聚乙二醇定點(diǎn)修飾集成干擾素突變體Ⅱ

中國生物工程雜志China Biotechnology ,2008 ,28(4):17 ~20聚乙二醇定點(diǎn)修飾集成干擾素突變體II *牛曉霞'，2周敏毅2劉金毅子”孫超鐘茜‘吳曉東'(1中國科學(xué)院研究生院北京100049 2 北京三元基因工程有限公司北京102600)(3中國藥科大學(xué)藥學(xué)院南京210000 4 中國藥科大學(xué)中藥學(xué)院南京20009)摘要目的:用聚乙二醇(PEG)修飾集成干擾素突 變體I ( IFN-Con-m2 ,IIFNm2) ,通過(guò)純化獲得新型修飾分子并對該分子進(jìn)行抗胰蛋白酶水解穩定性及初步藥代動(dòng)力學(xué)研究。方法:將mPEG2000定點(diǎn)偶聯(lián)到IlFNm2的第86位Cys殘基上,修飾后的產(chǎn)物經(jīng)CM層析后,以SDS-PAGE考察其純度,用WISH-VSV系統進(jìn)行生物活性測定;在0.1%胰蛋白酶條件下考察體外抗酶解穩定性;并以SD大鼠進(jìn)行初步藥代動(dòng)力學(xué)研究,繪制血藥濃度-時(shí)間曲線(xiàn)。采用3P87軟件進(jìn)行數據擬合,分析藥物動(dòng)力學(xué)參數。結果:干擾素修飾率約為50%,且絕大多數以單修飾體( mono-PEC- IIFNm2)形式存在;提純后mono-PEC-IIFNm2的純度大于98% ,比活性約為修飾前IIFNm2的1%?？挂鹊鞍酌杆庠囼灡砻?30min后，IFNm2抗病毒活性殘留為8%,mon-PEC-1IFNm2為41%。初步藥代動(dòng)力學(xué)研究顯示:lIFNm的消除半衰期為(1.57 +0. 34) h, mono-PEC-1IFNm2為(18.0土4.0)h。結論:成功地偶聯(lián)了PEC和IIFNm2,建立了mono-PEC-IIFNm2的純化工藝，PEG修飾能增加IIFNm2的體外抗胰蛋白酶水解穩定性,并顯著(zhù)延長(cháng)體內半衰期。關(guān)鍵詞集成干擾素突變體 定點(diǎn)聚乙二醇修飾 藥代動(dòng)力學(xué) 半衰期中圖分類(lèi)號Q503干擾素a能夠治療多種腫瘤和病毒性疾病,但是1材料與方法由于血清半衰期太短,病人需要隔日甚至每日注射才能達到理想的治療效果。1.1材料聚乙二醇化技術(shù)是一種改善治療性蛋白質(zhì)在體內人羊膜細胞( WISH)、水泡性口炎病毒(VSV),為運輸的有效方法,經(jīng)臨床驗證, PEG修飾可以顯著(zhù)改善三元基因工程有限公司保存。mPEC20000購自北京鍵蛋白質(zhì)類(lèi)藥物體內藥代動(dòng)力學(xué)性質(zhì)|1-3)。凱科技有限公司,IFNm2原液為三元基因工程有限公本課題組曾利用定點(diǎn)突變技術(shù),構建集成干擾素司生產(chǎn)。DEAE Sepharose FF, CM Sepharose FF購自突變體I (IFN-Con-m2 , IlFNm2),在86位引人一個(gè)Amersham Pharmacia公司。Cys,獲得兼具高活性和可用PEG定點(diǎn)修飾的干擾素新SD大鼠,250 ~300g,雌雄各半,購自協(xié)和動(dòng)物所實(shí)分子(。驗動(dòng)物中心。本文擬在上述研究的基礎上,利用馬來(lái)酰亞胺1.2方法PEG衍生物對多肽分子內的Cys殘基進(jìn)行定點(diǎn)修飾，1.2.1 IFNm2的濃縮將IFNm2用A液(25 mmol/并對修飾產(chǎn)物進(jìn)行抗胰蛋白酶穩定性研究和初步藥代L Tis-HCl pH8. 0)稀釋后上樣到DEAE Sepharose FP動(dòng)力學(xué)試驗,為臨床開(kāi)發(fā)提供試驗依據。層析柱,上樣量e 50mg/ml填料。先用2個(gè)柱體積的A液沖洗中國煤化工:(25 mmol/L PB收稿日期:2007-12-12修回日期2008-01-21pH7.6)HC NMH GPB + 0.5mol/L●北京市科技計劃重大專(zhuān)項( D0205001040321)資助項目NaCl pH 7.6)洗脫，收集洗脫峰即為濃縮的IIFNm2。#通訊作者,電子信箱:niuniuam@ sobu. com1.2.2IFNm2的PEG修飾反應體系中lIFNm2濃.18中國生物工程雜志China BiotechnologyVol. 28 No. 42008度約為10mg/ ml , IFNm2與mPEG20000摩爾比為1:2，于其理論分子量。計算并稱(chēng)量mPEG粉末溶入一-定體積的B液,迅速與__.2濃縮IFNm2混合,置于2~8C ,50r/min攪拌12h后，muli-PEClated IFNm2用D液( lmo/L醋酸溶液)調節反應體系pH到5.0以下以終止反應。用12% SDS-PAGE ,碘染和藍染兩種染mono-PEClated IFNn2mPEC20000色方法5.1)檢測修飾率。