WO1985003934A1 - Chemically modified protein and process for its preparation - Google Patents

Chemically modified protein and process for its preparation Download PDF

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Publication number
WO1985003934A1
WO1985003934A1 PCT/JP1984/000085 JP8400085W WO8503934A1 WO 1985003934 A1 WO1985003934 A1 WO 1985003934A1 JP 8400085 W JP8400085 W JP 8400085W WO 8503934 A1 WO8503934 A1 WO 8503934A1
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WIPO (PCT)
Prior art keywords
protein
hours
group
reaction
acid
Prior art date
Application number
PCT/JP1984/000085
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French (fr)
Japanese (ja)
Inventor
Osamu Nishimura
Masahiko Fujino
Original Assignee
Takeda Chemical Industries, Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to AU24950/84A priority Critical patent/AU2495084A/en
Application filed by Takeda Chemical Industries, Ltd. filed Critical Takeda Chemical Industries, Ltd.
Priority to PCT/JP1984/000085 priority patent/WO1985003934A1/en
Priority to AU33954/84A priority patent/AU3395484A/en
Priority to AU28287/84A priority patent/AU2828784A/en
Priority to AU28677/84A priority patent/AU2867784A/en
Priority to PCT/JP1984/000575 priority patent/WO1985003868A1/en
Priority to JP60027283A priority patent/JPH0676439B2/en
Priority to JP60037936A priority patent/JPH0696599B2/en
Priority to AT85102376T priority patent/ATE46349T1/en
Priority to EP85102376A priority patent/EP0154316B1/en
Priority to DE8585102376T priority patent/DE3572982D1/en
Priority to KR1019850001381A priority patent/KR920007681B1/en
Priority to CA000475743A priority patent/CA1255588A/en
Publication of WO1985003934A1 publication Critical patent/WO1985003934A1/en
Priority to US07/519,280 priority patent/USH1662H/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • 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
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • 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]
    • C07K14/57—IFN-gamma
    • 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
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a chemically modified protein and a method for producing the same.
  • Ii interface Xron
  • IL-2 interleukin-2
  • a method of chemically modifying bioactive protein is an extremely effective means, ie, the prolongation of the above-mentioned clearance in vivo, the reduction of anti-smallness, Furthermore, high bioactivity is expected, and the practical significance of chemical modification of bioactive proteins is extremely large.
  • a method of introducing a physiologically active protein into a physiological protein in the presence of a low-molecular aldehyde-boron-based reducing agent such as homadehyde Y, acetoadedo, pentadedeto, or pyridoxal [me, Knooped-in Energy, No.
  • the present inventors have conducted intensive research to solve these drawbacks and completed the present invention.
  • the present invention relates to at least one monoamino group of a bioactive protein,
  • the present invention provides a chemically modified protein directly linked with 3 ⁇ 4 (I: R is a terminal or elementary protecting group, n is a positive integer that can be arbitrarily changed), and a method for producing the same.
  • the protein may be a product derived from various animals including it engineering products, humans, a biological product, a plant product, or a synthetic product.
  • -a if K- ⁇ ), interferon-3 ( ⁇ ⁇ ⁇ ⁇ /?), interferon-r (IPH-r) »II» -2, growth hormone, insulin, perokina Ze, streptokinase, suunoxidase dismutase (S0D), asparaginase, various immunoglobulins, albumin, kata, -ze, tribune, tribune, quatribulin, lysozyme , l J Botakurea - Ze, Ca Bokishi Bae putida - peptidase, Amino Bae Buchidaze various cytochrome, Urika - Ze, papain, peptidase down, bromelain, insulin secretion-activating protein (IAP), Neoka Jinos
  • bioactive proteins human or human derived from animals, tyne phosphorus, egg white lysozyme, perokinase, etc., and various I (IFN- ⁇ ,, I I- ⁇ ) produced by genetic recombination technology. r), IL-2 and S0D derived from animals and microorganisms.
  • Primary amino groups of bioactive proteins include an at-amino group at the ⁇ -terminal and an e-amino group of a lysine residue.
  • Respect group represented by (I), is a protective group of the terminal element represented by R, A key, A Kanoiru 3 ⁇ 4 etc. fist up is, specifically as A key, C 1 - 1 8 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Etc.
  • n is preferably less than 500, particularly preferably 7 to t20.
  • the molecular weight of the group represented by the formula (I) is 25,000 or lower, and the molecular weight of the group is preferably 350 to 600.
  • the chemically modified protein of the present invention has a group represented by the formula (I) directly bonded to at least a part of a primary amino group of a bioactive protein. —If the amino group has only the IT terminal ⁇ -amino group, It has a group represented by the formula (I) directly bonded to a mino group.
  • the bioactive protein has one or more lysines one by one, it is directly associated with a part of the ⁇ -amino group, preferably with ⁇ 5 to 80 S6 (average) of the amino group.
  • the -amino group may or may not have a group represented by the formula (I) directly bonded.
  • the chemically modified protein of the present invention is, for example, a bioactive protein
  • It can be produced by reacting the indicated adduct with a reducing agent in the presence of a reducing agent.
  • Examples of the boron thread used in this reaction include sodium borohydride and sodium borohydride *. Of these, sodium borohydride is preferred because of its selectivity.
  • ad eide (B) is used for about 1-10,0 Q 0 mol of bioactive protein poor, and boron-based reduction is used for about ⁇ 10 for aldehyde (H).
  • the solvent used for the reaction may be any one that can interfere with the reaction ⁇ : for example, acid crushing solution, borate buffer solution * Either one can be used. Further, any organic solvent may be added, such as a lower alcohol which inactivates the protein and hinders the reaction (eg, methanol, methanol, i-propano, acetotri). 3 to 1 is a W capacity in a wide range of 4, I is near the neutral]? desirability.
  • reaction time 2 tf-50.
  • the range of c is more preferable.
  • the reaction time is G.5 to 72 hours, usually about 3 to 30 hours.
  • reaction solution was transparent, salted out, ion-exchange talomatography, and
  • the above-mentioned add ( ⁇ ) can be produced, for example, from an ethylene glyco-derivative represented by H ⁇ o-C3 ⁇ 4C3 ⁇ 43 ⁇ 4OH (summer: B and n are as defined above). Power * The composition of boric acid is low * advantageous * a production method.
  • the compound (a) is oxidized with chromium viridium in a halogenated ac solvent such as methylene chloride and chromium.
  • a halogenated ac solvent such as methylene chloride and chromium.
  • reaction is carried out at ⁇ 50, preferably at room temperature, for 1-30 hours.
  • reaction solutions were also extracted, extracted, re-distributed, re-precipitated, and chromatographed.
  • the chemically repaired protein according to the present invention has a corresponding known sarcotrophic bioactive protein.
  • OMPI-16PPOO It has the same usefulness: it has biological activity and is useful as a pharmaceutical product.
  • the chemically modified I protein poor of the present invention in which the in vivo clear lacsis is more advanced than the corresponding known non-modified bioactive protein poor, not only shows its activity for a long time, but also exhibits It has low antigenicity and can be safely used for the same purpose as known ⁇ !
  • the chemical protein of the present invention may be orally or non-peripherally administered to a mammal (sa, ita, pig, porcupine, mouse, human) as a universal pharmaceutical composition using a carrier, dilution or the like known per se. ) Can be administered.
  • the chemically modified I ⁇ of the present invention when used as an anti-dialysis agent, an adult, 1X once a day! 0 4 ⁇ 1 X 1 0 9 by the international unit to ⁇ 1? To administer.
  • FIG. 1 shows the clearance delay in pet plasma described in Example 3 (V).
  • ⁇ yeast ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • ⁇ enzyme immunotitration method
  • ⁇ antiviral activity controls the the r IFN - it shows it ⁇ results of beta a 0
  • the number of Phe in pig insulin is originally three, but the N-terminal Phe in the B chain is modified with polyethylene glycol methine and is 1 even less *.
  • the glucose lowering effect was about 50% of that of pig insulin.
  • Jinja example 2 Polyethylene glyric 3-meth; I-ail modified egg white lysozyme Preparation of
  • reaction solution was poured into a cephadex G-75 (3.0 dia., 43.0 «), and developed with 25 mM ammonium sulfate-laminate (pH 5.0) + 0.15 M soil. .
  • Example 3 A solution of the chemical condition IFH-e (IffA-3) of the present invention obtained in (1) was dissolved in 5 W of 250 W of blood s-abumin, etc. The solution was dissolved in a membrane filter ( Bore-size: 0.2 A tn), subdivide into 5 pellets. Aseptically freeze-dried and stored, and put in distilled water for injection, W immediately before use. Serve for use.
  • a membrane filter Bore-size: 0.2 A tn
  • M Polyethyleneglycol with a mean molecular weight of 750, 550, and 350-Methylate Derived to the corresponding aldehyde by the same method as above.
  • the chemical luxury decoration protein produced by the present invention is useful as a pharmaceutical or the like.

