TW202348623A - 葡萄糖醛酸化作為治療性單株抗體之新酸性轉譯後修飾 - Google Patents
葡萄糖醛酸化作為治療性單株抗體之新酸性轉譯後修飾 Download PDFInfo
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Abstract
提供用於鑑別葡萄糖醛酸化蛋白質藥物產品之組合物及方法。
Description
本發明概言之係關於鑑別治療性蛋白質之轉譯後修飾之系統及方法。
在製藥工業中,重組單株抗體(mAb)構成蛋白質治療劑之主要類別。mAb結構之改變可能會影響治療性mAb之治療效能、生體可用率及清除率以及免疫原性。此外,mAb之變化可改變藥物之安全性及效能。全面表徵mAb之一級結構、轉譯後修飾(PTM)及二硫鍵對於評估藥物效能及安全性以及理解結構/功能關係至關重要。
在用於確保產品及製程一致性之各種分析中,離子交換層析(IEX)為評價mAb分子之電荷異質性之常用技術。mAb電荷變體可能經常歸因於轉譯後修飾(PTM),其可改變mAb分子之表面電荷。由於LC-MS技術之不斷進步,業內已鑑別出許多已知或新穎之PTM。此外,已闡明該等PTM對電荷異質性之貢獻。儘管技術之進步已提供許多新穎之PTM,但業內仍需要進一步表徵及發現改變mAb活性及穩定性之PTM。
因此,本發明之目標係提供用於鑑別PTM之系統及方法。
提供用於鑑別葡萄糖醛酸化蛋白質藥物產品之組合物及方法。一個實施例提供用於鑑別蛋白質藥物產品之葡萄糖醛酸化之方法,其藉由使蛋白質藥物產品去醣基化並用酶(例如FabRICATOR®)處理去醣基化蛋白質藥物產品以產生一個Fc*片段(經由非共價相互作用結合在一起之兩個相同的Fc/2片段)及一個Fab
2。然後使Fc*及Fab
2片段分離成Fc*及Fab
2之酸性部分。在一個實施例中,Fc*及Fab
2片段係使用離子交換層析(例如強陽離子交換層析)來分離。分離後,收集酸性部分,乾燥並使其變性。使乾燥及變性之酸性部分烷基化且然後用胰蛋白酶消化以形成樣品。然後用NaBH
4處理樣品以形成還原樣品。然後對還原Fc*及還原Fab
2樣品進行逆相液相層析/質譜分析以鑑別蛋白質藥物產品之葡萄糖醛酸化。在一個實施例中,該方法包括比較還原樣品與非還原樣品之間的質譜結果以鑑別還原樣品與非還原樣品之間的質量差別之步驟。藥物產品可為單株抗體或融合蛋白。在一個實施例中,葡萄糖醛酸化係在蛋白質藥物產品之離胺酸殘基上檢測。
在一個實施例中,Fc*及Fab
2片段係使用以名稱FabRICATOR®出售之來自化膿性鏈球菌(
Streptocoocus pyogenes)之IdeS之重組修飾形式形成。
所揭示方法可用於監測蛋白質藥物產品(例如單株抗體或其他治療性蛋白質)之純度。一個實施例提供提高蛋白質藥物產品純度之方法,該方法藉由分析蛋白質藥物產品鑑別出蛋白質藥物產品之一或多種胺基酸之一級胺之葡萄糖醛酸化,並自蛋白質藥物產品去除葡萄糖醛酸化之蛋白質以產生經純化之蛋白質藥物產品。葡萄糖醛酸化可使用上文及實例中所述之方法來檢測。在一個實施例中,葡萄糖醛酸化蛋白質係藉由視情況與質譜耦聯之高效液相層析技術來檢測。代表性層析技術包括(但不限於)尺寸排阻層析、離子交換層析、親和層析、高效液相層析、超高效液相層析及其組合。通常,葡萄糖醛酸化發生在蛋白質藥物產品之離胺酸殘基上。
