TW202033536A - 製備生理活性多肽複合物之改良製程 - Google Patents
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- HGBOYTHUEUWSSQ-UHFFFAOYSA-N valeric aldehyde Natural products CCCCC=O HGBOYTHUEUWSSQ-UHFFFAOYSA-N 0.000 description 1
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Abstract
本發明揭示一種製備複合物之方法，其中該複合物中之生理活性多肽係經由非肽基連接體共價鍵結至免疫球蛋白恆定區。該方法之特徵在於使用還原劑，藉以克服傳統之低產率及多肽修飾問題。可製造具有高純度與高產率以及低成本之生理活性多肽-非肽基聚合物-免疫球蛋白恆定區複合物。因此該方法係適用於工業。此外，藉由表現延長作用型態，該生理活性多肽-非肽基聚合物-免疫球蛋白恆定區複合物可有效用於發展具有改良藥物順應性之生理活性多肽之長效型配方。
Description
本發明係有關一種製備複合物之方法，其中該複合物中之生理活性多肽係經由包含兩個或更多個醛作為官能基之非肽基聚合物連接體共價鍵結至免疫球蛋白恆定區。更具體而言，本發明係有關一種有效製備生理活性多肽複合物之方法，其特徵在於藉由在其製備期間調整還原劑之用量，來改良低產率及多肽試劑變形問題。
整體而言，生理活性多肽由於安定性低而容易變性，且容易在血液中及隨後之腎或肝清除過程中發生蛋白質水解性降解。因此，包含生理活性多肽作為醫藥成份之蛋白質藥物需要經常性投藥患者，以維持適當血清濃度與效價。然而，此等蛋白質藥物大多呈注射型式，之頻繁投藥，會造成患者疼痛，而且治療成本高。為了解決此等問題，業已致力改善蛋白質藥物之血清安定性，並使血液中之藥物長期維持在高濃度，以使藥物達到最高藥物效力。作為長效型製劑使用之需求，蛋白質藥物必需調配
成具有高安定性，且於不會引發患者免疫反應下維持足夠高濃度之效價。
傳統用於穩定蛋白質並防止酵素性降解及腎清除之方法為使用具高溶解度聚合物(如：聚乙二醇(下文稱為“PEG”)化學性修飾蛋白質藥物之表面。藉由結合至標靶蛋白質之專一性區域或各種區域，PEG可提高蛋白質溶解度，藉以穩定蛋白質並防止水解而不會引發嚴重副作用(Sada等人之J.Fermentation Bioengineering 71：137-139)。
例如：WO 2006/076471揭示一種藉由將B-型鈉利尿肽(BNP)與PEG共軛而達成長效性之方法，該PEG係與鈉利尿肽受體A(NPR-A)結合以啟動cGMP之合成，藉以降低動脈血壓。因此可用於治療充血性心臟衰竭。美國專利第6,924,264號說明一種藉由在其離胺酸殘基上進行PEG化，用來改善艾塞那肽(exendin-4)之活體內持續時間之方法。然而，此等方法中，儘管其有能力藉由提高PEG之分子量來增加肽藥物之循環時間，但PEG化仍有問題，如：大幅降低肽藥物之效價及降低產率，因為隨PEG分子量提高，肽之反應性下降。
WO 02/46227揭示一種融合蛋白質，其中GLP-1與艾塞那肽(exendin-4)或其類似物係與人類血清白蛋白或藉由基因重組法製成之免疫球蛋白片段(Fc)共軛。美國專利第6,756,480號係有關一種融合蛋白質，其中副甲狀腺激素(PTH)或其類似物係連接至Fc。雖然此等
方法可能被評估為解決PEG化之問題，如：低產率及無特異性，但該等增加標靶肽之活體內半衰期之效應仍不顯著，此點與預期結果相反，再者有些例子中之融合蛋白質具有低效價。雖然可利用各種不同肽基連接體以達到延長血清半衰期之最高效力，但其仍有可能引發免疫反應。再者，具有雙硫鍵之肽(如：BNP)仍有實際應用問題，因為其很容易引起摺疊錯誤。此外，無法經由基因重組法製造具有非天然胺基酸殘基之肽基連接體。
胰島素為一種由人類胰臟之β-細胞分泌之肽，主要用於調節體內血糖濃度。當胰島素之分泌不當或所分泌之胰島素無法在體內適當運作時，血糖濃度即會失控而且升高，而造成糖尿病。後者稱為第2型糖尿病。然而，若胰臟無法分泌胰島素而造成血糖濃度升高時，即造成第1型糖尿病。第2型糖尿病之患者以口服降血糖劑治療，其中有些人則以胰島素治療。而第1型糖尿病之患者則基本上需要注射胰島素。
通常，胰島素療法係藉由在每餐之前或之後經由注射法投藥胰島素，一天3次。然而，一天三次連續投藥胰島素令病患痛苦，且不方便。業已做出許多嘗試解決此等問題。其中一種設計在於提高生物膜中蛋白質藥物通透性之方法為經口或鼻吸入法傳送。然而，經口或鼻吸入法投藥之傳送效率顯著低於注射法，而且很難讓肽藥物維持在活體內活性所需之濃度。
另一種替代方法，可經皮下注射過量藥
物，並依延緩方式吸收至體內，用以即使每天注射一次，仍維持恆定之血中濃度。某些藥物(例如：蘭德仕注射劑(Lantus)，賽諾菲安萬特(Sanofi-aventis))已核准上市目前並投藥予患者。另外，已有進行以脂肪酸修飾胰島素之研究，以加強胰島素共軛物之結合力，並透過與注射位置或血液中之白蛋白之組合來延長其持續時間。某些藥物(例如：瑞密爾注射筆(Levemir)，諾和諾德(NovoNordisk))已核准上市。然而，此等藥物會在注射位置造成疼痛，而且必需一天注射一次，其對患者仍是一大負擔。