Abstract

Chemically modified proteins wherein a group R-(OCH2CH2)n- (wherein R represents a terminal oxygen-protecting group, and n represents an arbitrary positive integer) is directly bound to at least one primary amino group of a physiologically active protein. They are useful as medicines, etc.

Description

明 釉 書  Akira glaze book
化学修靡蛋白質およびその製造法  Chemically perturbed proteins and their production
技 術 分 野  Technical field
本発明は、化学修飾蛋白貧およびその製造法に関する。  The present invention relates to a chemically modified protein and a method for producing the same.
背 景 技 術  Background technology
t£来から、 インスリ ン ， インタ - フ Xロン（以下 I i" Nと骆記するこ とがある）あるいはィンタ—ロイキン- 2 (以下 I L - 2と略記 Tるこ とがある）など有用な生理活性蛋白貧が知られて たが、 近年、遺伝子 組め換え技^の発展にともな 、 これら生埋活性蛋白貧を大量に合成す ることが可能になってきた。 しかしながら、 生体に投与された生理活性 蛋白質の生体内におけるクリアランスは、一 に非常に早いことが知ら れている。 また生理活性蛋白貧が 種动物 ら得られたものである場合 には、場合により抗体が産生され、 重篤 症伏を引き起こす危険が予想 される。 従って、 これらを医薬として用いるに際しては、その活性を保 持したまま、 ク リ アランスを遷延させ、 さらにその抗原性を滅弱させる 技術の ϋ発が望まれている。 この目的を達成するために、生 活性蛋白 貧を化学的に修飾する方法はきわめて有効な手段である。 すなわち化学 修飾によって、上記の生体内におけるクリ 7ランスの遷延，抗朦性の滅 弱，さらには生理活性の增強が期待され、 生理活性蛋白質の化学修飾の 実用的意義はきわめて大き 。  Useful since the beginning of the year, such as insulin, interface Xron (hereinafter sometimes abbreviated as Ii "N) or interleukin-2 (hereinafter sometimes abbreviated as IL-2). However, in recent years, with the development of gene recombination techniques, it has become possible to synthesize a large amount of these bio-buried active proteins. It is known that the clearance of the biologically active protein obtained in vivo is very fast, and if the biologically active protein is obtained from a species, an antibody may be produced in some cases. Therefore, when using these as a medicine, the development of a technology that prolongs clearance and further reduces their antigenicity while maintaining their activity is expected. It is desired. In order to achieve the object of the present invention, a method of chemically modifying bioactive protein is an extremely effective means, ie, the prolongation of the above-mentioned clearance in vivo, the reduction of anti-smallness, Furthermore, high bioactivity is expected, and the practical significance of chemical modification of bioactive proteins is extremely large.
生理活性蛋白質の化学修飾を行なうにあたっては、それらの生理活性 を保持したまま、化学修飾を行 い得る方法が必要である。 ボリヱチレ ングリコ - メチ ェ -テ は、 このもの自体が抗原性を有し と考 えられているため、上記の目的の蛋白質の化学修鰊に用 られて る:^ 該物質の蛋白質への導入は 化シァヌルを用 る方法が一 的である。  In performing chemical modification of bioactive proteins, there is a need for a method capable of performing chemical modification while maintaining their bioactivity. Polyethylene glycol-methyate is used for chemical modification of the protein of interest because it is considered to have antigenicity by itself: ^ The introduction of this substance into proteins One method is to use cyanuric chloride.
OMPI しかしながら、 同時に結合基として導入される壌化 ァヌ はそれ自体 安全性に問題があ ϊ>、かつま 7tその生体内における分解物の安全性につ ても解明されておらず、その使用は慎重を期す必要がある。 また反応 に しても、 ア カリでの pH¾必要とし、 ア カリ性で失活しゃす 蛋白煑に しては、本法を適用でき： 4 欠点がある。 OMPI However, the use of Nylon guanine, which is simultaneously introduced as a linking group, is problematic in its own safety.> 7t has not yet been clarified as to the safety of its in-vivo degradation products. You need to be careful. In addition, this method can be applied to the reaction that requires a pH value of alkali and a protein that is inactivated and deactivated by alkali. There are four disadvantages.
また、生理雜蛋白質にホ ムア デヒ Y ,ァセ トァ デ ド ，ペン プア デ tド，ピリドキサ-ルなどの低分子のア デヒド¾ホウ素系還 元剤の存在下に導入する方法〔メ、ノプ ド イン ェンザィ ジ- ，第 In addition, a method of introducing a physiologically active protein into a physiological protein in the presence of a low-molecular aldehyde-boron-based reducing agent such as homadehyde Y, acetoadedo, pentadedeto, or pyridoxal [me, Knooped-in Energies, No.
47卷， 463 -478頁（ 1 977 ) 〕 ；特開昭 58 - 1 54596 号公報〕が知られて る。 しかしながらこれら公知の物貧によつては有 効なクリアランスの達延化は達成されず、抗原性の低下は期待され い のみるらず、導入され 子のァ デヒドがハプチンとして作用して 該蛋白貧に免疫慼性を与える可能注がある。 47, 463-478 (1977)]; Japanese Patent Application Laid-Open No. 58-154596] is known. However, effective known prolongation of clearance cannot be achieved by these known poverty, and no decrease in antigenicity is expected. However, the introduced aldehyde acts as a haptin to produce the protein. Poor immunity may be noted.
本発明者らは、 これらの欠点を解決すべく、 鋭意研究を行 、本発 明を完成した。  The present inventors have conducted intensive research to solve these drawbacks and completed the present invention.
発 明 の 開 示  Disclosure of the invention
本発明は、生理活性蛋白質の少 *くとも 1個の一鈒ァミノ基に、
Figure imgf000004_0001
¾( I ： Rは末端或素の保護基， nは任意に変わ うる正の整数）を直接結合してるる化学修飾蛋白質およびその 造法 を提供するものである。 The present invention relates to at least one monoamino group of a bioactive protein,
Figure imgf000004_0001
The present invention provides a chemically modified protein directly linked with ¾ (I: R is a terminal or elementary protecting group, n is a positive integer that can be arbitrarily changed), and a method for producing the same.
上記生理 @¾蛋白質に闋し、 該蛋白質は、 it 工学産物， ヒ ト 含 む各種動物由来のもの 生物由来のもの，植物由来のもの，合成品 ずれでもよ が、例えば各種 iFireインタ -フ ロン- a ( i ff K- α ) ， インターフェロン一 3 ( Ι ϊ· ΐϊ /? ) ， インターフェロン一 r ( I PH-r ) » I I»-2，生長ホルモン ，インス リ ン， ゥロキナ- ゼ ,ス トレブトキナーゼ ，ス一ノ<ーォキ ドジスムターゼ（ S 0 D ) , ァスパラギナ-ゼ，各種免疫グロブリン ，アルブミン ，カタ， -ゼ， ト リブ ン ， ト ブ ンィンヒビタ一 ,キ乇ト リブシン ， リゾチーム , lJ ボタクレア-ゼ ,カ ボキシぺプチダ-ゼ ，ァミノぺブチダーゼ，各種 チトクローム , ゥリカ -ゼ ,パパイン ，ペプ ン ，ブロメライン , イン スリン分泌活性化蛋白質（ I A P ) ,ネオカ ジノス チン等が挙げら れる。 と]?わけ好ましい生理活性蛋白質として、動物由来のヒトまたは プ、タイン リン ，卵白リゾチ-ム ，ゥロキナーゼ¾ど，遺伝子組換え技 術で生産される各種 I ( I F N - β , , I Ρ Ν - r ) , I L - 2 ¾どや鲂物及び微生物由来の S 0 D ¾どが挙げられる。 In contrast to the above-mentioned physiological protein, the protein may be a product derived from various animals including it engineering products, humans, a biological product, a plant product, or a synthetic product. -a (if K-α), interferon-3 (Ι ϊ · ΐϊ /?), interferon-r (IPH-r) »II» -2, growth hormone, insulin, perokina Ze, streptokinase, suunoxidase dismutase (S0D), asparaginase, various immunoglobulins, albumin, kata, -ze, tribune, tribune, quatribulin, lysozyme , l J Botakurea - Ze, Ca Bokishi Bae putida - peptidase, Amino Bae Buchidaze various cytochrome, Urika - Ze, papain, peptidase down, bromelain, insulin secretion-activating protein (IAP), Neoka Jinosu Chin like are found like . As particularly preferable bioactive proteins, human or human derived from animals, tyne phosphorus, egg white lysozyme, perokinase, etc., and various I (IFN-β ,, I I-Ν) produced by genetic recombination technology. r), IL-2 and S0D derived from animals and microorganisms.
生理活性蛋白質の一級ァミノ基として、 Η末端の at -アミノ基および リジン残基の e -ァミノ基が挙げられる。  Primary amino groups of bioactive proteins include an at-amino group at the Η-terminal and an e-amino group of a lysine residue.
