第10卷第3期過(guò)程工程學(xué)Vol 10 No.32010年6月The Chinese Joumal of Process Engineering胰高血糖素樣肽-1在乙醇中的聚乙二醇修飾王友傲1,翟艷琴‘,雷建都',馬光輝',蘇志國(guó)1(1.中國(guó)科學(xué)院過(guò)程工程研究所生化工程國(guó)家重點(diǎn)實(shí)驗(yàn)室,北京100190:2.中國(guó)科學(xué)院研究生院,北京10049)摘要:以乙醇為溶劑,單甲氧基聚乙二醇琥珀酰亞胺碳酸酯( mPEG-SO)為修飾劑,對(duì)胰高血糖素樣肽-1(GLP1)進(jìn)行了修飾反應(yīng)條件的優(yōu)化,同時(shí)對(duì)單修飾產(chǎn)物Mono- PEG-GLP-1進(jìn)行分離純化,并考察了其體外穩(wěn)定性和體內(nèi)活性.得77%采用冷凍離心方式對(duì) Mono-PEG-GLP1進(jìn)行初步分離和濃縮,然后經(jīng)反相液相色譜進(jìn)一步高效純化,純度可達(dá)97%以上體外實(shí)驗(yàn)表明, Mono-PEG-GLP1在血清中具有更好的穩(wěn)定性及更強(qiáng)的抗胰蛋白酶消化能力.體內(nèi)動(dòng)物實(shí)驗(yàn)表明, Mono-PEG-GLP1具有更好的降血糖作用關(guān)鍵詞:胰高血糖素樣肽-1:單甲氧基聚乙二醇琥珀酰亞胺碳酸酯;聚乙二醇修飾;乙醇中圖分類號(hào):Q5143;R915文獻(xiàn)標(biāo)識(shí)碼:A章編號(hào):1009606X(201003-0593-051前言率需加入過(guò)量修飾劑(修飾劑與GLP-1摩爾比達(dá)21甚至5:1以上),易導(dǎo)致 Mono-PEG-GLP.1繼續(xù)與胰高血糖素樣肽-1( Glucagon-like Peptide-1,GLPl) mPEG-SC反應(yīng)生成多修飾產(chǎn)物連接2個(gè)或2個(gè)以上聚是體內(nèi)L細(xì)胞分泌的葡萄糖依賴性腸降血糖多肽激素,乙二醇鏈的修飾產(chǎn)物),造成修飾反應(yīng)所得具有阻止胰腺細(xì)胞退化、刺激細(xì)胞的增殖和分化、促進(jìn) Mono-PEG-GLP的轉(zhuǎn)化率較低,最多僅為50%胰島素生成等作用.在2型糖尿病(Type2 Diabetes另一方面,由于修飾產(chǎn)物組分很復(fù)雜,含未修飾的Mellitus,T2DM治療方面有良好的應(yīng)用前景,成為近年GLP1、多修飾GLPl、單修飾GLP1及未反應(yīng)的修飾來(lái)糖尿病治療藥物的研究熱點(diǎn)但GLP1易被體內(nèi)二肽劑等組分,雖然可用反相液相色譜分離,但修飾反應(yīng)液基肽酶Ⅳ等酶降解,易被循環(huán)系統(tǒng)清除,半衰期很短是乙醇溶液,導(dǎo)致 Mono-PEG-GLP-I在色譜柱上不易吸(2min左右).另外,因具有免疫原性和抗原性,大大限附,因此不能直接將反應(yīng)液上樣,在采用反相液相色譜制了其臨床應(yīng)用14進(jìn)行分離前需將反應(yīng)液用超純水稀釋5倍,導(dǎo)致多次上蛋白質(zhì)或多肽類藥物的聚乙二醇[ Poly(ethylene樣分離,增加了分離純化的成本和時(shí)間,不利于放大制gyco,PG修飾技術(shù)是一種通過(guò)化學(xué)反應(yīng)將蛋白質(zhì)或備多肽分子與聚乙二醇大分子共價(jià)連接的技術(shù),其突出優(yōu)本研究以乙醇為反應(yīng)溶劑,采用分子量5kDa的單勢(shì)在于能降低藥物的免疫原性和抗原性,減少循環(huán)系統(tǒng)甲氧基聚乙二醇琥珀酰亞胺碳酸酯( mPEG-SC)修飾劑清除速率,從而有效延長(zhǎng)藥物體內(nèi)循環(huán)半衰期現(xiàn)已有對(duì)GLP1進(jìn)行修飾,考察了修飾反應(yīng)條件對(duì)10種長(zhǎng)效聚乙二醇藥物經(jīng)美國(guó)食品及藥物管理局批準(zhǔn) Mono-PEG-GLP1轉(zhuǎn)化率的影響.