNF-a(Tsutsumi et al. Jpn.J.Cancer Res., 85, 9， 1994)、 IL-6 (Inoue et al. J.Lab.Clin.Med., 124, 529, 1994) 和 CD4-IgG (Chamow et al. Bioconj.Chem., 5， 133， 1994) 中均有发现。
目前已有多种 PEG化治疗性蛋白质药物应用于临床。 1990年， ENZO 公司的第一种用聚乙二醇修饰的药用蛋白-聚乙二醇化牛腺苷脱 氨酶 （Adagen) 被 FDA批准上市， 用于治疗严重的复合免疫缺陷疾病
(pegfamg013102LB, http://www.fda.gov)； 1994年， 另一种用于治疗急 性淋巴细胞白血病的聚乙二醇修饰药用蛋白- PEG 化天冬酰胺酶
( pegasprgase ， Oncaspar ) 也在美 国 上 市 ( 10341 ls50521bl, http://www.fda.gov)； 2000年， Schering-Plough公司的 PEG修饰干扰素 a2b
(PEG IFN-a2b, PEG-Intron)得到 FDA批准上市； Hoffinan-la Roche公 司的另外一种 PEG化的 α干扰素（PEG IFN-a2a， Pegasys)也于 2002年 获得上市批准，二者均用于治疗肝炎（103964s50371bl， pegsche011901LB, http://www.fda.gov) o 2002年， Amgen公司的 PEG修饰人粒细胞集落刺 激因子（PEG-filgrastim ， Neulasta)也获得 FDA批准， 用于治疗迁移性 乳腺癌 （pegfamg013102LB, http://www.fda.gov); Pharmacia公司的 PEG 化人生长因子拮抗剂获 FDA申请受理； Celltech公司的 PEG结合 TNF-a 抗体片断、 Amgen公司的 PEG-TNF受体进入高级临床实验阶段；第一个 PEG—有机分子偶联物- -PEG化喜树碱也已经进入二期临床试验阶段。 2004年， FDA批准了 PEG修饰寡核苷酸（Pegaptanib, Macugen™) o 药 物中的 PEG部分（或 PEG)在体内的代谢过程已相当清楚， 已证实其是 一种良好的、 安全的、 无副作用的药物改性剂。 通常情况下， 聚乙二醇分子通过连接至蛋白质分子的 N端 α-氨基或 分子内部的赖氨酸的 ε -氨基而对蛋白质进行修饰。 已经用于修饰蛋白质 的聚乙二醇通常有三种形式： 直链分子形式 （ΕΡ 0593868)、 U形分支的 支链分子形式（ ΕΡ 0809996 )和 Υ形分支的支链分子形式（ CN1243779C )， 但是尚未报道 Υ形分支的支链 PEG修饰的干扰素 ot2a的制备及在不同的 氨基位点上单位点 PEG修饰的干扰素 ct2a的分离。已有文献报道支链 PEG 修饰的蛋白质的 pH抗性、热稳定性和抗蛋白酶酶解能力均明显强于直链 PEG修饰的蛋白质（ Monfardini et al. Bioconjugate Chem.， 6， 62, 1995)。
能与蛋白质药物结合的 PEG通常需经过衍生，使 PEG两端的一个或 二个端基被化学活化后具有适当的官能团， 该官能团对要结合的药物中 的至少一个官能团具有活性， 能与之形成稳定的共价键。 例如， PEG可 连接到蛋白质肽链中的 Lys残基的 £->¾2上， 或连接到蛋白质肽链 N端 氨基酸残基的 α-ΝΗ2上。欧洲专利 ΕΡ0809996所描述的 IFN-α的 PEG化， 即为 PEG-NHS通过亲核取代结合到 IFN-α的 N-端氨基酸的 α-ΝΗ2或 Lys 的 ε- Η2上。 该专利所述 PEG-NHS 为 U 型分支的 PEG 衍生物 (PEG2-NHS), 其分子式如下：
Figure imgf000004_0001
式中， R和 R，各自独立为低分子量烷基； n和 II'介于 600-1500之间； PEG 平均分子量介于 26KD-66 D。 IFN-α的 PEG2-NHS修饰产物分子式如下：
Figure imgf000004_0002
1个或多个 PEG2-NHS分子结合到 IFN-a的 N-端氨基酸的 a- H2或 Lys的 ε-ΝΗ2上，所得产物为非 PEG化、单位点 PEG化和多位点 PEG化 IFN-α的混合物； 通过已知纯化手段可将单位点 PEG化 IFN-α从反应产 物中分离出来。 由于 IFN-a具有 1个 N-端氨基酸和多个 Lys， 即有多个 PEG2-NHS反应位点， 因此分离得到的单位点 PEG化 IFN-a为多种单位 点 PEG化异构体的混合物。
欧洲专利 EP 0593868采用直链线性 PEG衍生物来修饰干扰素，修饰 产物的分子式如下-
Figure imgf000005_0001
其中， R为低分子量烷基； 、 R2、 R3和 R4为 H或低分子量垸基； m介于 1 到干扰素潜在 PEG修饰位点的数目之间o M； W为 O或 NH; X介于 1-1000， y 和 z介于 0-1000， + +∑介于3-1000； 、 R2、 R3和 至少其一为低分子量 焼基。 Yu-Sen Wang等 ( Yu-Sen Wang et al， Advanced Drug Delivery Reviews, 54： 547-570， 2002. Yu-Sen Wang et al, Biochemistry, 39， 10634-10640， 2000. ) 报道了 12KD 线性单甲氧基 PEG修饰的 rIFN-a2b (Peg-Intron) ， 通过 HPLC-IE分离分析证明其产物为 14个以上单位点修 饰异构体的混合物。 Yu-Sen Wang等采用的线性 PEG分子式如下：
0
0
„ 、 II
H3C-(0CH2 C¾)n -0-C-O-N
该 PEG平均分子量为 12KD( 发明概述
本发明采用的 PEG衍生物为 Y型分支， 是一种新型的分支型 PEG 衍生物， 其结构不同于 U型分支 PEG, 二者最大区别在于： 本发明采用 的 Y型 PEG衍生物的 2条 PEG分支链通过 N原子连接在一起， 而 EP0809996 U型 PEG衍生物 2条 PEG分支链通过 C原子连接在一起。本
Figure imgf000006_0001
其中， P nPb是相同或不同的聚乙二醇； j 为 1-12 的整数； 为 U、 C1-12 经取代或未经取代的烷基、 取代芳基、 芳垸基或杂烷基； Χ ΠΧ2分别独 立地是连接基团，其中 为 (CH2) n， X2为选自于以下组中的基团： (CH2) n、 (CH2) nOCO、 （C¾) n HCO、 (CH2) n CO, 而 n为 1-10的整数； F是选自 于以下组中的端基： 羟基、 羧基、 酯基、 酰氯、 酰肼、 马来酰亚胺、 吡 啶二硫化物， 可以与治疗药物或基体o c=上的氨基、 羟基或巯基反应形成共 价键。
在本发明一个优选的实施方案中， 所述 Y型 PEG衍生物分子如下式 所示： '
Figure imgf000006_0002
其中， R和 R'各自独立为 - 烷基， 优选甲基； m和 m'表征聚合度， 为任何整数； m+m，优选 600到 1500; 为11、 d_12经取代或未经取代的烷 基、 取代芳基、 芳烷基、 或杂垸基； j 为 1-12 的整数； F是选自于以下 组中的端基： 羟基、 羧基、 酯基、 酰氯、 酰肼、 马来酰亚胺、 吡啶二硫 化物， 可以与治疗药物或基体上的氨基、 羟基或巯基反应形成共价键。 优选地， 其中聚乙二醇的总平均分子量为约 10000— 60000道尔顿， 最优 选为约 40000道尔顿。
在本发明一个优选的实施方案中， Y型 PEG衍生物分子一种可能的 结构式如下式 （I)所示：
ROCH2CH2(OCH2CH2 )m-0-GH2CH OCH2 CH2(OCH2 CH2 )m-_o— CH.