上記（ I )で表わされる基 関し、 ； Rで示される末端 素の保護基と しては、 ア キ ，ア カノィル ¾どが拳げられ、 ア キ として具体 的には、 C 1 - 1 8のもの、 と わけメチ ，ェチ , プロビ ， i一ブ 口ビ ，ブチ ， i -ブチル， sec -ブチ ， t -プチ ¾ど低钹 Respect group represented by (I),; is a protective group of the terminal element represented by R, A key, A Kanoiru ¾ etc. fist up is, specifically as A key, C 1 - 1 8 ,,,,,,,,,,,,,,,,, Etc.
( C i _4 )ア キ が好ましい。 ア カノィルとして具体的には、 (C i — 4 ) space is preferred. Specifically, as A Canoil,
C，-8のもの、 と わけホ ミ ，ァセチ ， ブロビォ ブチリル f, iーブチリル *ど低級（ C 1 -4 )アルカノィ が好ましい。 nで表わさ れる正の整数は、 5 0 0 下、 と わけ 7 ~ t 2 0が好ましい。 C, - 8 ones, and Wakeho Mi, Asechi, Burobio butyryl f, is i Buchiriru * throat lower (C 1 -4) Arukanoi preferred. The positive integer represented by n is preferably less than 500, particularly preferably 7 to t20.
式（ I )で表わされる基の分子量として 2 . 5万 下、 と])わけ 350 - 6 0 0 0のものが好ましい。  The molecular weight of the group represented by the formula (I) is 25,000 or lower, and the molecular weight of the group is preferably 350 to 600.
本発明の化学修飾蛋白貧は、 生理活性蛋白質の一級ァミノ基の少 く とも一部に直接結合した式（ I )で表わされる基を有するものである。 —极ァミノ基として IT末端《 -アミノ基のみを有する場合は、そのァ ミノ基に直接綾合した式（ I )で表わされる基を有するものである。 ま た生理活性蛋白質中に 1個 1¾上のリジン ¾有する場合は、その β -アミ ノ基の一部に、 好ましくはそれら ァミノ基の Ϊ 5 ~ 8 0 S6 (平均〉 に、直接結会した式（ I )で表わされる基を有するものであ 1K の場 合、 -アミノ基は、直接結合した式（ I )で表わされる基を有 しても、 有しまくてもよい。 The chemically modified protein of the present invention has a group represented by the formula (I) directly bonded to at least a part of a primary amino group of a bioactive protein. —If the amino group has only the IT terminal <<-amino group, It has a group represented by the formula (I) directly bonded to a mino group. When the bioactive protein has one or more lysines one by one, it is directly associated with a part of the β-amino group, preferably with Ϊ5 to 80 S6 (average) of the amino group. In the case of 1K having a group represented by the formula (I), the -amino group may or may not have a group represented by the formula (I) directly bonded.
本発明の化学修鳙蛋白質は、例えば生理活性蛋白貧と  The chemically modified protein of the present invention is, for example, a bioactive protein
H- O ( JL： Rおよび nは前記と同意義）で
Figure imgf000006_0001
H-O (JL: R and n are as defined above)
Figure imgf000006_0001
示されるア デ ドとを還元剤の存在下反応させることによ 1?製造する ことができる。 It can be produced by reacting the indicated adduct with a reducing agent in the presence of a reducing agent.
本反応に用いるホウ素糸蘧元 としては、 水素化ホウ素ナト リウム ， ァノ水素化ホウ素ナトリウム *どが挙げられるが、 中でもシァノ水素 化ホウ素ナトリウムが反応の遴択性の点でよ U好まし 。  Examples of the boron thread used in this reaction include sodium borohydride and sodium borohydride *. Of these, sodium borohydride is preferred because of its selectivity.
反 に際しては、 ア デ eド（ B )を生理活性蛋白貧に対して、 1 ~ 1 0, 0 Q 0谙モル程度、ホウ素系還元 はア デヒド（ H〉に対して， - 1 0 程度用 ればよく、 蛋白質とア デヒド（ H )のモ 比 On the other hand, ad eide (B) is used for about 1-10,0 Q 0 mol of bioactive protein poor, and boron-based reduction is used for about −10 for aldehyde (H). The ratio of protein to aldehyde (H)
¾增»することによゥて修銻の接度 ¾任意に選択することができる。反 応に用 る溶媒は、反応 ¾:妨害し ¾ ものであれば ずれでもよ が、 例えば ン酸縵衝液，ホウ酸緩衝液 *どの瑷«が举げられる。 また、 蛋白質を失活さ 反応の支障に らな 低級ア 力ノ - （例、 メタノー ，工タノー , i一プロバノ ，ァセト-ト リ ¾どの 有機溶媒を添加してもよ 。反応の p Hは 3 ~ 1 4の広 範囲で W能で あるが、 中性付近がよ]?望まし 。反応温度は 0β〜 8 0。Cで蛋白貧が変 性し ¾ 温度であれば、 ずれでもよ が、 2 tf- 5 0。cの範囲がよ > 好まし 。 反応時間は G . 5 ~ 7 2時間、通常は 3 ~ 3 0時間程度で十 By changing the degree of repair, the degree of contact can be arbitrarily selected. The solvent used for the reaction may be any one that can interfere with the reaction 妨害: for example, acid crushing solution, borate buffer solution * Either one can be used. Further, any organic solvent may be added, such as a lower alcohol which inactivates the protein and hinders the reaction (eg, methanol, methanol, i-propano, acetotri). 3 to 1 is a W capacity in a wide range of 4, I is near the neutral]? desirability. if the reaction temperature is 0 β ~ 8 a ¾ temperature protein poor is denatured at 0.C, any deviation However, the reaction time is 2 tf-50.The range of c is more preferable.The reaction time is G.5 to 72 hours, usually about 3 to 30 hours.
O〜PI 分である。反応液は、透圻，塩析，イオン交換タロマトグラフィ- ，ザO to PI Minutes. The reaction solution was transparent, salted out, ion-exchange talomatography, and
Λ -過，高速液体ク マトグラフィ- ,電気泳!^通常の蛋白貧の精製 法で精製し、所望の化学修鍊蛋白貧を得ることができる。 またアミノ基 の修飾の程度は、例えぱ黢分解のあと、 アミノ黢分析を行 つて算出す ることができる。 過-Excess, high-performance liquid chromatography-, electric swimming! ^ It can be purified by the usual purification method of protein to obtain the desired chemically modified protein. The degree of modification of the amino group can be calculated, for example, by analyzing the amino group after decomposition.
前記したア デ ド（ Η )は、 例えば H~ o-C¾C¾¾«OH (夏： B よび nは前記と同意義）で示されるエチレングリコ - 誘導体から 製造できるが、下 ¾の方法は、対応する力 ボン酸の蠲成が少 *く有利 *製造法である。  The above-mentioned add (Η) can be produced, for example, from an ethylene glyco-derivative represented by H ~ o-C¾C¾¾OH (summer: B and n are as defined above). Power * The composition of boric acid is low * advantageous * a production method.
すなわち、 化合物（ a ) ¾ 化メチレン ，クロ ホ ム ¾どハロゲン 化ア キ 溶媒中、 クロ クロム ビリジ ^ムで酸化する。 この場合、  That is, the compound (a) is oxidized with chromium viridium in a halogenated ac solvent such as methylene chloride and chromium. in this case,
ヒ》リジ ^^ ^  Hi >> Rigi ^^ ^
ク a クロム酸 化合物（ X )に対し， ~ 3¾ 量用い、 - 1 0。~ 50 で、好ましくは室温で、 1 ~ 30時間反'応させる。  -A 10% less than chromic acid compound (X). The reaction is carried out at ~ 50, preferably at room temperature, for 1-30 hours.
また、 化 （ H ,但し n - i ) *t -ブタノ 中で力リウム ¾一 ブトキ^ドで処理した後、 プロ ¾ァセタ -ルを反応させ、ついで有機酸 ( ト フ 才ロ黥酸 *ど）または無機醸 ( ¾酸，硫黢 ど） ¾どの^で 処理することによ 1>化合物（ m )よ]? -OCH2CH2 - 鎖長の長 対応す るア デヒド（ H )を製造することができる。 この場会、 まず力リゥム t一ブトキシドを上記化合物（H )iC対し 1 0~30モ 量を加えて溶 解させ、 これにブ モアセター〃を化合物（ ffi) 対し 3~ 1 5¾Adt 加えて、 1 0"〜80¾で0 . 5 ~ 5時間反応させ、常法により後処理後、 上記黢の希薄水溶液に溶かし、 5分 ~ 2時間加熱する。 In addition, after treatment with potassium hydroxide in a chemical compound (H, where n-i) * t -butano, the reaction with procetal is carried out, followed by the reaction of an organic acid (such as tofuishiro * acid). 1) Compound (m) by treating with 醸) or inorganic brew (acid, sulfuric acid, etc.) ?? -OCH 2 CH 2- Long chain length Produce the corresponding aldehyde (H) can do. In this event, first, 10 to 30 moles of the potassium hydroxide was dissolved in the above compound (H) iC to dissolve it, and then the bumoresetter was added to the compound (ffi) 3 to 15¾Adt, and 1 The reaction is carried out for 0.5 to 5 hours at 0 "to 80 ° C, and after post-treatment by a conventional method, dissolved in the dilute aqueous solution of the above 1) and heated for 5 minutes to 2 hours.