1.2.3修飾產(chǎn)物的分離純化 將反應混合物用 E液(25 mmol/L AAB pH4. 5)稀釋20倍上樣到CMunnodifed 1IFNm2Sepharose FF層析柱。上樣結束后先用E液沖洗2~3個(gè)柱體積,再用F液(25 mmol/L AAB + 70 mmol/LNeCl pH4.5)沖洗8~10個(gè)柱體積,然后用C液(25圈1修飾產(chǎn)物的SDS-PAGE(碘染)Fig.1 SDS-PAGE analysis of conjugatesmmo/L AAB + 0.2 mol/L NaCl pH4. 5)洗脫，收集洗of PEG and IIFNm2( iodine staining)脫峰組分;最后用H液(25 mmol/L AAB + 0.5 mol/L1: Conjugate solution; 2: mPEG20000NaCl pH4. 5)洗脫,收集洗脫峰。洗脫產(chǎn)物用12% SDS-PAGE檢測純度。kDs1.2.4生物活性測定 用CPE法!"進(jìn)行生物活性測定,用Lowry法'"]進(jìn)行蛋白含量測定,兩者相除計算比nono-PEClated IFNm243.0活性。1.2.5體外抗胰蛋白 酶水解試驗分別取 IFNm2和31.0mono-PEG-IIFNm2各2ml ,加入0. 25%胰蛋白酶溶液使0.1其終濃度為0.1% ,混勻后置于37C,于0,2,5,10,15,unnodifed IFNm220 ,30 ,60 ,90min取樣,每次取樣后立即與含10%胎牛14.4血清的MEM培養基等體積混合均勻,凍存于-20C。待所有樣品取完,用CPE法檢測樣品中活性保留。繪圖2修飾產(chǎn)物的SDS-PAGE(藍染)制生物保留-時(shí)間曲線(xiàn)。Fig.2 SDS-PAGE analysis of conjugates of PEG1.2.6 初步藥代動(dòng)力學(xué)試驗 取SD大鼠8只,雌雄and IIFNm2( stained by coomassie brlliant blue)各半隨機分成2組,分別為IIFNm2組和mono-PEC-1: Conjugate eolution; 2: IFNm2; 3: Protein markerIIFNm2組,按臨床推薦人用劑量(3MIU ,5 x 10^IU/kg)2.2修飾產(chǎn)物的分離純化的約500倍皮下注射給藥。IlFNm2 組取血時(shí)間點(diǎn)為:修飾產(chǎn)物經(jīng)CM Sepharose FF層析柱進(jìn)行分離純化。0, 0.5, 1, 2, 4, 10, 24h;mono-PEG-IIFNm2組取血時(shí)洗脫先后經(jīng)F液G液和H液洗脫。其中F液主要用于間點(diǎn)為0, 0.5, 1,2,4,8, 12, 24, 48, 72, 96, 120,洗脫多修飾體( muli-BEC-IIFNm2) ,H液用于回收沒(méi)有144h。在設計的時(shí)間點(diǎn)眼底取血約0. 4ml,離心后血清修飾的千擾素,on-PE-IFNm2則主要存在于G液中，樣品放置在70C保存,CPE法[”檢測各血清樣品中千經(jīng)非還原性SDS-PACE檢測,純度高于98% (圖3)。擾素的濃度,繪制血藥濃度時(shí)間曲線(xiàn)。采用3P87軟件2.3生物活性測定進(jìn)行數據擬合，,分析藥物動(dòng)力學(xué)參數。采用CPE法,利用WISH-VSV系統測定修飾前后2結果干擾索的活性,結果見(jiàn)表1。表1 FNm2 和PEG-IIFNm2的生物活性2.1 IIFNm2 的PEG修飾L Im0no-PEG-IFNm2經(jīng)濃縮的IFNm2樣品，與mPEC20000在適宜的中國煤化工二t活性/(IU/mg)條件下進(jìn)行修飾反應,修飾后經(jīng)SDS-PAGE檢測發(fā)現TYHCNMH G410.73x10干擾素修飾率約為50%,修飾產(chǎn)物多以mono-PEG-mono-PEG-IIFNm25.61土0.59x106IIFNm形式存在(圖1,2)。修飾產(chǎn)物的表觀(guān)分子量大2008 ,28(4)牛曉霞等:聚乙二醇定點(diǎn)修飾集成干擾素突變體I19kDa .炙2 IIFNm2及mono-PEG-IIFNm2的藥代動(dòng)力學(xué)參數表生Table 2 Pharmacokinetic parameters43.031.0PK參數IIFNm2mono - PEC - IFNm2tl/2Ka(h)0.25 t0.1115.0+3.720.1t1/2Ke(h)1.57 +0.3418.0+4.0Tpeak(h)0.78+0. 1524.3+3.514.1Cpeak( IU/ml)23823.7 +3725.526941.7 +4853. 1AUC( IU●h/ml)76230.8士14407.3 1744758. 