分離純化過(guò)程中采用上市,取得了巨大的經(jīng)濟(jì)效益和社會(huì)效益56低溫冷凍離心方法對(duì)修飾反應(yīng)液進(jìn)行預(yù)處理,在去除未目前主要采用修飾劑單甲氧基聚乙二醇琥珀酰亞修飾GLP1的同時(shí)獲得高濃度的 Mono-PEG-GLP-1,進(jìn)胺丙酸酯 [Monomethoxy Poly(ethylene glycol而結(jié)合反相液相色譜獲得純度高達(dá)97%的單修飾產(chǎn)物succinimidyl propionic acid esther),mPEG-SPA、單甲最后,考察了 Mono-PEG-GLP在血清中的穩(wěn)定性、抗氧基聚乙二醇琥珀酰亞胺碳酸酯[ Monomethoxy胰酶酶解能力及降糖效果Poly(ethylene glycol succinimidyl carbonate), mPEG-SCI進(jìn)行GLP1的修飾研究,但還存在一些問(wèn)題,如二者2實(shí)驗(yàn)易水解,在pH80、溫度25℃條件下,其半衰期分別2.1實(shí)驗(yàn)材料與儀器僅有165和204min9為提高 Mono-PEG-GLP1轉(zhuǎn)化胰高血糖素樣肽-1(GLP-1)由北京中科亞光有限公收稿日期:201003-29,修回日期:201005-14基金項(xiàng)目:國(guó)家自然科學(xué)基金資助項(xiàng)目編號(hào):20976179):國(guó)家高技術(shù)研究發(fā)展計(jì)劃(863)基金資助項(xiàng)目編號(hào):2007AA021604:2006AA10Z437)北京市自然科學(xué)基金資助項(xiàng)目(編號(hào):2092027)作者簡(jiǎn)介:王友做(1986-),男,安徽省舒城縣人,碩士研究生,生物化工專業(yè):雷建都,通訊聯(lián)系人,Te:01082623692, F-mail:jdei@ home. ipeac cn.過(guò)程工程學(xué)報(bào)第10卷司提供,分子量5kDa的mPEG-SC由本實(shí)驗(yàn)室利用進(jìn)樣量100μ通過(guò)軟件計(jì)算GLP及mPEG活化而得,mPG( Polydispersity mPEG<1.08)Mono- PEG-GLP-1在色譜圖上對(duì)應(yīng)峰面積與初始峰面積購(gòu)自美國(guó) Union-Carbide公司,乙腈( Acetonitrile,ACN)、的比值,研究二者在大鼠血清中的保留行為三氟乙酸為色譜純,購(gòu)自北京 Dikma公司;乙醇、甘氨動(dòng)物體內(nèi)活性檢測(cè)2:將體重300-340g的SD大酸為化學(xué)純,購(gòu)自北京化學(xué)試劑公司.實(shí)驗(yàn)用水為鼠隨機(jī)分成3組,每組5只.實(shí)驗(yàn)前只給飲水,禁食過(guò)Millipore超純水機(jī)制備的電阻率182Mncm的超純水.夜,A組為空白對(duì)照,B組為GLP1對(duì)照,C組為實(shí)驗(yàn)用大鼠血清、SD大鼠均購(gòu)自北京大學(xué)醫(yī)學(xué)部實(shí)驗(yàn) Mono-PEG-GLP1實(shí)驗(yàn)組.3組均腹腔注射葡萄糖18動(dòng)物中心molko,A組腹腔注射生理鹽水作為對(duì)照,B組腹腔注IC20AT高效液相色譜儀(日本 Shimadzu公司),射18 nmolkg GLP1,c組腹腔注射18 nmol/kg高速離心機(jī)(美國(guó) Sigma公司)Mono-PEG-GLP-1.在0,5,60,150min時(shí)眼底取血,用2.2實(shí)驗(yàn)方法血糖儀測(cè)定血糖濃度PEG修飾反應(yīng):按一定摩爾比稱取GLP-1與mPEG-SC,溶解于乙醇中,37℃反應(yīng)一定時(shí)間,加入3結(jié)果與討論過(guò)量甘氨酸終止反應(yīng)3.1反應(yīng)條件對(duì)單修飾產(chǎn)物轉(zhuǎn)化率的影響Mono-PEG-GLP1的分離純化采用島津CBM20A聚乙二醇修飾中反應(yīng)條件的控制是影響目標(biāo)產(chǎn)物型液相色譜系統(tǒng),色譜柱為C18反相色譜柱(250mmx含量的重要因素.