上 ¾ ずれの反応液も、 抽出， ，再 勗，再沈殿， ク マトグラ The above reaction solutions were also extracted, extracted, re-distributed, re-precipitated, and chromatographed.
7ィ- ，蒸留 *ど通常の化学的 4 &理によ 精製する とができる。 7-, distillation * It can be purified by ordinary chemical 4 & process.
本発明の化学修餽蛋白質は、 对応する公知の菲修饑生理活性蛋白貧と  The chemically repaired protein according to the present invention has a corresponding known sarcotrophic bioactive protein.
OMPI WWIIPPOO 同様の有用 ¾生理活性を有し、医薬品 ¾どとして有用である。 OMPI WWIIPPOO It has the same usefulness: it has biological activity and is useful as a pharmaceutical product.
本発明の化学修 «I蛋白貧は、対応する公知の非修鍊生理活性蛋白貧に 比し、生体内におけるクリアラシスが遷½され、 長時 I»有効にその活性 を示すのみならず、 ，抗原性も低く、 公知の^!活性 白貧と同搛 の目的に、 同様の用法で安全に使用することができる。  The chemically modified I protein poor of the present invention, in which the in vivo clear lacsis is more advanced than the corresponding known non-modified bioactive protein poor, not only shows its activity for a long time, but also exhibits It has low antigenicity and can be safely used for the same purpose as known ^!
本発明の化学傪娣蛋白貧は、通常自体公知の担体，稀 翔等を用 遍 宜の医薬組成翁として経口的または非经ロ的に哺乳動物（サ ， イタ ， ブタ ， ゥサギ ，マウス ， ヒト）に投与することができる。  The chemical protein of the present invention may be orally or non-peripherally administered to a mammal (sa, ita, pig, porcupine, mouse, human) as a universal pharmaceutical composition using a carrier, dilution or the like known per se. ) Can be administered.
例えば、本発明の化学修銻 I βを抗ゥィ ス薊として使用する 場合、成人，日 1回 1 X ！ 04 ~ 1 X 1 09 国際単位を黪注によ 1?投与 するのがよ 。 For example, when the chemically modified Iβ of the present invention is used as an anti-dialysis agent, an adult, 1X once a day! 0 4 ~ 1 X 1 0 9 by the international unit to黪注1? To administer.
^細替中、 ァミノ黢に闋し赂号で表示する場合は、 I U. P A C - I ϋ B ( C ommi s s i on of Bi ologi cal iionieac lature) よる略号に基づくものである。  During the refining, the display of the amino name is based on the abbreviation of I U. P A C-I ϋ B (C ommi s sion of Biologi cal iionieac lature).
図 面 の ffi 单 ¾ 説 明  Ffi の explanation of drawing
第 1図は実施 « 3 «V)に記載したヲット血漿中のクリアランス遅延 ί 杲¾示す。〇（酵^ Λ疫濺定法 )および口（抗ウイルス活性）は実施调 3 ωで得た本発明の化学侈飾 I F IT - βの、 秦（酵素免疫漏定法）およ び鼴（抗ウイルス活性）は対照とした r I F N - β Aの溺定結果をそれ それ示す 0 FIG. 1 shows the clearance delay in pet plasma described in Example 3 (V). 〇 (yeast Λ Λ 濺 濺 濺 お よ び お よ び お よ び) and mouth (antiviral activity) were carried out 得 (enzyme immunotitration method) and 鼴 (antiviral activity) controls the the r IFN - it shows it溺定results of beta a 0
発明を実旌するための最良の形餞  The best form of gifting for the invention
以下実 ¾例および参考例によつて本発明を r ^具体的に説明するが、 本発明はこれら i 制限さ るものでは 。  Hereinafter, the present invention will be specifically described with reference to practical examples and reference examples, but the present invention is not limited to these examples.
実施 1 B 1 -ボ》 エチレング 9コ一 メチルエーテ 修鎵インス梦 ンの調製 f OMPI (I) ブクィンス リ ン 1 50 Wfr 20 Wの水に懸溽し、 れに—規定 ¾ 酸を—滴づっ加え、 ィンスリン *溶解させた。 そののち 0 . 4 Mリン酸 緩赚（PH7 . 0 ) 20W¾加え、最終的に 0 · 2Mのリン黢縵赚と した。 これに參考例 1 (i)で得たボリエチレングリコ -/^メチ ェ -チ ア デヒド（平均分^ 15000 ) 1 . 1 25 ^を加え、っ^で ァノ 水素化ホウ素ナト リウム 1 0 を加えて、 37°Cで 24時間かきまぜ to反応液を水に対して 1 2時間透析し、つ で内容物 ¾カ ボキ メ チ； Λセ 口ースのカラム（ 3 , 0 x23 . Q CB )に注 だ。 カラムを水 で洗ったのち、 水（ 500W)と 0 . 2 M静酸アン乇-ゥム瀠繊 (pH 6 . 8 )の間で、対数勾配をかけて溶出した。 主溶出幽'分 ( 320 -Practical 1 B 1-bo >> Preparation of ethylene glycol 9 methyl ether (I) Bouqinsulin 1 50 Wfr The suspension was suspended in 20 W water, and the specified acid was added dropwise thereto to dissolve Insulin *. After that, 0.4M phosphoric acid buffer (PH 7.0) 20W was added, and finally the phosphor was 0.2M phosphoric acid. To this, add the poly (ethylene glycol) obtained in Example 1 (i)-/ ^ methyl-thialdehyde (average min ^ 15000) 1.1 25 ^, and add the sodium borohydride 10 with ^^. In addition, stir the reaction mixture at 37 ° C for 24 hours, dilute the reaction solution against water for 12 hours, and then reconstitute the contents of the column (3, 0x23. Q CB). Note. After washing the column with water, the column was eluted with a logarithmic gradient between water (500 W) and 0.2 M ammonium hydrochloride (pH 6.8). Main elution
400 W )を集め凍結乾燥した。 つ でバィ才ゲ ： Ρ - 30のゲ 炉 ¾ に付し、 0 . 1規定齚黢で展開した。 主溶出画分（ 1 ， 0 ~ 1 5 400 W) was collected and lyophilized. By the way, it is attached to the Ρ-30 ゲ furnace and developed in 0.1 regulation. Main elution fraction (1, 0 to 15
を集めて 結乾燥した。 収量 1 m ^分解物（ 6 H 駿， 1 1 ere, Was collected and dried. Yield 1 m ^ decomposed product (6 H, 11 ere,
24時閼）中のアミノ靉分析値： Lys, 0.92(1); His, 2.12 (2), Arg, 1.08(1)； Aap, 3.20(3)； Thr , 2.11(2)； Ser,Amino Ay analysis value in the 24 hours): Lys, 0.92 (1); His, 2.12 (2), Arg, 1.08 (1); Aap, 3.20 (3); Thr, 2.11 (2); Ser,
2.86(3); Glu, 7.88(7)； Pro, 1.11(1); ( y, 3.78(4); Ala, 2.16(2)； Half Cys , 6.08(6)； Val , 3.67(4)； He, 1.78(2); Leu, 6-17(6)； Tjr , 4.04(4); Phe, 2.09(2) 2.86 (3); Glu, 7.88 (7); Pro, 1.11 (1); (y, 3.78 (4); Ala, 2.16 (2); Half Cys, 6.08 (6); Val, 3.67 (4); He , 1.78 (2); Leu, 6-17 (6); Tjr, 4.04 (4); Phe, 2.09 (2)
ブタインスリ ンの Phe は本来 3個であるが、 B鎖 N末端の Pheが、 ボリエチレングリコールメチ エーチ で修鰊され 1偶少 *く ¾つて る。 またグ コース低下作用はブタインスリ ンの約 50 %であった。 The number of Phe in pig insulin is originally three, but the N-terminal Phe in the B chain is modified with polyethylene glycol methine and is 1 even less *. The glucose lowering effect was about 50% of that of pig insulin.