0 +258623.465.59 +14.2115.47 *3.38圖3非還原 性SDS-PAGE檢測純化的3討論mono-PEG-IIFNmFig,3 Analysis of purified monopegylated目前，已有PEG-Intron ( Schering-Plough)和IIFNm2 by nonreductive SDS-PAGEPEGASYS( Roche)兩種氨基修飾的PEG干擾素a2產(chǎn)1 ;Protein marker; 2: mono-PEC-1IFNm2;品上市,PEG干擾素在臨床應用中已得到越來(lái)越廣泛3: muli-PEC-1IFNm2; 4: IFNm2的研究091。在新藥研發(fā)階段,與以往將Iys作為修mono PE-IFNm2的比活性大于5.0 x 10° IU/mg,飾位點(diǎn)的非定點(diǎn)修飾不同,因為具有工藝簡(jiǎn)單和質(zhì)量約為修飾前IIFNm2的1%。易控的優(yōu)點(diǎn),以Cye作為修飾位點(diǎn)的定點(diǎn)修飾受到越來(lái)越多的關(guān)注(2-14)。2.4 體外抗胰蛋白酶水解試驗酶解反應后,用CPE法檢測樣品的保留活性,繪制相對于Lys非定點(diǎn)修飾,Cys定點(diǎn)修飾條件較為固活性保留時(shí)間曲線(xiàn)(圖4)。試驗表明: 15min后,定,因此本研究沒(méi)有過(guò)多的進(jìn)行修飾條件摸索。我們IFNm2抗病毒活性保留為25% , mono-PE-IFNm2為注意到高濃度的PEG和IFNm2可以增加修飾率,但高75%;30min后,IFNm2抗病毒活性殘留為8%,mono-度的IlFN1m2也會(huì )使二聚體大大增加,可能是由于高濃PEC-1IFNm2 為41%。度增加了干擾素分子間的碰撞機會(huì )所致。20 [在多數PEG修飾蛋白藥物的研究工作中，因為PEG改變了蛋白的理化性質(zhì),其純化工藝的建立都是.0080 t困難的,本研究采用分步洗脫的方式得到高純的單修60 t飾體,方法簡(jiǎn)便,可操作性強。為考察藥物的半衰期,本研究進(jìn)行了體外抗胰蛋白酶水解試驗和大鼠初步藥代動(dòng)力學(xué)試驗。研究發(fā)20 t現:PEG修飾后,IFNm2體外抗胰蛋白酶水解能力顯著(zhù)206C8100增強;經(jīng)3P87軟件擬合發(fā)現IFNm2及其PEG衍生物時(shí)間/min在SD大鼠體內的代謝規律符合-級吸收的一房室模圖4活性保留-時(shí)間曲線(xiàn)型。PEG化可顯著(zhù)改善IFNm2的藥代動(dòng)力學(xué)性質(zhì),與Fig.4 Curve of retention ativity-timeIIFNm2相比,mPEG- IFNm2的吸收半衰期、達峰時(shí)間、一◆- :IFNn2;-I -: PC-1IFNm2消除半衰期顯著(zhù)延長(cháng);藥時(shí)曲線(xiàn)下面積顯著(zhù)增加;清除.5 初步藥代動(dòng)力學(xué)試驗率顯著(zhù)降低?？梢?jiàn),PEC化可以延長(cháng)IFNm2的體內平.以SD大鼠皮下注射給藥2.5x10'lU/kg,眼底取均存留時(shí)間,降低清除率。血,用CPE法檢測各血清樣品中干擾素的濃度,繪制血我們注意到修飾后比活性降低較多,約為修飾前藥濃度時(shí)間曲線(xiàn)(圖略)。采用3P87軟件進(jìn)行數據擬IFNm2中國煤化工重要的活性位合,分析藥物動(dòng)力學(xué)參數(表2)。研究顯示:單修飾體點(diǎn)”]。|Y片CNMHG活性較高,mono-的消除半衰期為修飾前的11. 5倍,清除率顯著(zhù)降低。PEC-IIFNm2仍有較高的比活性;而且IIFNm2分子內含有包括86位Cys在內的若干IFNalb的保守序列,我20中國生物工程雜志China BiotechnologyVol. 28 No. 42008們期待mono-PEC-1IFNm2有較低的臨床不良反應。2005. Appendix 56 ~57[8]中國藥典委員會(huì )編.中華人民共和國藥典.2005 版三部.北參考文獻京:化學(xué)工業(yè)出版社, 2005.附錄30Chinese Pharmacopoeia Comite. Chinese Pharmacopoeia,[1] Molineux G, Kinsler 0, Bridell B, et al. A new fom of2005 Edition Volume M. Beijing: Chemical Industial Pree,filgrastimn with sustained duration in rivo and enhanced abity to2005. Appendix 30mobilize PBPC in both mice and humans. Exp Hematol, 1999,[9] Lai L, Hui C K, Leung N, et al. Pegylated itereren alpha2a27 :1724 ~ 