為了克服先前技藝所遭遇的問題，本發明者已製備一種包含生理活性多肽與免疫球蛋白恆定區，且其藉由使用非肽基聚合物作為連接體連接之複合物，作為同時延長生理活性多肽(如：胰島素)之血漿半衰期及活體內持續時間之策略。然而，亦需要一種製備高產率及高純度複合物之方法，因為該複合物之組成份非常昂貴。考量此因素，本發明者已發展一種以降低成本、高產率及高純度製備生理活性多肽複合物之方法，其係藉由製造複合物之反應期間，於反應溶液中使用最佳濃度之適當種類之還原劑，藉以完成本發明。
本發明之目的係提供一種製備複合物之有效方法，其中該複合物係由生理活性多肽經由包含兩個或更多個醛作為官能基之非肽基聚合物連接體共價鍵結至免
疫球蛋白恆定區，其特徵在於在反應期間，於反應溶液中使用最佳濃度之適當種類之還原劑，改善低產率及多肽試劑變形之問題。
達成上述目的之一態樣中，本發明提供一種製備生理活性多肽-非肽基聚合物-免疫球蛋白恆定區之複合物的方法，其包括(1)將具有兩個或更多個醛作為官能基之非肽基聚合物與生理活性多肽或免疫球蛋白恆定區中之一者，於濃度為1至20mM之還原劑之存在下反應；以及(2)將(1)之反應混合物與生理活性多肽或免疫球蛋白恆定區中另一者於濃度為1至100mM之還原劑之存在下反應。
該反應混合物可包含非肽基聚合物與生理活性多肽之共軛物或非肽基聚合物與免疫球蛋白恆定區之共軛物，及/或仍保留未反應之反應物。因此，本發明之方法可進一步包括於步驟(1)後，自反應混合物中分離生理活性多肽-非肽基聚合物之共軛物或免疫球蛋白-非肽基聚合物之共軛物。
本文所採用術語“非肽基聚合物”係指由至少兩個重覆單元構成之生物相容性聚合物，該重複單元係藉由肽鍵以外之隨機共價鍵共同連接。適用於本發明之非肽基聚合物實施例包括聚乙二醇、聚丙二醇、乙二醇與丙二醇之共聚物、聚氧乙基化多元醇、聚乙烯基醇、多醣、葡聚醣、聚乙烯基乙基醚、生物降解性聚合物(如：聚乳酸
(PLA)與聚乳酸-羥乙酸(PLGA))、脂質聚合物、幾丁質、玻尿酸及其組合，其中以聚乙二醇(PEG)較佳。其技術領域習知之衍生物與易於使用技術領域已知之方法製備之衍生物均在本發明範圍內。
如上述，該非肽基聚合物可具有兩個或更多個醛作為官能基。因此上列非肽基聚合物本身可呈雙-或多-醛官能醛型，或較佳係在其兩個或更多個醇基上包含具有醛基之取代基。具有醛基之取代基可為烷基醛，如：丙醛或丁醛。於一較佳具體實施例中，非肽基聚合物可為在其各末端具有丙醛取代基之PEG。
藉由框內(in-frame)融合技術建構之融合蛋白質中所採用的傳統肽基連接體之缺點在於該等肽基連接體易於在活體內被蛋白酶裂解，因此無法藉由載劑保證延長血清半衰期，此與預期相反。然而，本發明中，使用抗蛋白酶之聚合物，因此肽之血漿半衰期可維持在與載劑類似之濃度。因此只要可抗活體內蛋白酶，任何非肽基聚合物均可用於本發明，沒有限制。非肽基聚合物之分子量範圍為1至100kDa，較佳為1至20kDa。此外，連接至生理活性多肽之非肽基聚合物可為個別聚合物或不同聚合物之組合。此外，適用於本發明之非肽基聚合物可在其兩個或三個末端具有可與生理活性多肽及免疫球蛋白恆定區偶合之官能基。該官能基較佳可為醛。
與常用於製備蛋白質藥物之長效型配方之PEG共軛之方法會增加蛋白質之安定性，但其中PEG之分
子量越高，其表現與蛋白質之反應性越低，因此減少產率。由於產率與製造成本及工業用途關係密切，增加產率極為重要。具有醛作為官能基之PEG可偶合至存在於生理活性多肽或免疫球蛋白恆定區之N-末端或Lys殘基側鏈上之胺基。在此方面，PEG化之產率可隨各種不同因素而定，包括PEG與蛋白質之莫耳比、反應溶液濃度、反應時間、pH、溫度等。Chem.Biol.Drug Des.2007；69；132-138說明藉由調整各種不同因素(包括莫耳比例、反應時間、pH等)使用5K醛mPEG在產率超過90%下進行胰島素PEG化，。在US 2009/0252703A1中提出，將有機溶劑添加至反應溶液可增加肽PEG化產率。WO 2010/089756A2揭示一種藉由將r-metHuG-CSF與PEG於碳水化合物存在下反應以改良PEG化產率。
然而，當採用包括具有兩個或更多個官能基之PEG之非肽基聚合物作為兩個不同多肽間之連接體時，反應中需要兩個或更多個步驟，因此會降低總產率。具體而言，觀察到第二反應之步驟(其中與具有兩個或更多個官能基之非肽基聚合物共軛之生理活性多肽或免疫球蛋白恆定區分別與免疫球蛋白恆定區或生理活性多肽反應，下文稱為“偶合反應”)，其產率顯著低於第一反應步驟(其中生理活性多肽或免疫球蛋白恆定區與具有兩個或更多個官能基之非肽基聚合物反應)。
本發明中，第一反應中之還原劑濃度已證實與第二偶合反應之產率具有相關性。第一反應中之還原
劑濃度越低，反應時間越短及反應溫度越低時，觀察到該偶合反應產率越高。