目前關(guān)于有機(jī)相如乙醇中的聚乙二醇46mmLD),洗脫液A為超純水+0.1%三氟乙酸,洗脫修飾研究報(bào)道較少,而GLP-l在乙醇中的修飾則未見報(bào)液B為乙腈∽0.1%三氟乙酸.反相液相色譜洗脫方法:道.為此,實(shí)驗(yàn)重點(diǎn)考察了 mPEG-SC與GLP-1摩爾比、0-5min35%B,5~25min35%55%B,25-30min35%B,GLP-濃度和溫度對(duì) Mono-PEG-GLP-l轉(zhuǎn)化率的影響B(tài)液的梯度變化由色譜系統(tǒng)按程序直接完成,流速13.1.1 mPEG-SC與GLP-1摩爾比對(duì) Mono-PEG-GLP1mL/min,檢測(cè)波長(zhǎng)280mm,進(jìn)樣量100μ.通過(guò)軟件轉(zhuǎn)化率的影響計(jì)算 Mono PEG-GLP-1峰面積占各組分吸收峰面積之圖1是 mPEG-SC與GLP1摩爾比對(duì)產(chǎn)物轉(zhuǎn)化率的和的百分比得 Mono-PEG-GLP-1轉(zhuǎn)化率影響,表明 mPEG-SC與GLP1摩爾比較低時(shí),采用島津CBM-20A型高效凝膠液相色譜系統(tǒng)檢測(cè)Mono- PEG-GLP-l轉(zhuǎn)化率隨摩爾比增大而升高;達(dá)到Mono- PEG-GLP.1純度,色譜柱為 uperdexTM75,流定水平后,隨摩爾比增大, Mono-PEG-GLP-1轉(zhuǎn)化率呈動(dòng)相為50mmo冮L(zhǎng)磷酸緩沖液+100mmol幾L硫酸鈉+0.5%逐漸下降趨勢(shì).這是由于 mPEG-SC與GLP-1摩爾比越疊氮化鈉,pH6.8,流速05 mL/min,檢測(cè)波長(zhǎng)280m.大,多修飾產(chǎn)物越易生成,從而降低了 Mono-PEG-GLP21l胰蛋白酶消化:按等摩爾比將一定濃度的GLP1轉(zhuǎn)化率.因此選擇 mPEG-SC與GLP1摩爾比為1進(jìn)行和 Mono-PEG-GLP.1溶于20mmoL的磷酸緩沖液中修飾研究文獻(xiàn)[8]在水溶液中采用 mPEG-SC進(jìn)行的pH7.0),根據(jù)一定摩爾比加入胰蛋白酶,37℃孵育,GLP-1修飾, mPEG-SC與GLPl摩爾比需達(dá)21甚至分別在0,15,30,60,120min取樣,置于4℃冰箱中終止更高,其 Mono-PEG-GLP.轉(zhuǎn)化率僅為50%反應(yīng).用反相液相色譜檢測(cè)反應(yīng)物的殘留量與加入量之比,考察胰蛋白酶對(duì)反應(yīng)物的消化降解作用GLP1及 Mono-PEG-GLP.1的穩(wěn)定性實(shí)驗(yàn):將1mL大鼠血清加入20mL20mmo/L的磷酸緩沖液(pH74)中,配成血清緩沖液.純化后的GLP1及Mono-PEG-GLP1按02mmoL溶于血清緩沖液,37℃孵育.定時(shí)取樣,離心取上清液,用反相液相色譜進(jìn)百行分析檢測(cè),色譜柱為C18反相色譜柱(250mm×4.6mmID),洗脫液A為超純水+0.1%三氟乙酸,洗脫液0.8B為乙腈+0.1%三氟乙酸.反相液相色譜洗脫方法:0~5Molar ratio of PEG to GLP-1min35%B,5~20min35%50%B,20-25min100%B圖1PEG與GLP1摩爾比對(duì) Mono-PEG-GLP1轉(zhuǎn)化率的影響25-30mi35%B,B液的梯度變化由色譜系統(tǒng)按程序直 Fig 1 Effect of molar ratio of PEG to GLP-I on the yield of接完成。流速1 mL/min,柱溫35℃,檢測(cè)波長(zhǎng)280m,Mono-PEG-GLP-I第3期王友傲等:胰高血糖素樣肽1在乙醇中的聚乙二醇修飾312GLP1濃度對(duì) Mono-PEG-GLP.1轉(zhuǎn)化率的影響3.2冷凍離心結(jié)合反相液相色譜對(duì) Mono-PEG-礎(chǔ)LP-1的在聚乙二醇修飾過(guò)程中,肽濃度對(duì)單修飾產(chǎn)物轉(zhuǎn)化分離純化率有重要影響,GLP-1濃度對(duì) Mono-PEG-GLP1轉(zhuǎn)化修飾反應(yīng)結(jié)束后,反應(yīng)液中除 Mono-PEG-GLP-1率的影響見圖2.