Figure imgf000006_0003
其中 R和 R'各自独立为 CVC4烷基，优选甲基； m和 m'表征聚合度， 为任何 整数； m+m，优选 600到 1500; j 为 1-12 的整数； 聚乙二醇的总平均分子 量为约 40000道尔顿。
本发明人采用 Y形分支的支链 PEG (YPEG) 衍生物修饰干扰素 c 2a (IFN-a2a) ， 并经 Q Sepharose FF离子交换层析分离得到单位点修饰的 YPEG-IFN-a2a。 进一步地， 分离的单位点修饰的 YPEG-IFN-a2a经 SP Sepharose FF层析拆分进一步得到 IFN-oc2a在相应于 SEQ ID NO: 1的 134位 赖氨酸的侧链 ε 氨基为主的连接 YPEG的 YPEG-IFN-a2a， 称为 YPEG-IFN-a2a ( 134 ) 。 经测定， YPEG-IFN-a2a ( 134) 的体外比活性显 著高于在其他氨基酸位点连接 YPEG的 YPEG-IFN-a2a， 并且血清药代半 衰期显著长于非修饰的 IFN-a2a。
因此，本发明提供了一种单位点修饰的聚乙二醇化干扰素 a2a，其具 有如下结构：
Figure imgf000007_0001
其中， Ρ^Π Pb是相同或不同的聚乙二醇； j为 1— 12的整数； 为 H、 经取代或未经取代的烷基、 取代芳基、 芳垸基、 或杂垸基； Χ^Π Χ2 分别独立地是连接基团，其中 为 (C¾) n， X2为选自于以下组中的基团： (CH2) n、 （CH2) n OCO、 （CH2) n HCO、 （C¾) n CO， 而 n为 1— 10的整 数。
在本发明一个优选的实施方案中， 本发明的聚乙二醇化干扰素 c 2a 具有如下结构式：
Figure imgf000007_0002
( II )
其中， R和 R'各自独立为 - 焼基， 优选甲基； j 为 1-12的整数； m 和 m'表征聚合度， 为任何整数， 可为相同或不同的整数； m+m'优选 600 到 1500。该结构中， Y分支 PEG通过单位点结合连接到干扰素 oi2a分子 上。式 II之 YPEG-IFN- a 2a的分子量主要取决于聚合度 m和 m'。 m+m' 优 选 600到 1500， 对应 YPEG的平均分子量为约 26000道尔顿到约 66000 道尔顿； m+m'优选 795到 1030，对应 YPEG平均分子量为 35000道尔顿 到 45000道尔顿； m+m'特别优选 885到 1030，对应 YPEG平均分子量为 39000道尔顿到 45000道尔顿； m+m'最优选 910， 对应 YPEG平均分子 量为 40000道尔顿。 m和 m'的比值范围可以为 0.5到 1.5，优选 0.8到 1.2。
在一个优选的实施方案中， 本发明的聚乙二醇化干扰素 a2a中聚乙二 醇与干扰素 a2a连接的酰胺键位于干扰素 a2a中相应于 SEQ ID NO: 1的 23、 31、 49、 70、 83、 112、 121、 131、 133、 134或 164位赖氨酸的侧链 ε氨 基或 Ν端氨基酸的 a氨基。
在一个更优选的实施方案中， 本发明的聚乙二醇化干扰素 a2a中聚乙 二醇与干扰素 a2a连接的酰胺键主要位于干扰素 c 2a中相应于 SEQ ID NO: 1的 134位赖氨酸的侧链 ε氨基。
任选地， 本发明的干扰素 a2a可以为从天然来源提取的或通过重组生 物技术获得的干扰素 a2a。 优选地， 所述干扰素 (x2a为从天然来源提取的 或通过重组生物技术获得的具有 SEQ ID ΝΟ: 1所示序列的人干扰素 ot2a ( WFN-a2a ) 。 更优选地， 所述人干扰素 cc2a是重组人干扰素 o 2a (rhIFN-a2a) 。 rhIFN-a2a可以是人工合成的， 也可以是原核系统如大肠 杆菌 E.col 表达的，也可以是真核酵母系统如毕赤酵母 Pichia pastoris) 表达的， 也可以是其它昆虫细胞系统或哺乳细胞系统如 CHO表达的。 制 备天然或重组 IFN-a2a的方法以及 IFN-a2a和其 YPEG修饰产物的活性检 测方法为本领域现有技术。
本发明所述 YPEG-IFN- a 2a与 IFN-a2a的临床用途相同， 均适用于 肿瘤和抗病毒感染治疗， 如肝炎、 毛细胞白血病、 细胞介导淋巴细胞溶 解、 Kapasi's肉瘤等。 临床使用时， 相对 IFN-ot2a， 本发明的 YPEG-IFN- α 2a在稳定性、 溶解度、 血清药代半衰期和临床疗效等方面均有明显改 进。 在给药方式中， 本发明的 YPEG-IFN- a 2a可以以组合物的形式给予 患者， 所述组合物中包含药物学有效剂量的 YPEG-IFN- a 2a和药物学可 接受的载体或赋形剂。 因此， 本发明另一方面提供了一种组合物， 其包 含药物学有效剂量的本发明的聚乙二醇化干扰素 a2a和药物学可接受的 载体或赋形剂。 优选地， 所述组合物包含甘露醇、 氨基酸、 氯化钠和醋 酸钠， 其中氨基酸优选天冬氨酸、 天冬酰胺和甘氨酸。
在另一方面， 本发明还提供了本发明的聚乙二醇化干扰素 a2a或包含 本发明的聚乙二醇化干扰素 o a的组合物在制备用于治疗需要用干扰素 (x2a治疗的疾病的药物中的应用。 优选地， 所述需要用干扰素 a2a治疗的 疾病选自病毒性感染如乙型肝炎、 丙型肝炎、 丁型肝炎、 尖锐湿疣等， 肿瘤如毛状细胞白血病、 慢性髓细胞性白血病、 低度恶性非何杰金氏白 血病、细胞介导淋巴细胞溶解、卡波氏（Kapasi's)肉瘤、 多发性骨髓瘤、 恶性黑色素瘤、 皮肤 T-细胞淋巴瘤、 喉乳头状瘤病、 复发性或转移性肾 细胞癌， 炎症反应异常性疾病如多发性硬化、 关节炎、 哮喘、 胆囊纤维 化和间质性肺病以及与骨髓增生性疾病相关的血小板增多。
为了获得 YPEG修饰的 IFN-a2a， 在本发明的一个实施方案中， 首先 将 YPEG经活化的衍生物、 如聚乙二醇琥珀酰亚胺酯（YPEG-NHS) ， 通 过亲核取代反应， 将 PEG部分共价结合在蛋白质的氨基 （-NH2) 上， 包 括蛋白质 N端的 a-氨基和赖氨酸残基的 ε-氨基。 IFN-a2a与 YPEG反应生成 YPEG-IFN-a2a的反应方程式如下：
Figure imgf000009_0001
i
Figure imgf000009_0002
反应条件温和， pH范围 4.5-9.5， 温度 0-25°C， 需搅拌或保持其它方 式的混匀。具体条件例如参见实施例。各种分子量的 YPEG均可通过本方 法连接到 IFN-a2a上， 反应产物包括单位点 （YPEG-IFN-a2a) 、 双位点 (YPEG2-IFN- 2a)和多位点 （YPEGn-IFN-a2a) 修饰产物， 可通过控制 反应条件使产物以单位点取代产物为主。