另一實施例提供用於鑑別轉譯後修飾之蛋白質藥物產品之方法,該方法藉由分析自哺乳動物細胞培養物獲得之蛋白質藥物產品之葡萄糖醛酸化來實施,其中蛋白質藥物產品之葡萄糖醛酸化之存在表明蛋白質藥物產品包含轉譯後修飾。哺乳動物細胞培養物通常含有中國倉鼠卵巢細胞。
另一實施例提供含有治療性蛋白質之蛋白質藥物產品,其中治療性蛋白質之少於1.0%、2.0%、3.0%、4.0%、5.0%、6.0%、7.0%、8.0%、9.0%或10%之胺基酸經葡萄糖醛酸化。在一個實施例中,胺基酸為離胺酸。通常,治療性蛋白質為單株抗體、重組蛋白或融合蛋白。
I. 定義
如本文所用術語「抗體」意欲表示具有「可變區」抗原識別位點之免疫球蛋白分子。術語「可變區」意欲區分免疫球蛋白之該域與抗體廣泛共享之域(諸如抗體Fc域)。可變區包括「超變區」,其殘基負責抗原結合。超變區包括來自「互補決定區」或「CDR」之胺基酸殘基(即通常在輕鏈可變域之大約殘基24-34 (L1)、50-56 (L2)及89-97 (L3)處及重鏈可變域之大約殘基27-35 (H1)、50-65 (H2)及95-102 (H3)處;Kabat等人,Sequences of Proteins of Immunological Interest,第5版,Public Health Service, National Institutes of Health, Bethesda, MD. (1991))及/或來自「超變環」之彼等殘基(即,輕鏈可變域之殘基26-32 (L1)、50-52 (L2)及91-96 (L3)及重鏈可變域之殘基26-32 (H1)、53-55 (H2)及96-101 (H3);Chothia及Lesk, 1987, J. Mol. Biol. 196:901-917)。「框架區」或「FR」殘基為除如本文所定義之超變區殘基以外之彼等可變域殘基。
如本文所用「單株抗體(mAb)」係指由相同之免疫細胞製成之抗體,該等免疫細胞皆為特異性結合靶物質之獨特親本細胞之純系。mAb作為多種疾病之治療劑越來越受歡迎,該等疾病包括(但不限於)癌症、關節炎、氣喘、結腸炎、自身免疫疾病及感染。mAb為分子量接近150 kDa之大蛋白質,且由兩條相同之約50 kDa重鏈(HC)及兩條相同之約25 kDa輕鏈(LC)構成。其亦含有至少16個二硫鍵,該等二硫鍵維持三維結構及生物活性。儘管共享相似之二級蛋白質結構,但不同之mAb之可變區、尤其互補決定區(CDR)之序列有很大不同,該等區域負責抗體-抗原結合之多樣性及特異性。
如本文所用術語抗體之「抗原結合片段」係指抗體中含有抗體之互補決定區(「CDR」)及視情況存在之包括抗體之「可變區」抗原識別位點之框架殘基且展現免疫特異性結合抗原能力之一或多個部分。該等片段包括Fab'、F(ab')2、Fv、單鏈(ScFv)及其突變體、天然變體及包括抗體之「可變區」抗原識別位點及異源蛋白質(例如毒素、不同抗原之抗原識別位點、酶、受體或受體配體等)之融合蛋白。