本文所採用術語“還原劑”係指其功能在於還原非肽基聚合物之醛基與多肽(生理活性多肽、免疫球蛋白恆定區)之胺基之間反應所形成之可逆性亞胺雙鍵而藉以形成共價鍵之化合物，並意欲包括技術領域已知的所有還原劑。為了本發明之目的，可添加還原劑至反應溶液中，其中非肽基聚合物與生理活性多肽或免疫球蛋白恆定區形成共價鍵。只要是技術領域常用之任何還原劑均可用於本發明。還原劑實例可包括，但不限於：氰基硼氫化鈉、硼烷吡啶複合物、硼氫化鈉、硼烷二甲基胺複合物、硼烷三甲基胺複合物、與三乙醯氧基硼氫化鈉。適當還原劑可依據生理活性多肽或免疫球蛋白恆定區與反應溶劑之種類選擇。
還原劑係用於生理活性多肽或免疫球蛋白恆定區與非肽基聚合物之共軛作用。反應溶液可包含還原劑，該還原劑用於生理活性多肽或免疫球蛋白恆定區與非肽基聚合物之間反應之濃度為1至20mM，及用於偶合反應之濃度為1至100mM。更佳者，該還原劑用於生理活性多肽或免疫球蛋白恆定區與非肽基聚合物之間共軛反應之濃度為1至20mM，及用於偶合反應之濃度為1至40mM。
生理活性多肽或免疫球蛋白恆定區與非肽基聚合物之間之共軛反應(步驟(1)反應)可於0至25℃溫度
進行1至16小時。此外，偶合反應(步驟(2)反應)可進行1至48小時。
較佳者，該步驟(1)反應可於0至25℃溫度，於濃度1至20mM之還原劑之存在下進行1至16小時，而步驟(2)反應可於濃度1至40mM之還原劑之存在下進行1至48小時。
在一較佳具體實施例中，在各種不同條件下使用氰基硼氫化鈉作為還原劑，以增加複合物之產率，於該複合物中，胰島素、具有兩個或更多個醛作為官能基之PEG連接體、與免疫球蛋白恆定區係連接在一起。已發現當該第一反應(其係進行連接生理活性多肽或免疫球蛋白恆定區與非肽基聚合物)在低溫下，於低濃度還原劑之存在下進行短時間時，可提高偶合反應產率(表1)。
在另一較佳具體實施例中，該反應係使用各種不同濃度之還原劑硼烷吡啶複合物進行，並在生理活性多肽或免疫球蛋白恆定區與非肽基聚合物之間使用較低濃度還原劑進行第一反應後，檢測到第二反應(偶合反應)之較高產率。在使用氰基硼氫化鈉作為還原劑的例子中顯示其產率高於使用硼烷吡啶複合物時之產率(表2)。
此外，已證實第二反應(偶合反應)之產率隨還原劑濃度提高而增加。
在一較佳具體實施例中，該偶合反應係在各種不同濃度之還原劑氰基硼氫化鈉進行，並在高濃度還原劑之存在下得到改良產率之偶合反應。然而，極高濃度
還原劑會引起免疫球蛋白恆定區出現異常。為了避免此異常，偶合反應係在20mM氰基硼氫化鈉之存在下進行13小時，並觀察維持高產率，而且使免疫球蛋白恆定區之異常降至最低(表3與4)。
本文所採用術語“生理活性多肽”係指一般概念上於活體內具有某種生理功能之多肽。其具有一般多肽基結構，並展現各種不同生物活性。當涉及某種功能之材料缺少或過量以致身體出現生物異常時，該生理活性多肽可調節基因表現或生理功能，藉以修正該異常。其典型實例為蛋白質藥物。
應用於本發明之生理活性多肽實例包括人類生長激素、生長激素釋放激素、生長激素釋放胜肽、干擾素、干擾素受體、群落刺激因子、類升糖素肽(GLP-1，等)、調酸素(oxyntomodulin)、G蛋白質-偶合受體、介白素、介白素受體、酵素、介白素-結合蛋白質、細胞素-結合蛋白質、巨噬細胞活化因子、巨噬細胞肽、B-細胞因子、T-細胞因子、蛋白質A、過敏抑制劑、細胞壞死醣蛋白質、免疫毒素、淋巴毒素、腫瘤壞死因子、腫瘤壓制劑、轉形生長因子、α-1抗胰蛋白酶、白蛋白、α-乳清蛋白、載脂蛋白-E、促紅血球生成素、糖基化促紅血球生成素、血管生成素、血紅素、凝血酶、凝血酶受體活化肽、凝血酶調節素、血液因子VII、VIIa、VIII、IX與XIII、纖維蛋白溶酶原活化劑、纖維蛋白結合肽、尿激酶、鏈球菌激酶、水蛭素、蛋白質C、C-反應性蛋白質、腎素抑制劑、膠原
酶抑制劑、超氧歧化酶、瘦素、血小板衍生之生長因子、上皮生長因子、表皮生長因子、血管生長抑制素、血管收縮素、骨生長因子、骨刺激蛋白質、降血鈣素、胰島素、心房肽(atriopeptin)、軟骨誘發因子、依降鈣素(elcatonin)、結締組織活化因子、組織因子途徑抑制劑、濾泡刺激激素、促黃體激素、促黃體激素釋放激素、神經生長因子、副甲狀腺激素、鬆弛素、分泌素、體介素、類胰島素生長因子、腎上腺皮質激素、升糖素、膽囊收縮素、胰多肽、胃泌素釋放肽、促腎上腺皮質激素釋放因子、甲狀腺刺激激素、自分泌運動因子(autotaxin)、乳鐵蛋白、肌肉生長抑制素(myostatin)、細胞表面抗原、病毒衍生之疫苗抗原、單株抗體、多株抗體與抗體片段。
用於本發明具體實施例之胰島素為一種當血糖濃度變高時從胰臟分泌之生理活性肽，其功能在於藉由引發肝、骨骼肌與脂肪組織吸收血液中葡萄糖並儲存成肝醣，並抑制脂肪分解(其係利用脂肪作為能量來源之代謝作用)，以控制血糖濃度。生理活性肽包括胰島素促效劑、前驅物、衍生物、片段、與變異體。以天然胰島素、速效型胰島素、與長效型胰島素較佳。
天然胰島素為一種胰臟分泌之激素，其藉由促進細胞吸收葡萄糖並抑制脂肪分解，而在控制血糖濃度中扮演重要角色。具有調節血糖濃度功能之胰島素係由不具調節血糖濃度功能之胰島素原前驅物經過一系列過程後產生。其胺基酸序列如下：