接下来利用阳离子交换层析、 阴离子交换层析和排阻层析等方法从 各种 YPEG修饰的 IFN-a2a混和物中分离单位点修饰的 YPEG-IFN-a2a， 并 对单位点修饰的 YPEG-IFN-(x2a进行进一步拆分得到在不同位点连接 YPEG的 YPEG-IFN-a2a。 常用方法如阳离子交换层析、 阴离子交换层析、 疏水层析和排阻层析等。 可通过本领域已知方法来进行性质分析， 如采 用质谱、 聚丙烯酰胺凝胶电泳、 高效液相色谱排阻层析等分析产物的分 子量， 从而将单位点修饰产物与双位点、 多位点修饰产物和未修饰底物 IFN-cc2a区分开， 并进而采用上述纯化方法将单位点修饰产物中的各种不 同位点异构体拆分开。 各种修饰产物的体外生物学活性检测根据干扰素 的已知活性检测方法进行，如细胞病变抑制法。对于 PEG单位点修饰 IFN， 不同的修饰位点异构体由于 PEG成分对 IFN活性结构域的保持的影响不 同，导致修饰位点异构体间的生物学活性有较大区别；从总体上说， PEG 修饰后， IFN的体外生物学活性均明显下降。而本发明对离子交换层析结 果中获得的 3个峰的分离物进行体外比活性测定， 结果显示第 3峰分离物 (SP2) 与其他峰的分离物以及 PEGASYS (瑞士巴塞尔豪夫迈，罗氏有限 公司）相比具有显著高的比活性， 并且比未修饰的 IFN-a2a具有显著更长 的血清药代半衰期。
在进一步的实施方案中， 分离获得 SP2的 Y分支 PEG连接的肽段， 利 用 Edman降解的方法对其进行氨基酸序列测定， 结果显示 SP2的主要成分 为 YPEG-IFN-a2a ( 134) 。
因此， 在另一方面， 本发明还提供了制备并纯化 YPEG-IFN-a2a的方 法， 包括：
(a) 在碱性条件下， 优选 pH值为 9.0， 将具有下式 （I)所示的 Y形分 支结构的 PEG与干扰素 (x2a溶液反应得到 PEG化干扰素 oc2a;
Figure imgf000010_0001
其中 R和 R，各自独立为 CrC4烷基，优选甲基; j 为 1-12 的整数; m和 m'表征聚合度， 为任何整数； m+m'优选 600到 1500;
(b)用阴离子交换树脂， 优选 Q Sepharose FF, 捕获步骤（a)获得的 反应产物， 并用阴离子梯度洗脱， 优选使用氯离子梯度， 得到修饰后 产物；
(c) 用阳离子交换树脂， 优选 SP Sepharose FF， 对步骤（b)捕获的 反应产物进行阳离子梯度洗脱， 优选使用钠离子梯度， 分别收集各个 峰；
(d)测量各个峰的活性， 取活性最高的峰对应的反应产物。 附图简述
图 1 : IFN-a2a三批次 Y-PEG (40KD)修饰反应 SDS-PAGE结果。 其中分离胶浓度为 12%，考马斯亮蓝 R-250染色。泳道 1-2: 20060804; 泳道 3-4: 20060807-1； 泳道 5-6: 20060807-2。 图 2: YPEG-IFN-a2a SP Sepharose FF纯化拆分修饰位点异构体图 谱。
图 3： YPEG-IFN-a2a SP Sepharose FF纯化样品 SDS-PAGE ( 12 % ) 电泳银染结果。 泳道 1 : 分子量参照； 泳道 2、 4、 6、 8： 空白； 泳道 3、 5、 7、 9： 分别对应于洗脱图谱之 1-4峰。
图 4: YPEG-IFN-a2a修饰位点异构体表观分子量的 SDS-PAGE电 泳银染检测结果。 泳道 1 : 分子量参照（GE Lifescience)； 泳道 2 : YPEG-IFN-a2a SP3 , 0Λμg^, 泳道 3 : YPEG-IFN-a2a SP2, 0Aμg^, 泳道 4： YPEG-IFN-a2a SP1 , 0 g。
图 5 : YPEG-IFN- 2a SP Sepharose FF纯化拆分样 MALDI-TOF MS 法分子量测定结果。 YPEG-IFN-a2a SP1对应于图 4之泳道 4样品， YPEG-IFN-a2a SP2对应于图 4之泳道 3样品， YPEG-IFN-a2a SP3对应于 图 4之泳道 2样品。
图 6: YPEG-NHS (40 D) MALDI-TOF MS法分子量测定结果。 图 7: 食蟹猴单次 s.c 30 g.kg-1 YPEG-rhIFN-a2a SP2后血清药物 浓度及 2'-5，A浓度变化情况比较
图 8: YPEG-IFN- 2a SP2胰酶酶解 Oh样 HPLC-RP C18胰酶肽图检 测结果。 YPEG-IFN- ct 2a SP2保留时间为 62.105min, 71.882min洗脱峰 为熔剂背景峰， 2-3min处洗脱峰为胰酶峰。
图 9: YPEG-IFN- a 2a SP2胰酶酶解 48h样 HPLC-RP C18胰酶肽图检 测结果。 捡出 71.581min溶剂峰， 该峰对应于酶解 Oh样品肽图谱之 71.882min溶剂峰； 在 59.5min-62.5min间无底物蛋白峰（62.105min)检 出， 说明样品基本酶解完全。
图 10 : YPEG-IFN-a2a SP2胰酶完全酶切样品 YPEG修饰肽段 Sephacryl S-100 HR分离图谱。 发明详细描述
本发明通过下述实施例进一步阐明， 但任何实施例或其组合不应 当理解为对本发明的范围或实施方式的限定。 本发明的范围由所附权 利要求书限定， 结合本说明书和本领域一般常识， 本领域普通技术人 员可以清楚地明白权利要求书所限定的范围。
实施例 1
Y型分支 PEG修饰重组人干扰素 a2a的制备
( 1 ) Y型分支 PEG修饰重组人干扰素 a2a的少量制备 取 166.3mg YPEG (式 I，平均分子量 40KD，等臂，批号 RD010P041 ) (北京键凯科技有限公司）溶解于 lml 2mM HCl (广东光华化学厂有限 公司），加入 40mg IFN-a2a (厦门特宝生物工程股份有限公司）和 50mM 硼酸一硼砂缓冲液 （pH9.0) (中国医药集团上海化学试剂公司） 使反 应总体积为 10ml。 该反应体系中， IFN-ct2a反应终浓度为 4mg/ml , IFN-oc2a与 YPEG反应摩尔比为 1 : 2。 搅拌条件下 0-25°C温浴 2h， 生成 PEG化的 IFN-a2a， 加入冰乙酸 （汕头市西陇化工厂） 使 pH<4.0以终止 反应， 取样进行 SDS-PAGE电泳。反应体系用水稀释 50倍， 0.2μπι过滤， 4°C放置备用。
使用 Q Sepharose FF层析分离残余 PEG和 PEG水解物、多位点 YPEG 化的 IFN-a2a、 单位点 YPEG化的 IFN-a2a和未修饰的 IFN-a2a。 Q Sepharose FF ( GE Healthcare ) 层析柱 （<D12mmx90mm， lCV=10ml) 用 20mM硼酸 /硼砂缓冲液（pH9.0) -1M NaCl (BBI)再生 3柱体积（CV)， 20mM硼酸 /硼砂缓冲液（pH9.0)平衡 5CV。设定紫外检测波长为 280nm。 4°C放置备用样品全部上样； 上样结束后， 20mM硼酸 /硼砂缓冲液