1734(40 kDa) in the treatment of chronic hepatis B. Int J[2] Bailon P, Plleromi A, Schaffer CA, et al. Rational design of aNanomedicine, 2006, 1(3) :255 ~262potent, long-acting form of interferon: a 40 kDa branched10] Sifman ML, SuterF, BeconBR, et alPeginterferon alfa-polyethylene eycal-conjugated iterferon a2a for the treatment2a and ribavirin for 16 or 24 weeks in HCV genospe2or3. Nof hepatitis C. Bioconjugate Chem, 2001, 12:195 ~202Engl J Med, 200, 357(2):124 -134[3] BellSJ, Fam C M, Chlipala E A, et al. 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LIU Jin-yi'SUN Chao' ZHONC Qian' WU Xiao dong'(1 Graduate Univesity of Chinese Academy of Sciences Bejing, 10049 , China)(2 Beijing Tri-prime Genetic Engineering Co. lId. , Beiing 102600, China)(3 Pharmaceutical Science Department, China Phamaceutical University, Nnjjing 210009, China)(4 Chinese Traditional Pharmacy Department, China Pharmaceutical University , Nanjing 210009, China)Abstract Recombinant interferon alpha ( IFN-alpha) has been proven useful for treating a variety ofhuman cancers and viral diseases. Because of its short circulating half-life IFN-alpha is administered to patientsby daily or thrice weekly injection for optimal effectiveness. Recombinant consensus interferon mutant I (IFN-Con-m2 , IIFNm2) was a mutant from IFN-Con with a free cysteine residues at position 86. The protein could bemodified with a 2 kDa maleimide PEG and the mono-PEGylated proteins were purified by CM Sepharose FastFlow column chromalography. Mono-PEGylated IIFNm2 could be separated from muli-PEGylated IIFNm2 andunmodified IIFNm2 by stepwise elution. After purifcation, the purity of mono-PEC-IIFNm2 was up to 98% , andthe biological activity was more than 5. 0 x 1061U/mg. The test of anti-typsin digestion suggested that PECylationcould improve the ability of avoiding the trypsin digestion. Pha中國煤化Its demonstraled thatthe circulating half-life of the PEGylated protein was up to 1;YHC N M H GIFNm2. These datacould demonstrate the utility of site- specific PEGylation for creating highly potent, long-acting IIFNm2.Key words Consensus interferon mutant Site-Specific Pegylated Pharmacokinetic Circulating half-life