-α鏈：
Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn(SEQ ID NO：1)
-β鏈：
本文所採用術語“胰島素促效劑”係指可以結合至活體內胰島素受體並表現與胰島素之相同生物活性但與胰島素結構無關之物質。
本文所採用術語“胰島素衍生物”係指一種功能在於控制活體內血糖濃度之肽，且其胺基酸序列與天然胰島素具有至少80%同源性。其中有些胺基酸殘基可有化學取代(例如：α-甲基化、α-羥基化)、刪除(例如：脫胺基化)或修飾(例如：N-甲基化、糖基化、脂肪酸)。
本文所採用術語"胰島素片段"係指一種功能在於控制活體內血糖濃度，且其藉由自胰島素之胺基或羧基末端添加至少一個胺基酸或刪除至少一個胺基酸後製成之肽。所添加之胺基酸可為非天然存在的胺基酸(例如：D-胺基酸)。
本文所採用術語“胰島素變異體”係指該肽之胺基酸序列與天然胰島素之差異在於一個或多個胺基酸殘基，但其仍具有控制活體內血糖濃度之功能。
此外，各別用於製備胰島素促效劑、片段
與變異體之方法可單獨採用或組合採用。例如：適用於本發明之胰島素肽可包括其胺基酸序列與天然胰島素之差異在於一個或多個胺基酸殘基，且N-末端殘基已經過脫胺基化，但仍具有控制活體內血糖濃度功能之肽。
本文所採用術語“免疫球蛋白恆定區”係指缺乏輕鏈與重鏈之可變區、重鏈之恆定區1(CH1)與輕鏈之恆定區(CL)之免疫球蛋白片段，亦即由重鏈恆定區2與3(CH2與CH3)(或包括重鏈之恆定區(CH4))構成之Fc區。免疫球蛋白Fc區可視需要進一步包含樞紐區(hinge region)。再者，本發明之免疫球蛋白恆定區可為只排除免疫球蛋白重鏈與輕鏈之可變區而包括一部份或全部重鏈(CH1)恆定區1及/或輕鏈(CL)恆定區之延長免疫球蛋白Fc區，只要其顯示實質上相同或優於彼等天然免疫球蛋白恆定區之效果者。此外，本發明免疫球蛋白恆定區可缺乏相當於CH2及/或CH3之主要部份胺基酸序列。因此本發明免疫球蛋白恆定區可包含(1)CH1功能域、CH2功能域、CH3功能域與CH4功能域，(2)CH1功能域與CH2功能域，(3)CH1功能域與CH3功能域，(4)CH2功能域與CH3功能域，(5)組合一個或多個恆定功能域與一個免疫球蛋白樞紐區(或一部份樞紐區)，或(6)重鏈之各恆定功能域與輕鏈之恆定區之二聚體。
包括Fc區之免疫球蛋白恆定區為生物可降解之多肽，其可在活體內代謝，因此可安全用作為藥物載劑。此外，免疫球蛋白Fc區在複合物之製造、純化與產率
上均優於完整之免疫球蛋白分子，因為其分子量相對較低。再者，由於其缺乏Fab(其由於在抗體與抗體之間的胺基酸序列差異而表現高度非同源性)，免疫球蛋白Fc單獨即可提供具有顯著加強同源性之複合物，並降低誘發血液抗原性之可能性。
免疫球蛋白恆定區可源於人類或動物，如：牛、山羊、豬、小鼠、兔、倉鼠、大鼠、天竺鼠，等，且以人類來源較佳。此外，免疫球蛋白恆定區可選自衍生自IgG、IgA、IgD、IgE、IgM、或其組合或雜交體之Fc片段。較佳者，該恆定區係衍生自IgG或IgM，其為血液中含量最高者，且以衍生自IgG最佳，其係已知改良配體結合性蛋白質之血清半衰期。
本文所採用術語“組合”意指來自相同來源之編碼單鏈免疫球蛋白恆定區(較佳係Fc區)之多肽連結至不同來源之單鏈多肽以形成二聚體或多聚體。亦即二聚體或多聚體可藉由選自：IgG Fc、IgA Fc、IgM Fc、IgD Fc與IgE Fc片段所組成群組之兩個或更多個片段組合而製成。
本文所採用術語“雜交體”意指編碼不同來源之兩個或更多個免疫球蛋白Fc片段之序列存在於免疫球蛋白恆定區(較佳係Fc區)之單鏈中。本發明中，可能有各種不同雜交體型式。例如：雜交體功能域可由選自IgG Fc、IgM Fc、IgA Fc、IgE Fc與IgD Fc之CH1、CH2、CH3與CH4所組成群組之1至4個功能域組成，且可包含樞紐
區。
IgG區分成IgG1、IgG2、IgG3與IgG4子類，本發明可包括其組合或雜交體。較佳係IgG2與IgG4子類，最佳係幾乎沒有效應子功能(如：補體依賴型細胞毒性(CDC))之IgG4之Fc區。
免疫球蛋白恆定區可能具有糖基化型式，其糖基化程度可能與天然型式相同、或更高或更低，或可能呈去糖基化型式。可藉由典型方法(例如：使用化學方法、酵素方法或使用微生物之遺傳工程法)提高或降低免疫球蛋白區之糖基化或去糖基化。其中當去糖基化時，與免疫球蛋白Fc區連結之補體(C1q)顯著減少，抗體依賴性細胞毒性或補體依賴性細胞毒性即下降或排除，因此不會誘發活體內不必要之免疫反應。本文中，去糖基化或非糖基化免疫球蛋白Fc區更符合藥物載劑之目的。因此，最適合作為本發明藥物載劑之免疫球蛋白Fc區為人類IgG4-衍生之非糖基化Fc區。人類衍生之Fc區比非人類衍生之Fc區更佳，後者會成為人體內之抗原並引發不意欲之免疫反應，如：產生對抗抗原之新抗體。
再者，本發明免疫球蛋白恆定區之範圍不僅包括具有天然胺基酸序列之免疫球蛋白恆定區，而且包括其胺基酸序列突變體。本文所採用術語“胺基酸序列突變體”係指其胺基酸序列已經過刪除、嵌插、一個或多個胺基酸殘基之保留性或非保留性取代或其組合而不同於野生型之多肽。例如：已知具有連接重要性之IgG Fc中位置214