(pH9.0) 平衡 3CV; 换 20mM硼酸 /硼砂缓冲液 （pH9.0) -12mM NaCl 洗脱至第一个峰完全洗出， 该峰为残留的 PEG; 换 20mM硼酸 /硼砂缓 冲液（pH9.0) -60mMNaCl洗脱， 收集洗脱峰样品即为以单位点 PEG化 产物 YPEG-IFN-a2a为主的修饰产物； 再换 20mM 硼酸 /硼砂缓冲液
(pH9.0) -500mM NaCl洗脱， 洗脱峰为未修饰的 IFN-a2a。
目的产物以单位点 PEG化的 YPEG-IFN-oc2a为主， 得率 20-40%。
(2) Y型分支 PEG修饰重组人干扰素 cx2a的大规模制备
取 4989.6mg YPEG (式 I，平均分子量 40KD，等臂，批号 RD010P041 )
(北京键凯科技有限公司）溶解于 25ml 2mM HC1,加入 1200mg IFN-a2a 和 50mM硼酸一硼砂缓冲液（pH9.0)使反应总体积为 200ml。 该反应体 系中， IFN-a2a反应终浓度为 6mg/ml， IFN-a2a与 YPEG反应摩尔比为 1 : 2。 搅拌条件下 0-25Ό温浴 2h， 加入冰乙酸使 pH<4.0以终止反应， 取样 进行 SDS-PAGE电泳。反¾体系用水稀释 50倍， 0.2μηι过滤， 4°C放置备 用。
使用 Q Sepharose FF层析分离残余 PEG和 PEG水解物、多位点 YPEG 化的 IFN-a2a、 单位点 YPEG化的 IFN-a2a和未修饰的 IFN-a2a。 Q Sepharose FF (GE Healthcare)层析柱（i>38mmx265mm， lCV=300ml) 用 20mM硼酸 /硼砂缓冲液 （ρΗ9·0) -1Μ NaCl再生 3CV， 20mM硼酸 /硼 砂缓冲液（pH9.0)平衡 5CV。 设定紫外检测波长为 280nm。 4°C放置备 用样品全部上样； 上样结束后， 20mM硼酸 /硼砂缓冲液（pH9.0) 平衡 3CV; 换 20mM硼酸 /硼砂缓冲液 （pH9.0) -12mM NaCl洗脱至第一个 峰完全洗出， 该峰为残留 PEG; 换 20mM硼酸 /硼砂缓冲液 （pH9.0) -60mM NaCl洗脱，收集洗脱峰样品即为以 YPEG-IFN-a2a为主的修饰产 物； 换 20mM硼酸 /硼砂缓冲液（pH9.0) -500mM NaCl洗脱， 洗脱峰为 未修饰的 IFN-a2a。
目的产物以单位点 PEG化的 YPEG-IFN-a2a为主， 得率 35-50%。 图 1示出了三批次 YPEG 0KD)修饰 IFN-a2a反应的 SDS-PAGE 结果。 从图 1 中的 SDS-PAGE 电泳结果可见， 在本反应条件下， rhIFN-a2a的 PEG修饰率介于 35-50%之间， 修饰率稳定； 修饰产物以 单位点修饰产物为主， 有多位点修饰产物。
实施例 2
YPEG-IFN- 2a SP Sepharose FF拆分
YPEG-IFN-a2a Q Sepharose FF捕获样用 20%乙酸调 pH值至 5.0， 用 5mM NaAc/HAc(pH5.0)(汕头市西陇化工厂）稀释 15倍；按 0.5mg/ml 载量上样 SP Sepharose FF 100ml (GE Healthcare) ( 18mmx394mm)， 5mM NaAc/HAc(pH5.0)平衡 3CV, 0%-30% 的 5mM NaAc/HAc-70mM NaCl (pH5.0)梯度洗脱 2.5CV, 30%-100%的5111]^ NaAc/HAc-70mM NaCKpH5.0)梯度洗脱 50CV。 SP Sepharose FF 100ml将 YPEG-IFN-a2a 拆分成 4个洗脱峰， 按峰收集样品， 分别取样进行 SDS-PAGE电泳， 采用银染显色。 对照电泳结果确定， SP Sepharose FF拆分 1峰主要为 YPEG多位点修饰产物 （YPEGn-IFN-a2a); SP Sepharose FF拆分 2峰 以单位点修饰产物（YPEG-IFN-a2a)为主，含有部分多位点修饰产物； SP Sepharose FF拆分 3峰、 4峰均为单位点修饰产物。 SP Sepharose FF 拆分 2-4峰为不同 YPEG修饰位点的单位点修饰产物异构体， 分别命 名为 YPEG-IFN-a2a SP1、 YPEG-IFN-a2a SP2和 YPEG-IFN-a2a SP3。 拆分图谱及电泳银染结果分别示于图 2和图 3。
YPEG-IFN-a2a SP1-3各样品分别补加氯化钠、 醋酸钠、 甘露醇、 门冬氨酸， 0.22μηι过滤除菌， 4°C放置备用。
实施例 3
YPEG-IFN-a2a修饰位点异构体的性质分析
( 1 ) 蛋白质浓度检测 凯氏定氮
YPEG-IFN-a2a的修饰位点异构体采用凯氏定氮法检测蛋白质浓度。
(2) 蛋白质表观分子量检测 采用 SDS-PAGE电泳检测 YPEG-IFN-a2a的修饰位点异构体的表观分 子量。 实验方法按 Laemmli (Nature 227:680, 1970)的报道进行， 凝胶浓 度 7.5%， 银染显色。 YPEG-IFN-a2a的修饰位点异构体的表观分子量基本 一致， 大小约 120KD (图 4) 。
(3) MALDI-TOF MS法检测分子量
采用德国 BRU ER公司 autoflex TOF/TOF质谱仪， MALDI-TOF MS 法测定 YPEG-rHuIFN-o^a的修饰位点异构体的分子量。 基质采用芥子酸 (Sinapinic acid, SA， CnH1205， MW 224.22； 批号 2006 236870 002,德 国 BRUKER公司）， 蛋白分子量标准品采用 BRUKER公司之 Protein Calibration Standard II ( Part No.207234 )， 分析软件为 flexAnalysis Ver.3.0.54.0.。 YPEG-IFN-a2a的修饰位点异构体的 MS分子量一致， 大小 约 59000道尔顿 （图 5) 。
(4) 蛋白质纯度检测
YPEG-IFN-a2a 的修饰位点异构体的纯度检测采用 HPLC-SE法。 HPLC分析柱采用 TSK G4000 SWXL ( 7.8mmx300mm) (TOSOH), 样品 上样体积 20μ1、 约 10μδ蛋白， 流动相为 0.1M PBNa-O.lM NaCl (pH7)， 洗脱流速 0.8ml/min， 检测波长 280nm。 YPEG-IFN-a2a SP2检测结果为 单一主峰， 纯度大于 99%。