結(jié)果表明,低濃度時(shí) Mono-PEG-GLP1外仍存在少量未修飾的GLP-1、多修飾產(chǎn)物及未反應(yīng)的轉(zhuǎn)化率隨GLP1濃度增大而增大,到一定水平后, mPEG-SC.為此,對(duì)少量經(jīng)甘氨酸終止反應(yīng)的修飾反應(yīng)Mono-PEG-GLP1轉(zhuǎn)化率隨GLP1濃度增大而逐漸減小.液用水稀釋后采用高效反相液相色譜法進(jìn)行分離,計(jì)算這是由于在高濃度下反應(yīng)溶液中生成的多修飾產(chǎn)物多.Momo- PEG-GLP.1轉(zhuǎn)化率.在C18色譜柱上GLP-保留因此GLP-1濃度取1mg/mL時(shí)間為152min,Mono- PEG-GLP-1保留時(shí)間為193313溫度與反應(yīng)時(shí)間對(duì) Mono-PEG-GLP.1轉(zhuǎn)化率的影響min,二者分離度大于2,分離效果好,結(jié)果見圖4(a)在 mPEG-SC與GLP1摩爾比為1和GLP1濃度1和圖4b但在制備Mono- PEG-GLP1時(shí),由于修飾反mg/mL的條件下,考察了溫度對(duì) Mono-PEG-GLP-1轉(zhuǎn)應(yīng)液為乙醇,若直接上樣 Mono-PEG-GLP-1,不易在反化率的影響,結(jié)果見圖3.溫度升高提高了修飾反應(yīng)的相液相色譜的色譜柱上吸附.為此,需將反應(yīng)液用超純速度,Mono- PEG-GLP1轉(zhuǎn)化率提高;但溫度進(jìn)一步升水稀釋5倍后才能上樣分離,這就需多次上樣分離,增高,GLP1中的手性氨基酸的旋光性易發(fā)生變化而導(dǎo)致大了分離純化成本和時(shí)間,實(shí)驗(yàn)中發(fā)現(xiàn),活性降低,因此選擇37℃作為修飾反應(yīng)的溫度.修 Mono-PEG-GLP.1、多修飾產(chǎn)物在乙醇中的溶解度隨溫飾反應(yīng)進(jìn)行24h后,Mono- PEG-GLP1轉(zhuǎn)化率達(dá)到最大度降低而下降,因此采用低溫冷凍離心方式初步分離值.因此選擇溫度37℃,反應(yīng)時(shí)間24h.Mono-PEG-GLP.1,將反應(yīng)液在4℃條件下高速(0000008GLP-1 concentration( mg/mL)圖2GLP1濃度對(duì)Mono- PEG-GLP-1轉(zhuǎn)化率的影響圖3溫度對(duì) Mono-PEG-GLP1轉(zhuǎn)化率的影響Fig 2 Effect of GLP-1 concentration on the yield ofFig 3 Effect of temperature on the yield ofMono-PEG-GLP-lMono-PEG-GLP-1Mono-PEG-GLP.1GLP-1圖4GLP1、反應(yīng)混合物和低溫離心沉淀物的反相液相色譜圖Fig 4 Reverse phase high performance liquid chromatography profiles of GLP-1reaction mixture and sediment of reaction mixture under frozen condition過(guò)程工程學(xué)報(bào)第10卷r/min)離心15min后,將上清液、沉淀分別進(jìn)行檢測(cè),在大鼠血清中的穩(wěn)定性及抗胰蛋白酶降解作用結(jié)果見表1,表明冷凍離心有效去除了大量未修飾3.31GLP-1及 Mono-PEG-GLP1在大鼠血清的穩(wěn)定性GLP1,實(shí)現(xiàn)了修飾產(chǎn)物的預(yù)分離.