至238、297至299、318至322、或327至331之胺基酸殘基可適用於修飾之位置。本發明可使用各種不同衍生物，如：彼等藉由移除天然Fc中可形成雙硫鍵之位置、移除數個N-末端胺基酸或在天然Fc之N-末端增加甲硫胺酸所製成者。此外，可自天然Fc區消除補體固定位置(例如：C1q固定位置)或ADCC位置，以移除效應子功能。製備免疫球蛋白恆定區之胺基酸序列突變體之技術已揭示於國際專利公開號第WO 97/34631與WO 96/32478號。
蛋白質或肽分子中不會改變分子活性之胺基酸取代係技術領域已知者(H.Neurath,R.L.Hill,The Proteins,Academic Press,New York,1979)。最常見之取代發生在Ala/Ser、Val/Ile、Asp/Glu、Thr/Ser、Ala/Gly、Ala/Thr、Ser/Asn、Ala/Val、Ser/Gly、Thr/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/Ile、Leu/Val、Ala/Glu、與Asp/Gly胺基酸殘基之間。胺基酸可視需要經磷酸化、硫酸化、丙烯酸化、糖基化、甲基化、法呢基化(farnesylation)、乙醯基化、與醯胺化修飾。
上述免疫球蛋白恆定區衍生物表現出與本發明免疫球蛋白恆定區相同之生物活性，但對熱、pH，等等具有改良之結構安定性。此等免疫球蛋白恆定區可從單離自人類或動物(如：牛、山羊、豬、小鼠、兔、倉鼠、大鼠、天竺鼠，等)之天然型所獲得，或可自轉化動物細胞或微生物之重組體或衍生物。可藉由蛋白酶分解自人類或動物樣本所單離之免疫球蛋白之整體領域所得的天然恆定
區。免疫球蛋白可被木瓜酵素裂解成Fab與Fc，被胃蛋白酶裂解成pF'c與F(ab)2，然後經過粒徑篩析層析法自其中分離Fc或pF'c。
較佳係得自微生物之重組人類免疫球蛋白恆定區。
如上述，可藉由本發明方法製得高純度與高產率以及低成本之生理活性多肽-非肽基聚合物-免疫球蛋白恆定區複合物。因此本發明方法適用於工業。此外，其可用於開發已改良藥物順應性之生理活性多肽之長效型配方。
可透過下列實施例進一步了解本發明，其僅供說明，但不構成本發明之限制範圍。
實施例1：使用氰基硼氫化鈉作為還原劑將胰島素PEG化與單-PEG化胰島素之純化
取胰島素粉末溶於10mM HCl，使用3.4K propion-ALD2 PEG(具有兩個丙醛基之PEG，韓國IDB)在β鏈之N-末端進行PEG化。此時，將5mg/ml胰島素與PEG依莫耳比1：2，於4℃至室溫下反應2小時。該反應於作為還原劑之2至20mM氰基硼氫化鈉之存在下，於活於45%異丙醇中之50mM檸檬酸鈉緩衝液(pH 6.0)中進
行。將反應混合物裝入SP-HP(GE Healthcare)管柱，隨後以含有檸檬酸鈉(pH 3.0)與45% EtOH之緩衝液，KCl之濃度梯度沖提以純化單-PEG化胰島素。
於製備包含胰島素與免疫球蛋白Fc區之複合物期間，根據還原劑氰基硼氫化鈉條件下之胰島素PEG化產率係概述於下表1。
實施例2：根據PEG化中所使用氰基硼氫化鈉作為還原劑之條件下之單-PEG化胰島素-免疫球蛋白Fc區複合物產率之變化
為了檢測胰島素-PEG-免疫球蛋白Fc區複合物之產率，將實施例1製備之單-PEG化胰島素依莫耳比1：1與免疫球蛋白Fc於25℃下反應13小時，總蛋白質濃度設定在20mg/ml。此偶合反應係於100mM HEPES緩衝液(包含22mM磷酸鉀與10%乙醇)(pH 8.2)中且於20mM氰基硼氫化鈉作為還原劑存在下進行。
將反應混合物裝入Source 15Q(GE Healthcare)管柱，隨後使用Tris-HCl(pH 7.5)緩衝液並使用NaCl濃度梯度沖提以分離且純化未反應之胰島素、未反應之免疫球蛋白Fc區、胰島素-PEG-免疫球蛋白Fc區複合物、及與兩個或更多個單-PEG化胰島素(胰島素-PEG)部分偶合之免疫球蛋白Fc區。經過層析法純化後，以280nm之UV吸光度測定胰島素-PEG-免疫球蛋白Fc區複合物之產率。
表1中，根據胰島素PEG化作用中使用氰
基硼氫化鈉作為還原劑之條件概述說明與免疫球蛋白Fc區之偶合反應產率。
表1
實施例3：使用硼烷吡啶複合物作為還原劑之胰島素PEG化與單-PEG化胰島素之純化
取胰島素粉末溶於10mM HCl，使用3.4K propion-ALD2 PEG(具有兩個丙醛基之PEG，韓國IDB)在β鏈之N-末端進行PEG化。此時，將5mg/ml胰島素與PEG依莫耳比1：2，於4℃下反應2小時。該反應於3至20mM硼烷吡啶複合物作為還原劑之存在下，於活於45%異丙醇中之50mM檸檬酸鈉緩衝液(pH 6.0)中進行。將反應混合物裝入SP-HP(GE Healthcare)管柱，隨後以包含檸檬酸鈉(pH 3.0)與45% EtOH之緩衝液，及使用KCl之濃度梯度沖提以純化單-PEG化胰島素。