(5) 内毒素含量检测
采用鲎试剂法 （《中华人民共和国药典》 2005年版三部附录 XC)测 定 YPEG-IFN-a2a的内毒素含量， 各样品的检测结果均 <5.0EU/mg。
(6) YPEG-IFN-a2a SP2的动物体内活性及药物代谢动力学研究 ① YPEG-IFN-a2a SP2的动物体内活性研究
在 IFN作用机制中， 部分是由于促使机体产生 2'-5'AS (2'5'-寡腺苷 合成酶）， 从而进一步发挥其抗病毒等作用。用1251作为示踪， 通过检测 体内 2'-5'AS活性， 反映 IFN的药效学指标。 2'-5'AS在 poly(I)poly(C) 琼脂存在时能够催化 ATP生成 2'-5，A (2'-5'AS的活性由产生的 2，-5'A (2'5'-寡腺苷）浓度表示） 。 首先， 样品中的 2'-5'AS被 poly(I)poly(C) 琼脂糖吸附并激活后， 催化底物 ATP生成 2'-5，A。 加入含 1251标记的 2，-5，A、 2，-5，A抗血清和二抗的混合物， 孵育， 离心分离混合物， 去除上 清， 用 Y计数仪测定沉淀物中的放射性， 计算与最初加入的 1251标记 2'-5'A的结合率，并用四参数 Logistic回归作出标准曲线，计算未知样品 中 2，-5，AS诱导产生的 2，-5，A浓度。
表 1及图 7是 2'-5，A法测得的食蟹猴（食蟹猴 15只， 雌 7雄 8。 军 事医学科学院实验动物中心（合格证编号 SCXK- (军） 2002-001 )， 体重 2.5〜3.7kg。分笼喂养， 饲以标准猴饲料， 自由饮水）单次皮下注射（sx) 30μ^-1 YPEG-IFN- a 2a SP2后血清中 2，-5，A的浓度，从图 5中可以看 出， 给药后血清中 2，-5，AS的活性明显升高， 血清中 2，-5，A的达峰时间 比 YPEG-IFN-ct2a SP2的达峰时间后延。平均达峰时间为 24±18.33 h, 达 峰浓度为 K S ltSSjg Pmol-dl o
表 1.食蟹猴 s.c 30 g'kg-l YPEG-rhIFN-a2a SP2后血清内 2，-5，A浓度随 时间变化情况 （Pmo dL— 4
time(h) - 食蟹猴编号 Mean sd
1# 2# 3#
0 16.08 19.01 42.91 26.00 土 14.72
1 39.04 ― 16.19 27.61 士 16.16
2 48.21 16.90 20.20 28.44 土 17.21
4 55.22 36.09 74.16 55.15 土 19.04
8 32.04 59.69 99.52 63.75 + 33.92
10 13.52 41.21 51.85 35.53 士 19.79
12 37.35 53.32 1 19.76 70.14 + 43.71
24 58.29 167.22 87.42 104.31 士 56.39
48 77.50 160.67 71.41 103.19 + 49.87
72 62.88 165.97 58.52 95.79 士 60.82
96 73.53 119.79 90.85 94.72 士 23.37
168 45.41 135.26 68.92 83.20 + 46.60
240 48.14 102.61 73.97 74.90 士 27.25
312 93.23 21.69 62.84 59.26 土 35.90
② YPEG-IFN-a2a SP2与 rhIFN-a2a在食蟹猴体内的药物代谢动力学研究 食蟹猴单次皮下注射 YPEG-IFN-a2a SP2 7.5、 30和 120 g'kg— 给 药组于药前、 药后 1、 2、 4、 8、 10、 12、 24、 48、 72、 96、 168、 240和 312h 取注药对侧后肢静脉血 lmL ; rhIFN-a2a 单次皮下给药组 (7.5 g'kg_1) 于药前、 药后 0.5、 1、 2、 3、 4、 5、 6、 8、 24h取血 lmL, 血样于 4°C放置 30 min后， 2000转低温离心 10 min,立即分离血清于 -20 °C保存待分析。
应用定量双夹心饼干免疫技术。将一个对重组人干扰素 α特异的单抗 预先包被在微孔板上。 将标准品和样品吸入至孔内， 其中的 rhIFN-a2a 或 YPEG-IFN-a2a SP2将与固化抗体结合。洗掉所有未结合的物质后，将 抗人干扰素 α的 IgG (二抗）加至孔内， 反应完全后， 洗板并将辣根过氧 化物酶 （HRP)加至孔内， 在洗掉所有未结合的酶试剂后， 每孔内加入 HRP 的底物溶液而产生的颜色与最初一步结合的 IFN-a2a 或 YPEG-IFN-cc2a SP2量成比例。 终止反应并测定颜色强度。 吸光度 OD值 越高，则样品中 IFN-a2a或 YPEG-IFN-a2a SP2浓度越高。分别作两种样 品的标准曲线以测定各血样的血清药物浓度。
按照试剂盒（美国 Biomedical公司， Lot# 3271 )说明书的要求操作。 每孔加入 100 μΐ标准品或血清样品， 并以平板混匀器轻轻混匀， 根据未 知样品预期浓度， 用稀释液将样品稀释至标准曲线的浓度范围。 各板都 设置 rhIFN-c 2a或 YPEG-IFN-a2a SP2标准曲线，用以计算该板未知样品 浓度。 室温孵育 lh。 以洗板液洗板 1次， 每孔加入 100 μΐ二抗， 继续室 温反应 lh。洗涤 3次， 每孔加入 100 μΙ ΗΚΡ结合物， 室温反应 lh。洗板 4次， 每孔加入 100 μΐ ΤΜΒ底物， 室温避光放置 15min。 每孔加 ΙΟΟμΙ 终止液， 轻轻混匀， 终止反应。 在 5min内用酶标仪于 450 nm波长读取 光吸收 OD值。 测定各样品浓度。
食蟹猴单次皮下注射 YPEG-rhIFN-a2a 7.5、 30和 120 g'kg'1后， 末 端半衰期分别是 35.81 ±2.50、 31.38± 11.84和 36.77±2.24 h; 食蟹猴单 次皮下注射 rhIFN-cc2a 7.5 g'kg 后， 末端半衰期为 3.02 ± 0.55h。 rhIFN-a2a经 PEG化后， 末端半衰期显著延长。
(7) 体外比活性检测
采用细胞病变抑制法检测 YPEG-IFN-cc2a的各修饰位点异构体的体 外生物学活性。 按照 《中华人民共和国药典》 2005 版三部附录 XC《干 扰素效价测定法》中所述方法，根据干扰素可以保护人羊膜细胞（WISH) 免受水泡性口炎病毒 （VSV) 破坏的作用， 用结晶紫对存活的 WISH细 胞染色， 于 570nm处检测吸光度， 绘制干扰素对 WISH细胞的保护效应 曲线， 以此测定干扰素的体外生物学活性。 各样品体外生物学活性检测 结果见表 2， 每个样品均同步检测 3个平行样。 YPEG修饰后， 单位点修 饰产物的各修饰位点异构体中 SP2样品的体外比活性最高，比 SP1和 SP3 高 1-2倍， 比未拆分样以及 PEGASYS (瑞士巴塞尔豪夫迈 ·罗氏有限公司 制造， 上海罗氏有限公司分装， 产品批号 B1016, 分装批号 SH0020)亦 高 1-2倍。