將離心所得沉淀用GLP1與 Mono-PEG-GLP-1在大鼠血清中的穩(wěn)定性水溶解后,采用反相液相色譜進(jìn)行純化,結(jié)果見圖4(c),見圖6,顯示GLP-1在血清中很快被水解成無(wú)活性的收集 Mono-PEG-GLP-1[收集區(qū)間為圖4(c)中GLP1(9-36)NH2,而修飾后的 Mono-PEG-GLP1的184-20.36min]后用高效凝膠液相色譜檢測(cè),結(jié)果見圖穩(wěn)定性顯著增強(qiáng),在血清中可長(zhǎng)時(shí)間保持穩(wěn)定,孵育135,單修飾產(chǎn)物純度為97%,收率達(dá)95%以上h后仍保留90%,未發(fā)生明顯降解,表明表1 Modified妣LP1和P1在修飾反應(yīng)液、Mono-PEg-GLP-1比GLP1在血清中的穩(wěn)定性顯著提高離心沉淀和上清液中的含量332胰蛋白酶酶解穩(wěn)定性檢測(cè)Table 1 Modified-GLP-I and GLP-I contents in the reaction圖7是GLP-1與 Mono-PEG-GLP-1胰蛋白酶酶解動(dòng)力學(xué)曲線結(jié)果顯示,酶解05h后,GLP-l在酶解反應(yīng)液中僅有5%殘留,而 Mono-PEG-GLP-1仍有23.6%Modifed-GLP-1(%,保留,這表明修飾后的GLP-1能顯著增強(qiáng)抗胰蛋白酶降140解的能力Mono-PEG-GLP.11008000●- Mono-PEG-GLP1Elution volume(mL)圖5單修飾產(chǎn)物的高效凝膠液相色譜檢測(cè)圖0005152Fig 5 High performance gel filtration liquid chromatographyTimeof purified Mono-PEG-GLP-l by reverse phase high圖7胰蛋白酶酶解GLP1和Mono- PEG-GLP.1動(dòng)力學(xué)曲線performance liquid chromatographyFig 7 Digestion kinetics of GLP-I and Mono- PEG-GLP-l in3.3體外穩(wěn)定性檢測(cè)yasin solutionGLP1無(wú)復(fù)雜的二級(jí)結(jié)構(gòu),極易被酶解而失去活3.4體內(nèi)活性檢測(cè)性,在體內(nèi)的半衰期極短,僅2min.聚乙二醇修飾后,為了考察 Mono- PEG-GLP1與GLP1的體內(nèi)活性,能否抑制蛋白酶降解就成了檢定聚乙二醇修飾是否有將15只大鼠隨機(jī)分成3組,按18 mmol/kg劑量給大鼠效的主要方式,因此對(duì)比了 Mono-PEG-GLP.1和LP1腹腔注射葡萄糖溶液,并定時(shí)觀察老鼠的血糖變化,結(jié)果見表2.在給藥150min后, Mono-PEG-GLP.1的降血糖效果明顯優(yōu)于未修飾GLP-1,結(jié)果見圖8.由于GLP1在體內(nèi)半衰期短,所以其促胰島素分泌作用時(shí)間短,而mPEG-SC修飾后所得Mono- PEG-GLP1在體內(nèi)的半衰GLP-1期大幅度延長(zhǎng),促胰島素分泌作用時(shí)間長(zhǎng),因而降血糖ono-PEG-GLP-1效果更好表2P-1和 Mono-PEG叫LP1的降血糖效果比較(血糖濃度mo|/)Table 2 Effects of GLP-1 and Mono-PEG-GLP-l on plasma1012glucose homeostasis(blood ghsalino633±0.3424.28±0圖6GLP1和 Mono- PEG-GLP1在大鼠血清中穩(wěn)定性GLP-I6490451702±14511341078060.54Fig 6 Stability of GLP-I and Mono-PEG-GLP-l in rat plasma Mono-PEG-GLP-1 6.08#0.32 16.77:0.88 9.95+1.01 6.47#0.76第3期王友傲等:胰高血糖素樣肽1在乙醇中的聚乙二醇修飾Glucagon-like Peptide-1(7-37)in Diabetic and Nondiabetic Subjects[3]Nauck M A, Wollschlager DGlucagon-like