於製備包含胰島素與免疫球蛋白Fc區之複
合物期間，根據還原劑硼烷吡啶複合物條件下之胰島素PEG化產率係概述於下表2。
實施例4：根據PEG化中所使用硼烷吡啶複合物作為還原劑之條件下之單-PEG化胰島素-免疫球蛋白Fc區複合物產率之變化
為了檢測胰島素-PEG-免疫球蛋白Fc區複合物之產率，將實施例3製備之單-PEG化胰島素依莫耳比1：1與免疫球蛋白Fc於25℃下反應13小時，總蛋白質濃度設定在20mg/ml。此偶合反應係於100mM HEPES緩衝液(包含22mM磷酸鉀與10%乙醇)(pH 8.2)中，於20mM氰基硼氫化鈉作為還原劑之存在下進行。
將反應混合物裝入Source 15Q(GE Healthcare)管柱，隨後使用Tris-HCl(pH 7.5)緩衝液及使用NaCl濃度梯度沖提以分離與純化未反應之胰島素、未反應之免疫球蛋白Fc區、胰島素-PEG-免疫球蛋白Fc區複合物、及與兩個或更多個單-PEG化胰島素(胰島素-PEG)部分偶合之免疫球蛋白Fc區。經由層析法純化後，以280nm之UV吸光度測定胰島素-PEG-免疫球蛋白Fc區複合物之產率。
表2中，根據胰島素PEG化中使用硼烷吡啶複合物作為還原劑之條件下概述說明與免疫球蛋白Fc區之偶合反應產率。
表2
實施例5：根據氰基硼氫化鈉濃度與反應時間之偶合反應產率與免疫球蛋白Fc異常之形成
為了檢測根據偶合反應中還原劑濃度與反應時間之免疫球蛋白Fc異常之形成，將單-PEG化胰島素依莫耳比1：1與免疫球蛋白Fc於25℃下反應13至43小時，總蛋白質濃度設定在20mg/ml。此偶合反應係於100mM HEPES緩衝液(包含22mM磷酸鉀與10%乙醇，pH 8.2)中，於5至40mM氰基硼氫化鈉之存在下進行。
將反應混合物裝入至Source 15Q(GE Healthcare)管柱，隨後使用Tris-HCl(pH 7.5)緩衝液及使用NaCl之濃度梯度沖提，以分離與純化未反應之胰島素、未反應之免疫球蛋白Fc區、胰島素-PEG-免疫球蛋白Fc區複合物、及與兩個或更多個單-PEG化胰島素(胰島素-PEG)部分偶合之免疫球蛋白Fc區。經由層析法純化後，以280nm之UV吸光度測定胰島素-PEG-免疫球蛋白Fc區複合物之產率。
表3中，根據偶合反應中使用氰基硼氫化
鈉作為還原劑之濃度概述說明包含胰島素與免疫球蛋白Fc區複合物之偶合反應產率。
表3
藉由Propac SAX-10(DIONEX)管柱之LC，以Tris-HCl(pH 8.0)緩衝液並使用NaCl之濃度梯度沖提，偵測偶合反應中根據還原劑濃度之免疫球蛋白Fc異常形成。
表4中，根據用以製備包含胰島素與免疫球蛋白Fc區之複合物之偶合反應中氰基硼氫化鈉濃度與反應時間，給出免疫球蛋白Fc異常之產率。
表4
實施例6：使用氰基硼氫化鈉作為還原劑之免疫球蛋白Fc之PEG化與單-PEG化免疫球蛋白Fc之純化
使用5K propion-ALD2 PEG(具有三個丙醛基之PEG，日本NOF)將免疫球蛋白Fc之N-末端進行PEG化。此時，將10mg/ml免疫球蛋白Fc與PEG依莫耳比1：2，於4℃至室溫下反應4.5小時。該反應係於100mM磷酸鉀緩衝液(pH 6.0)中，於20mM氰基硼氫化鈉作為還原劑之存在下進行。將反應混合物裝入至Source 15Q管柱，隨後以Tris-HCl(pH 7.5)緩衝液及使用NaCl之濃度梯度沖提，以純化單-PEG化胰島素。
實施例7：使用氰基硼氫化鈉作為還原劑製備單-PEG化免疫球蛋白Fc區-胰島素複合物
為了製備胰島素-PEG-免疫球蛋白Fc區複合物，將實施例6製備之單-PEG化免疫球蛋白Fc依莫耳比1：4與胰島素於4℃下反應13小時，總蛋白質濃度設定在20mg/ml。此偶合反應係於100mM磷酸鹽緩衝液(pH 6.0)中，於20mM氰基硼氫化鈉作為還原劑之存在下進行。
將反應混合物裝入至Source 15Q(GE Healthcare)管柱中進行初次純化。另以Source 15ISO(GE Healthcare)管柱進行第二次純化，以獲得胰島素-PEG-免疫球蛋白Fc區複合物。
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Claims (23)
- 一種製備生理活性多肽-非肽基聚合物-免疫球蛋白恆定區複合物之方法，其包括：(1)將具有兩個或更多個醛作為官能基之非肽基聚合物與生理活性多肽或免疫球蛋白恆定區中之一者於濃度1至20mM之還原劑之存在下反應；以及(2)將步驟(1)之該反應混合物與該生理活性多肽或免疫球蛋白恆定區中另一者於濃度1至100mM之還原劑之存在下反應。
- 如申請專利範圍第1項所述之方法，其進一步包括於步驟(1)之後，自該反應混合物中分離生理活性多肽-非肽基聚合物之共軛物或免疫球蛋白恆定區-非肽基聚合物之共軛物。
- 如申請專利範圍第1項所述之方法，其中該還原劑之功能在於還原該非肽基聚合物之醛基與該生理活性多肽或該免疫球蛋白恆定區之胺基之間鍵結所形成之可逆性亞胺雙鍵以形成共價鍵。
- 如申請專利範圍第1項所述之方法，其中該還原劑係選自下列者所組成群組：氰基硼氫化鈉、硼烷吡啶複合物、硼氫化鈉、硼烷二甲基胺複合物、硼烷三甲基胺複合物與三乙醯氧基硼氫化鈉。