(«) 参考^ 1で得た平均分子量 1 900 よび 750のボリエチレン グリコ - メチ〃エー ァ デヒドを用 Tブタインス 9ンを同様に 処理し、上記 (i)と同じく B鎖の If末端 Pheの β -ァミノ基が平均分子  («) Reference ^ Use the poly (ethyleneglyco-methadhydealdehyde) with an average molecular weight of 1900 and 750 obtained by T1 in the same manner as for T-butadiene 9 to obtain β -Amino group is the average molecule
C PI 量！ 300 よび 750のボリエチレングリコ一 メチ エーテ で修 飾されたィンスリン歸導体が得られ Λ：。 平均分子量 1 900のボリェチ レングリコー メチ ェーテ/修 j»インスリンの酸分解物（ 酸，C PI amount! 300 and 750 polyethylene glycol-modified insulin are obtained. Boletti with an average molecular weight of 1900
1 10¾ , 24畤閱）中のアミノ黢分析僮： rs, 0.98(1)ϊ His, 2.09(2){ Arg, 1.09(1)} Αβρ, 3.17(3)； Thr, 2.04(2)ϊ Ser , 2.89(3)； (ϊΐη, 7.60(7) ί Pro, 1.09( 1) &ly, 3.76(4) 5 Ala, 2.03(2)； Half Cys， 3.93(6)； Val , 3.27(4)； lie, 1.54(2)1 Leu, 5.87(6)； T7r , 3.88(4); Phe , 2.00(2) Amino analysis in 1 10¾, 24 閱): rs, 0.98 (1) ϊ His, 2.09 (2) {Arg, 1.09 (1)} Αβρ, 3.17 (3); Thr, 2.04 (2) ϊ Ser , 2.89 (3); (ϊΐη, 7.60 (7) ί Pro, 1.09 (1) & ly, 3.76 (4) 5 Ala, 2.03 (2); Half Cys, 3.93 (6); Val, 3.27 (4); lie , 1.54 (2) 1 Leu, 5.87 (6); T 7 r, 3.88 (4); Phe, 2.00 (2)
平均分子量 750のボリエチレングリコ一 メチ エーテ 修籙ィンス リンの酸分解物 ( 6 if塩酸中， 1 1 0 , 24時間）中のァミノ黢分析 值： Lys, 1,03(U; His, 2.18(2)； Arg, 1.12(1)； Asp, 3.30(3)； Thr, 2.15(2) J Ser, 3.13(3)； Glu, 7.83(73i Pro, 1.17(1)i (Jly. 4.04(4) j Ala, 2.24(2)； Half Cys, 5.43(6)} Val, 3.26(4)； He, 1.58(2) ; Leu, 6.30(6)； Tyr, 4.03(4) ί Phe, 2.23(2) Amino acid analysis in acid digests of poly (ethylene glycol) methacrylate phosphate with an average molecular weight of 750 (in 6 if hydrochloric acid, 110, 24 hours): Lys, 1,03 (U; His, 2.18 ( Arg, 1.12 (1); Asp, 3.30 (3); Thr, 2.15 (2) J Ser, 3.13 (3); Glu, 7.83 (73i Pro, 1.17 (1) i (Jly. 4.04 (4) j Ala, 2.24 (2); Half Cys, 5.43 (6)} Val, 3.26 (4); He, 1.58 (2); Leu, 6.30 (6); Tyr, 4.03 (4) ί Phe, 2.23 (2)
(I) グ コ -ス低下 用は平均分子量 1900のボリエチレングリコ 一 メチ ェ-チ 修鎵ィンス » ンがブ ィンスリンの 85 ，平均分 ¾750のボリエチレングリ 3一 メチ エーテ 修練ィンスリンが 1 00 であった。 ま 上記 ）^ ¾および《ί)で得た 3種のィシスリン靜 導体をラジ トに投与し、血中クリアランスを測定すると、 ブタインスリ ンに比較して、 明らか *運 化が認められた。 ブタインスリ ン抗体に対 する反応性も、 ボリエチレングリコ - メチ エーテ の分子量が大き <¾るほど、滅弱する煩向が認められた。  (I) For the reduction of glucose, a polyethylene glycol-modified silicone with an average molecular weight of 1900 is 85 in Binsulin, and a polyethylene glycol 31-methylated modified insulin with an average molecular weight of 750 is 100. there were. In addition, when the three isocyanins obtained in (1) and (2) above were administered to radiate and the blood clearance was measured, a clear * kinetics was observed as compared to that of porcine insulin. The reactivity with the porcine insulin antibody was also found to diminish as the molecular weight of polyethylene glycol-methate increased.
実艨例 2 ポリエチレングリ 3 - メチ; Iエー ル修飾卵白リゾチーム の調製 Jinja example 2 Polyethylene glyric 3-meth; I-ail modified egg white lysozyme Preparation of
iij 卵白リゾチーム 1 50^*0 .2 Mリン酸緵衡液 3 にとかレ 参考例 1で得た平均分子量 550のボリエチレングリコ - メチ エ- テ アルデヒド 1 1 6 加え、つ で ァノ水素化ホウ素ナト リウム 40 加え、 37 で 24時 Wかきまぜた。 つ で反応液を 4¾で 水に対して、 1 2時間透析した。透析した液をカルボキ メチ〃セ 攀スのカラム ( 3 . 0 23 . 0»)に注 だ。 カラムは水でよく洗つ たのち、 水（ 500W)と 0 · 7 M酢酸アン ¾ -ゥム（pHS · 8 ) ( 500 W )の間で直線勾 S3をかけて港出し 主溶出画分（ 550 ~ 70 *集め、 凍結乾^した。 凍結乾燥粉末はバイオゲ P - 30 のカラム（ 2 . 5 X 1 0 ，展開液 0 . 1 黢）でゲ 遏を行 精製した。 収量 1 1 酸分解物（ 6Η嵬酸， 1 1 CTC , 24時闞） 中のアミノ^^柝儘： ： Lys， 4.79} His , 0.97(1)； Arg, 11.07(11)； Trp, 分解（6); Asp, 21.68(21)； Thr , 7.25(7)； Ser, 9.96(10); Glu, 5.08(5)； Pro, 2.04 (2)； Gly, 12.62(12); Ala, 12,38(12); Half C73 » 8.47(8)； Val, 5.33(6)； Met, 1.94(2)； He, 5.48 (6)； Leu, 7.92(8) Tyr, 3.12(3)； Phe, 3.03(3) この結果は、 i rs の ァミノ基の約 20 itがボリエチレングリコ- メチ ェ-チ で修飾されて ることを示して る。 本品のミクロコ ッカスルチウスの乾燥菌体を用 る溶菌活性は卵白リゾチ-ムの 4 1 % であづた ο  iij Egg white lysozyme 1 50 ^ * 0.2 M Phosphoric Acid Solution 3 To be added to Polyethylene glyco-methyl aldehyde with an average molecular weight of 550 obtained in Reference Example 1 1 16 Sodium sodium boron was added, and the mixture was stirred at 37 hours for 24 hours. The reaction solution was then dialyzed against water at 4 1 for 12 hours. The dialyzed solution was applied to a column (3.0 23.0 ») of the carbohydrate. The column is washed well with water, and then a linear gradient S3 is applied between water (500 W) and 0.7 M ammonium acetate (pHS · 8) (500 W), leaving the port. 550 ~ 70 * Collected and lyophilized ^ The lyophilized powder was purified using a Bioge P-30 column (2.5 X 10, 0.1 L developing solution). Amino in ^^^ acid, 11 CTC, 24h): Lys, 4.79} His, 0.97 (1); Arg, 11.07 (11); Trp, decomposition (6); Asp, 21.68 (21); Thr, 7.25 (7); Ser, 9.96 (10); Glu, 5.08 (5); Pro, 2.04 (2); Gly, 12.62 (12); Ala, 12,38 (12); Half C73. »8.47 (8); Val, 5.33 (6); Met, 1.94 (2); He, 5.48 (6); Leu, 7.92 (8) Tyr, 3.12 (3); Phe, 3.03 (3) This shows that about 20 it of the amino group of i rs is modified with polyethylene glycol-methycolysis The lysing activity of this product using dried micrococcal sultius cells is 4% of that of egg white lysozyme. 1% ο
(Β) 上記の反応でボリエチレングリコ -〃メチ ェ-チ ァ 1デヒ の量を 385 W , 77 と增加させると、 修餽の割合は各 56 〔酸分解物（ 8¾Γ攙黢， 1 1 0eC , 24時間）中のアミノ酸分析値： Lya, 2.67； His, 1.06(1) J Arg , 11.50( 11)； Trp, 分 解（6); Asp, 22.13(21)1 Thr, 7.54(7)5 Ser, 10.62 (10); G-iu, 5.32(5) J Pro, 2.15(2) J dlj * 12.82 (12)J Ala, 12.49(12)； Half Cys, 7.20(8) i al , .82(6)； Met, 1.81(2)1 lie, 5.11(6)1 Leu, 8.86(8); T7r, 3.81(3)； Pie, 3.10(3) 〕， (Β) When the amount of polyethyleneglycol-dimethyl-demethyl was increased to 385 W and 77 in the above reaction, the ratio of the repair was 56 [acid decomposition products (8¾Γ 攙 黢, 11) 0 e C, 24 hours) amino acid analysis: Lya, 2.67; His, 1.06 (1) J Arg, 11.50 (11); Trp, decomposition (6); Asp, 22.13 (21) 1 Thr, 7.54 (7) 5 Ser, 10.62 (10); G-iu , 5.32 (5) J Pro, 2.15 (2) J dlj * 12.82 (12) J Ala, 12.49 (12); Half Cys, 7.20 (8) ial, .82 (6); Met, 1.81 (2) 1 lie, 5.11 (6) 1 Leu, 8.86 (8); T 7 r, 3.81 (3); Pie, 3.10 (3)],
T 4 〔酸分解物（ S ϋ植酸， 1 ! 0*C , 24時閬）中のアミノ酸分柝 値： Lys， 1.59； His, 1.09(1); Arg, 11.36(11)； rp, 分解（6)i Asp, 21.92(21); Thr, 7.35(7); Ser, 9.99 (10)i Giu, 5.45(5)； Pro, 2.05(2) ; iy, 12.67(12)i Ala, !2.22( 12) i Half Cys , 3.04C8); Val, 5.14(6) J Met, 1.72(2)； lie, 5.79(6) ; Leu, 8.26(8) Tyr， 3.91(3)! Phe, 3.14(3)  T 4 [Acid degradation product (S @ folic acid, 1.0 * C, 24h)] Amino acid analysis value: Lys, 1.59; His, 1.09 (1); Arg, 11.36 (11); rp, degradation (6) i Asp, 21.92 (21); Thr, 7.35 (7); Ser, 9.99 (10) i Giu, 5.45 (5); Pro, 2.05 (2); iy, 12.67 (12) i Ala,! 2.22 (12) i Half Cys, 3.04C8); Val, 5.14 (6) J Met, 1.72 (2); lie, 5.79 (6); Leu, 8.26 (8) Tyr, 3.91 (3)! Phe, 3.14 (3 )
と増加し、溶菌活性は！ 4 , 7 と抵下した。 And increase the lytic activity! Defeated 4 and 7.