表 2. YPEG-IFN-a2a的各修饰位点异构体的体外生物学活性检测结果 （三次测定)
Figure imgf000016_0001
( 8 ) YPEG-IFN- 2a SP2修饰位点解析 YPEG-IFN-a2a SP2用 5K超滤器（Millipore, 聚醚砜材质） 将溶剂 体系超滤替换成 50mM H4HC03 (pH8.0 ) , 紫外扫描确定蛋白浓度 4.02mg/ml。 TPCK TrypsinC Promega)用商品附带的溶解液配制成 0.5 μβ/μ1。 按表 3加样：
表 3. YPEG-IFN- a 2a SP2胰蛋白酶酶解反应组分
反应组分 体积
50mM H4HCO3, pH8.0 7.15ml
PEG-IFN-a2a SP2 (4.02mg/ml) 1.25ml
Trypsin (0.5μ^μ1) 0.2ml
反应总体积 8.6ml
反应体系 37°C水浴 48h, 加入 1.52ml 20%乙酸终止反应。 取小样做 HPLC-RP C18肽图检测，检测用仪器为 Waters HPLC系统，主机型号 600， 2487双波长检测器， 数据处理软件 Empower 2。 HPLC分析柱为 Jupiter C18, 粒径 5μηι, 孔径 30θΑ， O4.6xl50mm, 美国 Phenomenex公司出产。 流动 A相为 0.1%TFA/H2O， 流动 B相为 0.1%TFA/90%ACN/H2O; 检测 流速 lml/min， 检测波长 214nm。 洗脱梯度见表 4， 结果见图 8-9。
表 4. YPEG-IFN- a 2a SP 2 HPLC-RP C18胰酶肽图检测洗脱梯度
Figure imgf000017_0001
根据检测结果确定样品已基本酶解完全。 反应终止产物进行 DTT还 原处理。 Sephacryl S-lOOHR (<D18x255mm， lCV=64ml; GE Healthcare) 预先用 20mM PBNa-400mM aCl (pH7)平衡 3CV，将 YPEG-IFN-a2a SP 2 之 TPCK Trypsin 完全酶解样用静水压按 3%CV 上样， 20mM PBNa-400mM NaCl (pH7)洗脱， 检测波长 280nm。 收集第一个洗脱峰 样品 （样品编号： YPEG-IFN-a2a S100-l， 图 10)， 5K超滤将溶剂体系替 换为 5mM PBNa (pH7)， 真空冷冻千燥。 冻干样采用 Edman降解法测定 N端氨基酸序列， 样品的 N端 7个氨基酸序列为 XYSPXAW (表 5 )， 其 中 X指 Edman降解法无法检出的 α氨基酸的半胱氨酸（Cys)、非 α氨基 酸和其它修饰氨基酸。 根据与 SEQ ID ΝΟ:1 所示序列进行比对， 确定 YPEG-IFN- 2a SP2主要为 Lysl34的 YPEG修饰产物。
表 5. YPEG-IFN-a2a SI 00-1 N端氨基酸序列测定结果
Figure imgf000018_0001
注： X指 Edman降解法无法检出的 a氨基酸的半胱氨酸 （Cys)、 非 a氨基酸和其 它修饰氨基酸。

Claims

权利要求
1、 一种聚乙二醇化干扰素 a2a， 其具有如下结构：
Figure imgf000019_0001
其中-
P^n Pb是相同或不同的聚乙二醇；
j为 1— 12的整数；
Ri为 H、 .12经取代或未经取代的浣基、 取代芳基、 芳烷基、 或杂 焼基；
和 X2分别独立地是连接基团， 其中 为^！！ ， X2为选自于以 下组中的基团： （CH2)n、 （CH2)nOCO、 （CH2)nNHCO、 (CH2)nCO, 而 n为 1一 10的整数。
2、如权利要求 1所述的聚乙二醇化干扰素 (x2a，其具有如下结构式：
Figure imgf000019_0002
其中 R和 R，各自独立为 CrC4烷基， 优选甲基； j 为 1-12 的整数； m和 m，表征聚合度， 为任何整数； m+m，优选 600到 1500， 优选聚乙二 醇的总平均分子量为约 10000— 60000道尔顿，最优选为约 40000道尔顿。
3、权利要求 1或 2所述的聚乙二醇化干扰素 (x2a， 其中聚乙二醇与 干扰素 (x2a连接的酰胺键位于干扰素 a2a中相应于 SEQ ID ΝΟ:1的 23、 31、 49、 70、 83 s 112、 121、 131、 133、 134或 164位赖氨酸的侧链 ε氨 基或 Ν端氨基酸的 a氨基， 优选位于干扰素 a2a中相应于 SEQ ID ΝΟ:1 的 134位赖氨酸的侧链 ε氨基。
4、 权利要求 1-3任一项所述的聚乙二醇化干扰素 ct2a， 其中所述干 扰素 a2a为从天然来源提取的或通过重组生物技术获得的干扰素 a2a，优 选具有 SEQ ID ΝΟ:1所示序列， 最优选为重组人千扰素 a2a。
5、权利要求 4的聚乙二醇化干扰素 a2a，其中所述重组人干扰素 a2a 是人工合成的或由选自如下一组的表达系统表达： 原核系统如大肠杆菌、 真核酵母系统如毕赤酵母、昆虫细胞系统和哺乳动物细胞系统如 CHO细 胞。
6、一种组合物，其包含药物学有效剂量的权利要求 1一 5任一项的聚 乙二醇化干扰素 a2a和药物学可接受的载体或赋形剂。
7、权利要求 6的组合物，其包含甘露醇、氨基酸、氯化钠和醋酸钠， 其中氨基酸优选天冬氨酸、 天冬酰胺和甘氨酸。
8、权利要求 1一 5任一项的聚乙二醇化干扰素 cx2a或权利要求 6或 7 的组合物在制备用于治疗需要用干扰素 a2a治疗的疾病的药物中的应用， 所述优选选自病毒性感染如乙型肝炎、 丙型肝炎、 丁型肝炎、 尖锐湿疣， 肿瘤如毛状细胞白血病、 慢性髓细胞性白血病、 低度恶性非何杰金氏白 血病、细胞介导淋巴细胞溶解、 Kapasi's肉瘤、 多发性骨髓瘤、恶性黑色 素瘤、 皮肤 T-细胞淋巴瘤、 喉乳头状瘤病、 复发性或转移性肾细胞癌， 炎症反应异常性疾病如多发性硬化、 关节炎、 哮喘、 胆囊纤维化和间质 性肺病以及与骨髓增生性疾病相关的血小板增多。
9、 一种制备和纯化权利要求 1-5任一项的聚乙二醇化千扰素 a2a的 方法， 其包括如下步骤：
(a)在碱性条件下，优选 pH值为 9.0，将具有下式 Y形分支结构的 PEG 与干扰素 a2a溶液反应得到 PEG化干扰素 a2a;
Figure imgf000020_0001