- 如申請專利範圍第1項所述之方法，其中於該步驟(2)中，該還原劑之使用濃度為1至40mM。
- 如申請專利範圍第1項所述之方法，其中該步驟(1)之 反應係進行1至16小時。
- 如申請專利範圍第1項所述之方法，其中該步驟(2)之反應係進行1至48小時。
- 如申請專利範圍第1項所述之方法，其中該步驟(1)之反應係於0至25℃之溫度下進行。
- 如申請專利範圍第1項所述之方法，其中該步驟(1)之反應係於0至25℃，於濃度為1至20mM之該還原劑之存在下進行1至16小時，且該步驟(2)之反應係於濃度1至40mM之該還原劑之存在下進行1至48小時。
- 如申請專利範圍第1項所述之方法，其中該非肽基聚合物係經由其兩個或更多個醛官能基共價鍵結至該生理活性多肽與該免疫球蛋白恆定區之各者。
- 如申請專利範圍第1項所述之方法，其中該非肽基聚合物之該官能基係鍵結至該生理活性多肽之胺基與該免疫球蛋白恆定區之各者之胺基，其中該胺基係存在於N-末端或Lys殘基之側鏈上。
- 如申請專利範圍第1項所述之方法，其中該非肽基聚合物係選自下列者所組成群組：聚乙二醇、聚丙二醇、乙二醇與丙二醇之共聚物、聚氧乙基化多元醇、聚乙烯基醇、多醣、葡聚醣、聚乙烯基乙基醚、聚乳酸(PLA)、聚乳酸-羥乙酸(PLGA)、脂質聚合物、幾丁質、玻尿酸及其組合。
- 如申請專利範圍第1項所述之方法，其中該非肽基聚合物為聚乙二醇。
- 如申請專利範圍第1項所述之方法，其中該非肽基聚合物之分子量為1至100kDa之範圍。
- 如申請專利範圍第1項所述之方法，其中該免疫球蛋白恆定區係非糖基化。
- 如申請專利範圍第1項所述之方法，其中該免疫球蛋白恆定區係由選自CH1、CH2、CH3與CH4功能域所組成群組之1至4個功能域所組成。
- 如申請專利範圍第1項所述之方法，其中該免疫球蛋白恆定區進一步包含樞紐區。
- 如申請專利範圍第1項所述之方法，其中該免疫球蛋白恆定區係選自衍生自IgG、IgA、IgD、IgE、IgM之恆定區、或其組合或其雜交體所組成群組。
- 如申請專利範圍第1項所述之方法，其中該免疫球蛋白恆定區係選自IgG1、IgG2、IgG3、IgG4之恆定區、或其組合與雜交體所組成群組。
- 如申請專利範圍第1項所述之方法，其中該免疫球蛋白恆定區為IgG4 Fc區。
- 如申請專利範圍第20項所述之方法，其中該免疫球蛋白恆定區為非糖基化人類IgG4 Fc區。
- 如申請專利範圍第1項所述之方法，其中該生理活性多肽係選自下列者所組成群組：人類生長激素、生長激素釋放激素、生長激素釋放胜肽、干擾素、干擾素受體、群落刺激因子、類升糖素肽(GLP-1，等)、調酸素(oxyntomodulin)、G蛋白質-偶合受體、介白素、介 白素受體、酵素、介白素-結合蛋白質、細胞素-結合蛋白質、巨噬細胞活化因子、巨噬細胞肽、B-細胞因子、T-細胞因子、蛋白質A、過敏抑制劑、細胞壞死醣蛋白、免疫毒素、淋巴毒素、腫瘤壞死因子、腫瘤壓制劑、轉形生長因子、α-1抗胰蛋白酶、白蛋白、α-乳清蛋白、脂蛋白-E、促紅血球生成素、糖基化促紅血球生成素、血管生成素、血紅素、凝血酶、凝血酶受體活化肽、凝血酶調節素、血液因子VII、VIIa、VIII、IX與XIII、纖維蛋白溶酶原活化劑、纖維蛋白結合肽、尿激酶、鏈球菌激酶、水蛭素、蛋白質C、C-反應性蛋白質、腎素抑制劑、膠原酶抑制劑、超氧歧化酶、瘦素、血小板衍生之生長因子、上皮生長因子、表皮生長因子、血管生長抑制素、血管收縮素、骨生長因子、骨刺激蛋白質、降血鈣素、胰島素、心房肽(atriopeptin)、軟骨誘發因子、依降鈣素(elcatonin)、結締組織活化因子、組織因子途徑抑制劑、濾泡刺激激素、促黃體激素、促黃體激素釋放激素、神經生長因子、副甲狀腺激素、鬆弛素、分泌素、體介素、類胰島素生長因子、腎上腺皮質激素、升糖素、膽囊收縮素、胰多肽、胃泌素釋放肽、促腎上腺皮質激素釋放因子、甲狀腺刺激激素、自分泌運動因子(autotaxin)、乳鐵蛋白、肌肉生長抑制素(myostatin)、細胞表面抗原、病毒衍生之疫苗抗原、單株抗體、多株抗體、與抗體片段。
- 如申請專利範圍第1項所述之方法，其中該生理活性 多肽為胰島素。
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2013
- 2013-03-07 AR ARP130100755A patent/AR090281A1/es unknown
- 2013-03-08 KR KR1020130025344A patent/KR101679612B1/ko active IP Right Grant
- 2013-03-08 JP JP2014560857A patent/JP6383667B2/ja active Active
- 2013-03-08 CN CN201380023673.2A patent/CN104271604A/zh active Pending