(W) このよクにして得られた修錄リゾチ-ムをゥサギょ U得 與白リ ゾチ-ム抗体 ¾用 てその扰原性をしらべると修麴の程度が蔺く ¾るほ ど、卵白リゾチ-ム抗体との反応性が抵下する鎮向が認められ 。  (W) The repaired lysozyme obtained in this manner is used as a heron to obtain a lysozyme antibody. On the other hand, reversal of the reactivity with the egg white lysozyme antibody was observed.
IV) 76 修銪されたものをラットに授与すると、 卵白リダチ-ムに 比較して、 明らかるクリア，ンスの還珐化が認められ、 ゥサギに投与し ても、 抗体の産生はほとんど認められ *かった。  IV) When the 76-modified rat was given to rats, clear clearing and reduction of the protein were observed as compared with egg white rhidachime, and almost no antibody production was observed even when administered to rabbits. *won.
(VJ 平均分子量 5000 , 1 300 , 750 ， 350のポリエチレン グリコ-〃メチルエーテ ァ 1デヒドを用 て、上記と同様に反応し、 Ly sの *ーァミノ基の内約 20 が 鎵されたリゾチ-ムを得る とが できたが、それらの溶菌活性は各^平均分 ¾ 5 Q 00のもの 3 %， 1 300のもの 5 ， 750のもの 23¾ί , 350のもの 34 Siであつ 笑施例 3 ボ エチレング 9コ チ ェ-尹 修鑲 I FN - βの綢 (VJ average molecular weight 5000, 1300, 750, 350 polyethylene glycol-dimethyl ether 1 dehyde using the same reaction as above, the lysozyme in which about 20 of the Lys * -amino group was found However, their lytic activity was as follows: the average lysis activity was 3% for each of 5Q00, 5 for 1300, 23 for 750, and 34Si for 350. Laughing example 3 boys 9-尹 F I FN-β silk
(j) I PH -« ( r I FN- A )の溶液5«^(¾白質量にして4.8 ^)をと 、 0 · 2Mリン酸緩衝液（pH7 . 0 ) + 0. I 511食¾に对 し、 4eCで 1 2特閟透析した。 透析液を取]?出し、 れに參考例 1で胬 たボリ Xチレングリコ一 メチ Λェ-， ァ /1 デヒ ド（平均分子 Jt(j) A solution of I PH- «(r I FN-A) 5« ^ (4.8 ^ in white mass) and 0.2M phosphate buffer (pH 7.0) + 0.1 I 511 meals and对to and 1 2 Toku閟dialyzed against 4 e C. Take out the dialysate], and refer to it. Reference Example 1
1 900 ) ( 260V) ，つ でシァノ水素化ホウ素ナトリウム 1 900) (260V), which is sodium cyanoborohydride
( 140W)を加え、 37¾で 40時間かきまぜた。 反応液をセフアデ ックス G - 75のカヲム（ 3 . 0ズ43 . 0«)に注ぎ込み、 25 m M 齚酸アン ¾ -ゥム緩 欲（pH5 . 0 ) + 0 . 15 M食壤で展開した。 5 づっ分取し、 目的物を含むフタタ 《ン（ 100-15 を集め 7 0 のフ ク 轚ンの蛋白煑含量は牛血淸アルブミ ン 機準として ーリ - ( Lowry ) 法で澜定すると 84 g/Wであった。 また黢分解 物（ 逾酸， 1 10 'C , 24時閱）中のアミノ酸分析値は以下の如く であった。 ： Asp, 12.2( 12)； T r, 10.4( 10)； Ser, 16.0 (14); Giu, 24.8(26)； Pro, 6.0(5)； ai7» 6.3(5)} Ala, 8.6(8)； Val , 6.5(7) i Met. 4.0(5) I lie, 7.8 (8); Leu, 21.0(21); Tyr, 5.2(5)； Phe , 9.9(10) { Lya, 6.5； His, 3.8(3)； Arg , 9.1(9)； Cya , Trp , 分 解、 r I FN- β Aには 1 1 IS Lya が含まれて るので、上纪の結 果から、 インター 7Λロン a中の Lysの * -ァミノ基の約 41 リエチレングリコ- メチ ;uェ-テ （平; 00 )で條銪さ れて る 21とが分った。 本品は酵素免疫測定法〔メソ ク ド イ ン ェン ザィモロジ- ,第 73卷， 583 -535¾( 1 381 )〕で溯定した 結果 1 · 5 Iズ 1 07 国際单位 で、 ジャーナ ォブ ビ ロジー， 第 37卷 755 -758頁（ 1981 )に記載の方法 後 溯定した 抗ウイ ス活性は 3 .57 X ί 07国際单位 Ζ であ，た。本 (140W) and stirred at 37¾ for 40 hours. The reaction solution was poured into a cephadex G-75 (3.0 dia., 43.0 «), and developed with 25 mM ammonium sulfate-laminate (pH 5.0) + 0.15 M soil. . Was collected 5 Dzu' min, over Li protein煑含amount of off click轚N of collected 7 0 Futata "emissions (100-15 as Ushichi淸albumin machine level containing the desired product - when澜定in (Lowry) method The analytical value of amino acids in the hydrolyzate (puric acid, 110 ° C, 24 hours) was as follows: Asp, 12.2 (12); Tr, 10.4 Ser, 16.0 (14); Giu, 24.8 (26); Pro, 6.0 (5); ai 7 »6.3 (5)} Ala, 8.6 (8); Val, 6.5 (7) i Met. 4.0 (5) Ilie, 7.8 (8); Leu, 21.0 (21); Tyr, 5.2 (5); Phe, 9.9 (10) {Lya, 6.5; His, 3.8 (3); Arg, 9.1 (9); cya, Trp, decomposition, the r I FN- β a contains 1 1 iS Lya Runode, from the results of the above Chronicles, inter 7 lambda Ron a solution of the Lys * - about 41 Riechiren of Amino groups Glyco-methine was determined to be 21 as defined by U-tete (flat; 00) .This product was determined by enzyme-linked immunosorbent assay [Methods Enzymology, Vol. 73, Vol. -535¾ (1 381)] Results 1 · 5 I 1 0 7 International单位, journal O Bed bi biology, anti Huy scan activity as Sakanobojo after process according to paragraph 37 Certificates 755 -758 (1981) is 3 .57 X ί 0 7 International It was 单 rank Ζ. Book
一 3 )¾後述のラプト J おけるクリアランスの突 I C供した。  1 3) (4) Provided a clearance IC at Lapto J described later.
(I) 参考例 1で得た平均分子量 750のボリヱチレングリコ- メチ エーテ ァ/ uデヒド 1 00^， ァノ水素化ホウ素ナト リウム 1 00 ^を用 て、（ と同様に r I S - Aを耀すると 1 30 も, ml 蛋白質量のボリエチレングリコ - メチルェ -テ Ηίίϋ I F Ν - βの潘 液 3 が得られた。 黢分解物（ S Η檯酸， 1 1 (PC， 24時間）中の アミノ鑀分析値は J5TFの如くであった。 ： Asp, 12.1(12)1 Tiir, 10.1(10); Ser, 13,6(14) J aiu, 26.7(26) \ Pro ,5.5 (5)； Sly, 5.6(5)；· Ala, 8.4(8)； Val, 6,7(7 Met, 5,5(5); He, 7.4(8); Leu, 21.0X21) ! Tyr, 5. l(5)i Phe, 9.6(10)； Lys, 4.7； His, 3.5(3); Ar , 9.1 (9)i Trp, 1.8(2)； Cjrs, 分^ この結果から、本品は約 57 の Lys の《 -ァミノ基が修飾されてお]?、 Π)と同接に酵素免疫法で 測定した結果 5 10β 国際単位 で、抗ウイ ス活拄は 0 . 14 X 1 08国凝単位 であった。 (I) Using the poly (ethylene glycol-methacrylate / u-dehyde 100 ^ with an average molecular weight of 750 obtained in Reference Example 1 and the sodium borohydride 100 ^, similarly to (r-IS-A As a result, 1-30 ml of poly (ethyleneglycol-methyl-te) -IF-β-Ban solution with a protein content of 30 ml was obtained. 黢 Decomposed product (S Η 檯 acid, 11 (PC, 24 hours) Amino-analytical value of was as J5TF: Asp, 12.1 (12) 1 Tiir, 10.1 (10); Ser, 13,6 (14) Jaiu, 26.7 (26) \ Pro, 5.5 (5) Aly, 8.4 (8); Val, 6,7 (7 Met, 5,5 (5); He, 7.4 (8); Leu, 21.0X21)! Tyr, 5. l (5) i Phe, 9.6 (10); Lys, 4.7; His, 3.5 (3); Ar, 9.1 (9) i Trp, 1.8 (2); Cjrs, min ^ Lys 《-amino group is modified] ?, Π) as measured by enzymatic immunoassay 5 10β international units and antiviral activity is 0.14 X 10 8 national units there were.