如本文所用「離子交換層析(IEX)」係指基於可電離分子之總電荷分離可電離分子之方法。蛋白質由帶正電荷及帶負電荷之化學基團組成。端視環境之pH,蛋白質可攜帶淨正電荷、淨負電荷或不帶電荷。分子不帶淨電荷時之pH稱為等電點(pI)。所關注蛋白質之淨電荷係藉由組合等電點及緩衝液之pH來計算,等電點可基於分子之一級序列來計算。當IEX管柱在特定pH下裝載樣品時,所有適當帶電之蛋白質將結合管柱中之樹脂。舉例而言,帶淨負電荷之蛋白質將由陰離子交換樹脂管柱捕獲。
如本文所用「電荷變體」係指與未修飾之形式相比等電點(pI)或電荷改變之同種型及蛋白質變體。pI相對較低之電荷變體稱為「酸性變體」,而pI相對較高之電荷變體稱為「鹼性變體」。電荷變體可影響抗體之特性,包括改變mAb結合蛋白質或細胞膜靶之能力。此會影響抗體之組織穿透、組織分佈及藥物代謝動力學。電荷變體之實例包括(但不限於)去醯胺、N末端焦麩胺酸鹽之形成、聚集、異構化、唾液酸化之聚醣、抗體片段化及離胺酸殘基上之醣化。
如本文所用「轉譯後修飾(PTM)」係指蛋白質生物合成後,蛋白質上一或多個胺基酸發生之生物化學修飾。PTM經由調節蛋白質摺疊、使蛋白質靶向特定細胞區室或經由調節配體與其他蛋白質之間之相互作用在細胞功能中發揮重要作用。最常見之修飾為特異性裂解前體蛋白質;形成二硫鍵;或共價添加或去除低分子量基團,從而產生諸如以下之修飾:乙醯化、醯胺化、生物素化、半胱胺酸化、去醯胺化、法尼醯化、甲醯化、香葉基化、麩胱甘肽化、醣化(與碳水化合物之非酶偶聯)、醣基化(與碳水化合物之酶偶聯)、羥基化、甲基化、單ADP-核醣基化、肉豆蔻醯化、氧化、棕櫚醯化、磷酸化、聚(ADP-核糖基)化、硬脂醯化或硫酸化。泛素化為在蛋白質降解路徑中非常重要之另一種常見之PTM。一些PTM可藉由解偶聯酶之作用逆轉。
如本文所用「N-連接醣基化」係指轉譯後蛋白質修飾。N-連接醣基化為寡醣與氮原子、通常為天冬醯胺殘基之N4之連接。所有N-連接之碳水化合物係經由N-乙醯葡萄糖胺及胺基酸天冬醯胺連接。
如本文所用「肽圖譜(peptide mapping)」係指表徵蛋白質並闡明其一級胺基酸結構之技術。其係廣泛使用之表徵單株抗體及其他重組蛋白藥品之技術。
如本文所用「葡萄糖醛酸化」係指其中衍生自輔因子UDP-葡萄糖醛酸之葡萄糖醛酸共價連接至含有親核官能團之受質的結合反應。
II. 用於鑑別葡萄糖醛酸化之方法及其使用方法 A. 鑑別葡萄糖醛酸化
提供用於鑑別葡萄糖醛酸化蛋白質藥物產品之組合物及方法。實例1-3提供用於檢測及鑑別治療性蛋白質中之葡萄糖醛酸化胺基酸之方法之詳細描述。通常,一種用於鑑別蛋白質藥物產品之葡萄糖醛酸化之方法包括使蛋白質藥物產品去醣基化並用FabRICATOR®處理去醣基化蛋白質藥物產品以產生一個Fc*片段(經由非共價相互作用結合在一起之兩個相同的Fc/2片段)及一個Fab
2片段。在一個實施例中,Fc*及Fab
2片段係使用以名稱FabRICATOR®出售之來自化膿性鏈球菌之重組IdeS酶產生。
然後使Fc*及Fab
2片段分離成Fc*及Fab
2之酸性部分。在一個實施例中,Fc*及Fab