其中 R和 R，各自独立为 CrC4烷基， 优选甲基； j 为 1-12 的整数； m和 m'表征聚合度， 为任何整数； m+m'优选 600到 1500;
(b)用阴离子交换树脂捕获步骤 （a) 获得的反应产物， 并用阴离子梯 度洗脱得到修饰后产物， 优选阴离子交换树脂为 Q Sepharose FF及阴离 子梯度为氯离子梯度； ( c ) 用阳离子交换树脂对步骤 （b ) 捕获的反应产物进行阳离子梯度洗 脱， 分别收集各个峰， 优选阳离子交换树脂为 SP Sepharose FF及阳离子 梯度为钠离子梯度；
( d) 测量各个峰的活性， 获得活性最高的峰所对应的反应产物。
10、 权利要求 9的方法， 其中所述 PEG分子量为 40KD, 优选为等 臂 YPEG, 更优选 IFN-cc2a与 YPEG的反应摩尔比为 1 : 2。
11、 一种治疗患有需要用干扰素 a2a治疗的疾病的患者的方法， 所 述方法包括给予所述患者治疗有效量的权利要求 1-6 任一项的聚乙二醇 化干扰素 a2a或权利要求 7或 8的组合物， 其中所述需要用干扰素 cc2a 治疗的疾病优选选自病毒性感染如乙型肝炎、 丙型肝炎、 丁型肝炎、 尖 锐湿疣， 肿瘤如毛状细胞白血病、 慢性髓细胞性白血病、 低度恶性非何 杰金氏白血病、 细胞介导淋巴细胞溶解、 Kapasi's肉瘤、 多发性骨髓瘤、 恶性黑色素瘤、 皮肤 T-细胞淋巴瘤、 喉乳头状瘤病、 复发性或转移性肾 细胞癌以及与骨髓增生性疾病相关的血小板增多， 炎症反应异常性疾病 如多发性硬化、 关节炎、 哮喘、 胆囊纤维化和间质性肺病。
PCT/CN2007/002643 2007-09-04 2007-09-04 聚乙二醇修饰的干扰素α2a及其制备方法和应用 WO2009030065A1 (zh)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8143214B2 (en) 2007-08-16 2012-03-27 Pharmaessentia Corp. Protein-polymer conjugates
US8597635B2 (en) 2007-09-04 2013-12-03 Biosteed Gene Expression Tech. Co., Ltd. Interferon alpha-2B modified by polyethylene glycol and methods of preparation thereof
US8597634B2 (en) 2007-09-04 2013-12-03 Biosteed Gene Expression Tech. Co., Ltd. Interferon alpha-2a modified by polyethylene glycol and methods of preparation thereof
US11559567B2 (en) 2014-11-06 2023-01-24 Pharmaessentia Corporation Dosage regimen for pegylated interferon
CN116120424A (zh) * 2022-06-06 2023-05-16 江苏靶标生物医药研究所有限公司 一种干扰素α2b可溶性重组表达和分离纯化方法及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112014005091A2 (pt) * 2011-09-05 2017-06-13 Beijing Hanmi Pharmaceutical Co Ltd composição farmacêutica para o tratamento do câncer compreendendo conjugado de interferon alfa

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
EP0593868A1 (en) 1992-08-26 1994-04-27 F. Hoffmann-La Roche Ag PEG-interferon conjugates
EP0809996A2 (en) 1996-05-31 1997-12-03 F. Hoffmann-La Roche Ag Interferon conjugates
CN1711109A (zh) * 2002-11-15 2005-12-21 霍夫曼-拉罗奇有限公司 PEG干扰素α－2a的位置异构体
CN1243779C (zh) 2002-03-13 2006-03-01 北京键凯科技有限公司 具有y形分支的亲水性聚合物衍生物、其制备方法、与药物分子的结合物以及包含该结合物的药物组合物

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6610830B1 (en) * 1980-07-01 2003-08-26 Hoffman-La Roche Inc. Microbial production of mature human leukocyte interferons
US5951974A (en) * 1993-11-10 1999-09-14 Enzon, Inc. Interferon polymer conjugates
TW426523B (en) * 1995-04-06 2001-03-21 Hoffmann La Roche Interferon solution
CN1243779A (zh) 1998-07-31 2000-02-09 东莞中镇鞋材有限公司 纤维板及其制造方法
US6887462B2 (en) 2001-04-09 2005-05-03 Chiron Corporation HSA-free formulations of interferon-beta
AU2002356422A1 (en) * 2002-11-01 2004-05-25 Cadila Healthcare Limited Mthod for producing recombinant human interferon alpha 2b polypeptide in pichia pastoris
MX2010002557A (es) 2007-09-04 2011-12-16 Biosteed Gene Expression Tech Co Ltd Interferon alfa 2b modificado por glicol de polietileno, la preparacion y el uso del mismo.