Peptide-1 (GLP-1 [7-36 amide) in Patients withia,1996,39(12)1546-1553[4]Arulmozhia D K, Porthab B, GLP-1 Based Therapy for Type DiabetesEur. J. Pharm. Sci,2006,28(1/2):96-108[5]Crawford J. Pegfilgrastim Administered Once per Cycle Reduces62(Sl):8989[6]Zhai Y Q, Zhao Y J, Lei J D, et al. Enhanced Circulation Half-life ofSite-specific PEGylated rhG-CSF: Optimization of PEG Molecular圖8腹腔注射藥150min后大鼠體內(nèi)的血糖濃度Weight [] J BiotechnoL, 2009, 142(3/4): 159-166.Fig 8 Blood glucose concentration of the rats after[7]Lee S, Youn Y S, Lee S H, et al. PEGylated Glucagon-like Peptide-1drug injection for 150 minDisplays Preserved Effects on Insulin Release in Isolated Pancreatic結(jié)論lets and Improved Biological Activity in Db/Db Mice p]Diabetologia, 2006, 49(7): 1608-1611[8]張磊,翟艷琴,雷建都,等.胰高血糖素樣肽1的聚乙二醇定點(diǎn)(1)以分子量5kDa的 mPEG-SC為修飾劑,在乙醇修飾[過(guò)程工程學(xué)報(bào)2009,9(6:1169-1173.溶液中進(jìn)行GLP1聚乙二醇修飾,優(yōu)化反應(yīng)條件為:[9] Roberts M J, Bentley M D, Harris J M. Chemistry for Peptide andGLP-1濃度1mgmL,mPEG- SC/GLP-1摩爾比1:1,37℃Protein PEGylation [J]. Adv. Drug Del. Rev, 2002, 54(4): 459-476.[10] Zalipsky S, Barany G Facile Synthesis of a-Hydroxy-oo-反應(yīng)24h.該條件下 Mono-PEG-GLP1轉(zhuǎn)化率可達(dá)77%,carboxymethyipolyethylene Oxide. Bioact. Compat. Polym.遠(yuǎn)高于文獻(xiàn)報(bào)道的水溶液中的轉(zhuǎn)化率,且 mPEG-SC199052)27-231與GLP1摩爾比僅需1:1,降低了 mPEG-SC用量[ll遲玉石,黃文龍,張惠斌,等胰高血糖素樣肽1類似物的合成(2)通過(guò)低溫冷凍離心方法對(duì) Mono-PEG-GLP1進(jìn)12)ou1.cmi2H,LL,sa. Preparation and PEGylation of行預(yù)分離和濃縮,大大減少了后續(xù)分離純化難度和工作Exendin Peptide Secreted from Yeast Pichia pastoris U]. Eur. J.量,結(jié)合反相液相色譜法實(shí)現(xiàn)了 Mono-PEG-GLP1高效Pharm Biopharm,2009,72(2):412-417純化, Mono-PEG-GLP.1純度達(dá)97%,收率達(dá)9%以上[13] Wang Y S, Youngster S, Grace M, et al. Structural and BiologicalCharacterization of PEGylated Recombinant Interferon Alpha-2b and(3 Mono-PEG-GLP1在血清中的穩(wěn)定性遠(yuǎn)高于未Its