- 2013-03-08 MX MX2014010734A patent/MX369613B/es active IP Right Grant
- 2013-03-08 EP EP13757904.1A patent/EP2822972A4/en active Pending
- 2013-03-08 US US14/383,334 patent/US11168109B2/en active Active
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- 2013-03-08 IN IN7956DEN2014 patent/IN2014DN07956A/en unknown
- 2013-03-08 UA UAA201410250A patent/UA114192C2/uk unknown
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- 2013-03-08 RU RU2014138621A patent/RU2639256C2/ru active
- 2013-03-08 CA CA2866473A patent/CA2866473A1/en not_active Abandoned
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- 2013-03-08 AU AU2013228144A patent/AU2013228144B2/en not_active Ceased
- 2013-03-08 CN CN201910479805.1A patent/CN110339369B/zh active Active
- 2013-03-08 WO PCT/KR2013/001885 patent/WO2013133659A1/en active Application Filing
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- 2014-09-08 PH PH12014502004A patent/PH12014502004A1/en unknown
-
2015
- 2015-03-10 HK HK15102405.8A patent/HK1201858A1/zh unknown
-
2018
- 2018-03-13 JP JP2018044927A patent/JP2018104465A/ja active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| CN110339369A (zh) | 2019-10-18 |
| CN104271604A (zh) | 2015-01-07 |
| JP6383667B2 (ja) | 2018-08-29 |
| MX369613B (es) | 2019-11-14 |
| HK1201858A1 (zh) | 2015-09-11 |
| RU2639256C2 (ru) | 2017-12-20 |
| US11168109B2 (en) | 2021-11-09 |
| TW201341395A (zh) | 2013-10-16 |
| MY165211A (en) | 2018-03-05 |
| JP2015514690A (ja) | 2015-05-21 |
| MX2014010734A (es) | 2014-12-05 |
| KR101679612B1 (ko) | 2016-11-25 |
| UA114192C2 (uk) | 2017-05-10 |
| KR20130103447A (ko) | 2013-09-23 |
| CN110339369B (zh) | 2024-01-23 |
| EP2822972A1 (en) | 2015-01-14 |
| IN2014DN07956A (zh) | 2015-05-01 |
| PH12014502004A1 (en) | 2014-11-24 |
| JP2018104465A (ja) | 2018-07-05 |
| SG11201405537UA (en) | 2014-11-27 |
| WO2013133659A1 (en) | 2013-09-12 |
| EP2822972A4 (en) | 2015-12-16 |
| RU2014138621A (ru) | 2016-04-27 |
| AU2013228144A1 (en) | 2014-09-25 |
| US20150299247A1 (en) | 2015-10-22 |
| AR090281A1 (es) | 2014-10-29 |
| CA2866473A1 (en) | 2013-09-12 |
| AU2013228144B2 (en) | 2017-08-17 |
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