WO2009030065A1 (zh) 2007-09-04 2009-03-12 Biosteed Gene Expression Tech. Co., Ltd. 聚乙二醇修饰的干扰素α2a及其制备方法和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
EP0593868A1 (en) 1992-08-26 1994-04-27 F. Hoffmann-La Roche Ag PEG-interferon conjugates
EP0809996A2 (en) 1996-05-31 1997-12-03 F. Hoffmann-La Roche Ag Interferon conjugates
CN1243779C (zh) 2002-03-13 2006-03-01 北京键凯科技有限公司 具有y形分支的亲水性聚合物衍生物、其制备方法、与药物分子的结合物以及包含该结合物的药物组合物
CN1711109A (zh) * 2002-11-15 2005-12-21 霍夫曼-拉罗奇有限公司 PEG干扰素α－2a的位置异构体

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
CHAMOW ET AL., BIOCONJ. CHEM., vol. 5, 1994, pages 133
DELGADO ET AL., CRITICAL REVIEWS IN THERAPEUTIC DRUG CARRIER SYSTEMS, vol. 9, 1992, pages 249
HENCO K.; BROSIUS F.J. ET AL., J. MOL. BIOL., vol. 185, 1985, pages 227 - 260
INADA ET AL., J. BIOACT. AND COMPATIBLE POLYMERS, vol. 5, 1990, pages 343
INOUE ET AL., J. LAB. CLIN. MED., vol. 124, 1994, pages 529
KATRE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 84, 1987, pages 1487
KATRE, ADVANCED DRUG DELIVERY SYSTEMS, vol. 10, 1993, pages 91
KENJI ORITANI; PAUL W KINCADE ET AL.: "Type I interferon and limitin: a comparison of structures, receptors, and functions", CYTOKINE AND GROWTH FACTOR REVIEWS, vol. 12, 2001, pages 337 - 348
LAEMMLI ET AL., NATURE, vol. 227, 1970, pages 680
MONFARDINI ET AL., BIOCONJUGATE CHEM., vol. 6, 1995, pages 62
PHARMACOPOEIA OF THE PEOPLE'S REPUBLIC OF CHINA, vol. 3, 2005
SATAKE-ISHIKAWA ET AL., CELL STRUCTURE AND FUNCTION, vol. 17, 1992, pages 157 - 160
TSUTSUMI ET AL., JPN. J. CANCER RES., vol. 85, 1994, pages 9
YU-SEN WANG ET AL., ADVANCED DRUG DELIVERY REVIEWS, vol. 54, 2002, pages 547 - 570
YU-SEN WANG ET AL., BIOCHEMISTRY, vol. 39, 2000, pages 10634 - 10640
YU-SEN WANG; STEPHEN YOUNGSTER ET AL.: "Structural and biological characterization of PEGylated recombinant interferon alpha-2b and its therapeutic implications", ADVANCE DRUG DELIVERY REVIEWS, vol. 54, 2002, pages 547 - 570

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8143214B2 (en) 2007-08-16 2012-03-27 Pharmaessentia Corp. Protein-polymer conjugates
US8597635B2 (en) 2007-09-04 2013-12-03 Biosteed Gene Expression Tech. Co., Ltd. Interferon alpha-2B modified by polyethylene glycol and methods of preparation thereof
US8597634B2 (en) 2007-09-04 2013-12-03 Biosteed Gene Expression Tech. Co., Ltd. Interferon alpha-2a modified by polyethylene glycol and methods of preparation thereof
US11559567B2 (en) 2014-11-06 2023-01-24 Pharmaessentia Corporation Dosage regimen for pegylated interferon
CN116120424A (zh) * 2022-06-06 2023-05-16 江苏靶标生物医药研究所有限公司 一种干扰素α2b可溶性重组表达和分离纯化方法及其应用

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US8597634B2 (en) 2013-12-03
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US20100239532A1 (en) 2010-09-23
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BRPI0721988B1 (pt) 2021-07-27
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BRPI0721988A2 (pt) 2016-10-18
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