(¾ 参考例 1で得た平均分子量 750のボリエヂレングリコ- メチ エーテルァ デヒド 2 , ^ァノ水素化ホウ素ナトリウム 2  (¾ Polyethylene glycol-methyl ether aldehyde 2 with an average molecular weight of 750 obtained in Reference Example 1, ^ sodium borohydride 2
用 t(i)と同様に処理すると、 45 <gZWの蛋白貧量のボリエチレン グリコ一 メチ エーチ V修鳒 I PIT - a 5 が得られた。 黢分解物 ( δΐτ瘇黢， 1 10*0 , 24時間）中のアミノ酸分析値は以下の如くで あった。 ： Asp, 13.6(12); Thr, 10.4(lQ)t Ser , 1 .9(14); &l , 26.6(26)； Pro, 5.5(5) J Oly, 6.1(5)； Ala, When treated in the same manner as in t (i), poly (ethylene glycol) methylated V-modified I PIT-a5 having a protein content of 45 <gZW was obtained.ア ミ ノ 酸 Analytical values of amino acids in the degradation product (δΐτ 瘇 黢, 110 * 0, 24 hours) were as follows. Asp, 13.6 (12); Thr, 10.4 (lQ) t Ser, 1.9 (14); & l, 26.6 (26); Pro, 5.5 (5) J Oly, 6.1 (5); Ala,
O PI 8.3(3)； Val, 6.6(7) J Met, 5.2(5)； lie, 7.4(8)； Leu, 21.0(21) ? Tyr, 5.3(5)1 Phe , 10.2(10) 1 Lye, 9.0; His, 3.6(3); Arg, 9.1(9); Trp, 2.3(2)i Cys, 分解 この锗果から、本品は約 1 8%の l rs の ァミノ基が修駕さ れてお]?、（i)と同様に酵素免疫法で溯定した錶果 1 · 09 X ί 08国際 単位 で、 抗ウィ ス活性は〗 . 53 X ， 08国際単位 であつ 7 = 上記 (i)で得た本発明の化学修鍊 Ι Ρ Η - « ( Ι ϊ"Α - 3 )を、 1 . 274 X 1 0s 単位づっ、 雌性 7週令の s Dラ トの大腿部筋肉に 1群 3匹づっ注射した。 一定時間後に、 尾部縿撇より採血し、 血 中の I Fir - β力価を実施例 3 記載の酵素免投法および抗ウィ ス活性 により澜定した。 非修飾のインタ -フ ロン ( Γ ΐ ΕΊΓ - α Α )¾: 1 . 259 ^ 1 0° 単位づ.つを投与しえ群に比較して明らか *クリてラ ンスの遅延化が認められた。 O PI 8.3 (3); Val, 6.6 (7) J Met, 5.2 (5); lie, 7.4 (8); Leu, 21.0 (21)? Tyr, 5.3 (5) 1 Phe, 10.2 (10) 1 Lye, 9.0 ; His, 3.6 (3); Arg, 9.1 (9); Trp, 2.3 (2) i Cys, decomposition From this result, it is found that this product has about 18% of l rs amino groups surpassed. ]?, in錶果1 · 09 X ί 0 8 international units was Sakanobojo by enzyme immunoassay method in the same manner as in (i), the anti-Wie scan activity〗. 53 X, 0 8 Atsu 7 = above in international units (i ) the chemical Osamu鍊iota [rho Eta of the present invention obtained - «(Ι ϊ" Α - . a 3), 1 274 X 1 0 s units Dzu', the thigh muscles of s D rat-female 7-week-old Injections were made in groups of three per animal After a certain period of time, blood was collected from the tail 縿 撇, and the I Fir-β titer in the blood was determined by the enzyme-free method and the anti-viral activity described in Example 3. Interferon (Γ ΐ ΕΊΓ-αΑ) ¾: 1.259 ^ 10 ° units per dose. * Compared to the control group, * Crying was delayed.
れらの結果を第 1図に示す。  Figure 1 shows the results.
^施例 4 ^ Example 4
実施例 3 (ϋで得た本発明の化学條飾 I FH - e ( I ff A- 3 )の溶液 5 Wに 250 Wの ト血淸ア ブミン 如えて溶かす。 本溶液をメンブ ヲンフィ タ- (ボア-サイズ： 0 . 2 A tn )で泸過し、 5個のパイァ ルに小分けする。.無菌的に凍糠乾燥して保存し、 使用直前に注射用蒸留 水， Wに港かして使用に供する。  Example 3 (A solution of the chemical condition IFH-e (IffA-3) of the present invention obtained in (1) was dissolved in 5 W of 250 W of blood s-abumin, etc. The solution was dissolved in a membrane filter ( Bore-size: 0.2 A tn), subdivide into 5 pellets. Aseptically freeze-dried and stored, and put in distilled water for injection, W immediately before use. Serve for use.
参考例 \ ボリエチレングリコー メチ ! /エーテ /« ァ デヒドの合成 Reference example \ Polyethylene glycol methyl! / Ate / «Synthesis of aldehyde
(I) ボリエチレングリコールメチ エーチ/ u( 59，平均分子 4  (I) Polyethylene glycol methacrylate / u (59, average molecular weight 4
5,000 ) *¾化メチレン（ 1 0 に溶かし、 ク》 ク ム酸ビリ ジ-ゥム （ 330，）を加え、 室温で， 2時 Wかきまぜた。 反応液を2 倍量の ¾化メチレンでうすめて、 フロ リジ の力，ム （ 6x 1 0α·)に 一"一 5,000) * Methylene dioxide (dissolved in 10 and added) Bi-dicum-peroxide (330,) was added, and the mixture was stirred at room temperature for 2 hours W. The reaction solution was diluted with twice the amount of methylene peroxide. To the power of Florisi, 6x10α · One "one
注ぎ込み、 カラムを 化メチレン ，つ でクロ ホ ムで洙つたのち、 メタノー —タ cr isホ ム ( f ： 9 )で溶出した。 2 , 4 -ジ-トロプ ド，ジンテストで攝性の画分 *集めて、溶媒を滅圧留去し、. I* 晶性の 7プクスを得 。 収量， . 5 , ( 30 ) ，薄層ク マトグラフ ィー ： Bf» 0.08 (クロ口ホ ム ： メタノー〃：齚酸霍 9 : 1 ：After pouring the column, the column was washed with methylene chloride and chloroform, and then eluted with methanol (cris) (f: 9). Collectable fractions in 2,4-di-troped and gin test * were collected and the solvent was decompressed and distilled off to obtain 7 pixs of .I * crystallinity. Yield, .5, (30), thin-layer chromatography: Bf »0.08 (black mouth: methanol: 9: 1)
0.5. リカゲ ） ，13C -N KRT3 6.2 P M に水和した型 (一 CH( 0H)2) でアルデヒ ド基の吸収を認め 7 e 0.5. Rikage), 13 C -N KRT3 6.2 7 observed the absorption of aldehyde groups in hydrated mold PM (one CH (0H) 2) e
(ί) ボリエチレングリ =t一 メチ ェ-尹〃（ 1 0 f ，平均分子量 5,000 )を三綾ブタノール（ 1 00 に溶かし、 力リウム Ξ級ブト キ ド（ 4► 1 了 ) ¾加え、 つ でブロムァセター  (ί) Polyethylene glycol = t-methy-yin (10 f, average molecular weight 5,000) is dissolved in triabutanol (100), and potassium hydroxide is added to the tributane (4►1 end). In Bromaceter
加え、 4 (TCで 2時間かきまぜた。 三級ブタノ- を滅圧下曹去し、 残留物に水を加え、つ でクロ ホ ム（ 200 2 )で抽出した。 水で洗 、無水铳酸ナト リウムで乾燥した。 クロ口ホルムを滅圧下留去 し、残留物に石油ベンジンを加え、 生ずる緒晶性残渣を泸取し、 エーチ で洗浄 ¾；对応するボリエチレングリコ - メチ エーテ ジェチ ァ セター 9.5 f ( 95 % )が得られた。 この内 5 ί1を取])、 0.0 Ηトリフ ォ ^睁黢 5 に溶かし、 瀕とう水中で 30分膊 理したあ と凍結乾澡し、（1)で得たものよ 1?も一 0 - CH2CH2だけ鎖長の長 ボリェ チレングリコ - /Vメチ； I エーチルア デヒ ド ^得られた。 In addition, the mixture was stirred for 4 hours with TC (tertiary butano) was removed by soaking in sodium carbonate under decompression, water was added to the residue, and the mixture was extracted with chloroform (200 2). The chloroform was distilled off under reduced pressure, petroleum benzene was added to the residue, and the resulting crystalline residue was collected and washed with H.sub.2; the corresponding polyethylene glycol-methyl methacrylate 9.5 f (95%) was obtained. of these preparative 5 ί 1]), dissolved in 0.0 Eta triflumizole O ^睁黢5, and 30 minutes膊sense in danger shaking water frozen Inui澡and Taha, (1 ) obtained in those by 1 also Ichi 0 - CH 2 CH 2 only chain length of the long Borye Chirenguriko - / V-methylol;? I Echirua de human de ^ obtained.