Therapeutic Implications [] Adv Drug Del. Rev, 2002, 54(4)修飾GLP1,對(duì)胰蛋白酶有更好的抗酶解能力,比末修547-570飾GLP-1具有更好的降血糖作用[14] Senderoff R l, Kontor K M, Kreiigaard L, et al. Consideration ofConformational Transitions and Racemization during Process參考文獻(xiàn)Development of Recombinant Glucagon-like Peptide-1 [] J[] Kieffer T J, Habener J F. The Glucagon-like Peptide [ EndocrinePharmacol sci.,1998,87(2)183-189Reviews,1999,205)876-913Modification of Glucagon-like Peptide-1 in Ethanol with Polyethylene GlycolWANG You-ao"2, ZHAI Yan-qin 2, LEI Jian-du, MA Guang-hui,SU Zhi-guo(1. State Key Lab. Biochem. Eng, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China,2. Graduate University of Chinese Academy of Sciences, Beijing 100049, China)Abstract: Glucagon-like peptide-l(GLP-l)was modified with monomethoxy polye(ethylene glycol succinimidyl carbonate) by usingethanol as a reaction medium. The reaction conditions were investigated and optimized, and the separation procedures of productMono-PEG-GLP-I were developed. After that, the stability in vitro and activity in vivo of Mono-PEG-GLP-l were investigated. Theoptimized reaction conditions were obtained as follows: concentration of GLP-1 1.0 mg/mL, molar ratio of PEg to GLP-1 1: l, andreaction time 24 h at 37C. Under the optimized conditions, the yield of Mono-PEG-GLP-l was up to 77%. The Mono-PEG-GLP-I waseparated from reaction mixture by a preliminary centrifugation under frozen temperature and successive purification by reverse phasechromatography. The purity of Mono-PEG-GLP-l was higher than 97%, as characterized by size exclusion chromatography. TheMono-PEG-GLP-l showed better stability in plasma, decreased proteolysis to trypsin and better activity in vivoey words: glucagon-like peptide-1; monomethoxy poly(ethylene glycol succinimidyl carbonate); PEGylation; ethanol