m ボリエチレングリコー メチ エーテ （ 5 . 7 f ,平均分子：！ 1 900 )を¾化メチレン（ 1 00W)に溶かし、 クロ^ク ム酸ビリ ジ-ゥム（ 97 を加え、 室温で， 2時間かきまぜた。 反応液を ¾ 化メチレンで希釈し、 プ ジ のカラム（ 6 . 0 x 1 0 . 0«)に注 ぎ込み、 カラム！: ¾化メチレン ，つ でクロ口ホ ムで洗ったあと、 13 %メタノー /ク Pホ ムで溶出した Λ 2 , 4-ジ-トロフエ- 一 IS— m Polyethylene glycol methylate (5.7 f, average molecular weight: 1900) is dissolved in methylene chloride (100 W), and added with bis-dimethyl chromate (97, at room temperature for 2 hours) Dilute the reaction mixture with methylene chloride, pour it into a Puzi column (6.0 x 10.0 «), and wash with column! , and eluted with 13% methanol / click P ho arm lambda 2, 4-di - Torofue - One IS—
ドラジンテストで陽性の函分を集めて、溶媒 ¾留去すると結晶住の ワックスを得 Tto 収 i 1 . f ( 30 % ) '薄層ク Pマトグラフィー： Rf-0.10(ク ホ !ム ：メタノ - ：齚黢- 9 ： 1 ： 0 · 5 ， リ 力ゲル） 1SC - 1TMRで S 6 . 2 PPM に;^した形（一 £3(0H)2 ) でァ !デヒド基の吸収 ¾認めた。After collecting the components positive in the drazine test and removing the solvent by distillation, a wax of crystalline form is obtained. Tto yield i 1. f (30%) 'thin layer P-Patography: Rf-0.10 (K: Methano-: 齚 黢 -9: 1: 0 · 5, resilience gel) 1S C-1TMR to S 6.2 PPM; absorption of aldehyde group in ^^ form (one £ 3 (0H) 2 ) ¾ Admitted.
v) ボリエチレングリコー； メチ^ヱ一チ /1 1 9 . 5 / »平均分子量 1 900 )を三級ブタノ - （ 00W)に溶かし、 力リウム三級ブト キ ド（ ， 0 . 4 f )を、 つ でブロムァセター （ 6 . 4W)を加え、 4 (3 ¾で 2時間かきまぜた。 三緣ブタノ -ル¾滅圧で留去し、 残留物に 水 *加え、ついでクロ ホ ム（ 20 OWx 2 )で抽出し 7t。反応液を 水洗，つ で無水硫鬚ナト リウムで乾^した。 クロ ホ; tムを滅圧下留 去し、幾留物に石油ペンジンを加え、生ずる結 A 物を^取し、'ェ 一テ で洗浄しァセタール 8 . 5 ( 89 .536 ) 得た。 この内 39 *0 . 05ϋトリプ 才ロ酢黢に溶かし、 沸とう水中で 30分閼処理し たあと、 凍结乾； 8*し、（》)で得たものよ も- 0 - CS2C¾ -だけ鎖長の長 ボリエチレングリコ - メチルェ-テ ア デヒドが得られ 。 v) Dissolve polyethylene glycol; methyl / 1 チ 19.5 / »average molecular weight 1900) in tertiary butano- (00W) and add potassium tertiary butoxide (, 0.4 f). Then, bromaceter (6.4 W) was added, and the mixture was stirred for 4 hours (3¾) for 2 hours. Distillation was performed at a pressure of 30 butanol, and water * was added to the residue, and then chloroform (20 OWx 2 ), And the reaction mixture was washed with water, and then dried over anhydrous sodium sulfate.The chloroform was distilled off under reduced pressure, and petroleum pentane was added to the distillate. Then, it was washed with water and obtained acetal 8.5 (89.536), of which 39 * 0.05 was dissolved in tryptic vinegar, treated in boiling water for 30 minutes and frozen.结 Dry; 8 *, and the chain length is longer than that obtained in (>>) by -0-CS 2 C¾. Polyethyleneglycol-methyletheraldehyde is obtained.
M 平均分子量 750 ， 550 ， 350のボリエチレングリコ - メ チ エ-テ ¾：上 ¾と同捸の方法で対応するア デヒドに導 。  M Polyethyleneglycol with a mean molecular weight of 750, 550, and 350-Methylate: Derived to the corresponding aldehyde by the same method as above.
産 業 上 の 利 用 可 能 性  Industrial availability
本発明で餺造される化学侈飾蛋白質は、医薬品等として有用である。  The chemical luxury decoration protein produced by the present invention is useful as a pharmaceutical or the like.

Claims

一 1β— 請 求 の 範 囲 One 1β—Scope of billing
1. 生理活拴蛋白質の少¾くとも 1價の一綾ァミフ基に、  1. At least one price of the bioactive protein
R"^0 - C¾C¾ 基（ IIは末禱酸素の保 , nは任意に変わ j>うる 正の整数） 直接豬会して *る化学修鋒蛋白 ΙΤβ R "^ 0 - C¾C¾ group (II coercive youngest禱酸element, n represents optionally River j> ur positive integer) directly豬会to * Ru chemical Osamukissaki protein Iotatau beta
2. 生理活性蛋白 ¾と CHO( B よび nは2. Bioactive proteins ¾ and CHO (B and n
Figure imgf000018_0001
Figure imgf000018_0001
前 ¾と同意義 )で示されるア デヒドを還元菊の存在下反応させる と を特徼とする請求の範囲第 1項記載の化学修鎵蛋白 JTの製造法 β Before ¾ chemical Osamu鎵蛋white JT process for producing a range described first of claims to Toku徼the when the A-dehydro reacting the presence of a reducing chrysanthemum represented by same meaning) beta
E¾E¾
Ο ΡΙ Ο ΡΙ
つ J One J
" "
PCT/JP1984/000085 1983-02-07 1984-03-06 Chemically modified protein and process for its preparation WO1985003934A1 (en)

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AU24950/84A AU2495084A (en) 1983-02-07 1984-01-23 Takeout tong head
PCT/JP1984/000085 WO1985003934A1 (en) 1984-03-06 1984-03-06 Chemically modified protein and process for its preparation
AU33954/84A AU3395484A (en) 1983-03-25 1984-03-26 Kornige, freifliessende waschmittelkomponente und verfahren zu ihrer herstellung
AU28287/84A AU2828784A (en) 1983-04-09 1984-04-09 Vorrichtung zum aufbereiten von fliesfahigen materialien
AU28677/84A AU2867784A (en) 1983-05-12 1984-05-14 Aircraft undercarriage assemblies
PCT/JP1984/000575 WO1985003868A1 (en) 1984-03-06 1984-12-05 Chemically modified lymphokine and process for its preparation
JP60027283A JPH0676439B2 (en) 1984-03-06 1985-02-13 Chemically modified peptide hormone and method for producing the same
JP60037936A JPH0696599B2 (en) 1984-03-06 1985-02-26 Chemically modified lymphokine and method for producing the same
AT85102376T ATE46349T1 (en) 1984-03-06 1985-03-02 CHEMICALLY MODIFIED LYMPHOKINES AND PROCESSES FOR THEIR PRODUCTION.
EP85102376A EP0154316B1 (en) 1984-03-06 1985-03-02 Chemically modified lymphokine and production thereof
DE8585102376T DE3572982D1 (en) 1984-03-06 1985-03-02 Chemically modified lymphokine and production thereof
KR1019850001381A KR920007681B1 (en) 1984-03-06 1985-03-05 Process for production of chemically modified lymphokine
CA000475743A CA1255588A (en) 1984-03-06 1985-03-05 Chemically modified lymphokine and production thereof
US07/519,280 USH1662H (en) 1984-03-06 1990-04-05 Chemically modified lymphokine and production thereof

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