2片段係使用離子交換層析(例如強陽離子交換層析)來分離。如實例1中所述,將一等份去醣基化mAb樣品(約50 µg)注射至YMC-BioPro SP-F強陽離子交換(SCX)管柱(100 × 4.6 mm)上用於質量量測,該管柱耦聯至Thermo Exactive Plus EMR質譜儀或Thermo Q Exactive plus質譜儀。使樣品分離並經20分鐘pH梯度用基於乙酸銨之緩衝液(緩衝液A:20 mM乙酸銨,pH 5.8;緩衝液B:200 mM乙酸銨,pH 7.6)溶析。在SCX管柱之後連接有分析型分離器(約200:1比率)以使分析流在質譜儀之前減小至約2 µL/min用於質量檢測。使來自分離器之高流轉向Waters ACQUITY光二極體陣列(PDA)檢測器以進行同步UV檢測(280 nm)。因此,檢測到酸性肩峰且歸於質量增加約176 Da之抗體變體。
分離後,收集酸性部分,乾燥並使其變性。在一個實施例中,首先在SpeedVac™中乾燥收集的酸性部分,且然後藉由在50℃下加熱30分鐘在20 µL含有5 mM二硫蘇糖醇(DTT)、8 M尿素及100 mM Tris-HCl (pH 7.5)之溶液中變性並還原。應意識到,熟習此項技術者應理解,可使用其他還原劑、變性劑及溫度來乾燥酸性部分及使其變性。
使乾燥及變性之酸性部分烷基化且然後例如用胰蛋白酶酶消化,以形成樣品。酸性部分可藉由將其在室溫下在黑暗中在10 mM碘乙醯胺(IAA)中培育30分鐘來烷基化。該等部分之消化可藉由將其用175 µL 100 mM Tris-HCl (pH 7.5)稀釋並在37℃下以1:10 (w/w)之酶對受質比率添加胰蛋白酶保持4小時來完成。
然後在37℃下將胰蛋白酶消化之酸性部分與50 mM NaBH
4一起培育1小時,隨後用10%甲酸(FA)淬滅以形成還原樣品。然後對還原樣品進行逆相液相層析/質譜分析以鑑別蛋白質藥物產品之葡萄糖醛酸化。在一個實施例中,該方法包括比較還原樣品與非還原樣品之間的質譜結果以鑑別還原樣品與非還原樣品之間的質量差別之步驟。藥物產品可為單株抗體或融合蛋白。在一個實施例中,葡萄糖醛酸化係在蛋白質藥物產品之離胺酸殘基上檢測。
B. 用於提高蛋白質藥物產品之純度之方法
所揭示方法可用於監測蛋白質藥物產品(例如單株抗體或其他治療性蛋白質)之純度。一個實施例提供提高蛋白質藥物產品純度之方法,該方法藉由分析蛋白質藥物產品鑑別出蛋白質藥物產品之一或多種胺基酸之一級胺之葡萄糖醛酸化,並自蛋白質藥物產品去除葡萄糖醛酸化之蛋白質以產生經純化之蛋白質藥物產品。葡萄糖醛酸化可使用上文及實例中所述之方法來檢測。在一個實施例中,葡萄糖醛酸化蛋白質係藉由視情況與質譜耦聯之高效液相層析技術來去除。代表性層析技術包括(但不限於)尺寸排阻層析、離子交換層析、親和層析、高效液相層析、超高效液相層析及其組合。通常,葡萄糖醛酸化發生在蛋白質藥物產品之離胺酸殘基上。
另一實施例提供用於鑑別轉譯後修飾之蛋白質藥物產品之方法,該方法藉由分析自哺乳動物細胞培養物獲得之蛋白質藥物產品之葡萄糖醛酸化來實施,其中蛋白質藥物產品之葡萄糖醛酸化之存在表明蛋白質藥物產品包含轉譯後修飾。哺乳動物細胞培養物通常含有中國倉鼠卵巢細胞。
C. 蛋白質藥物產品
另一實施例提供含有治療性蛋白質之蛋白質藥物產品,其中治療性蛋白質之少於1.0%、2.0%、3.0%、4.0%、5.0%、6.0%、7.0%、8.0%、9.0%或10%之胺基酸經葡萄糖醛酸化。葡萄糖醛酸化%可使用所揭示方法測定。在一個實施例中,胺基酸為離胺酸。通常,治療性蛋白質為單株抗體或融合蛋白。
蛋白質藥物產品可為抗體或其抗原結合片段、重組蛋白或融合蛋白。抗體較佳為單株抗體。
實例 1 :在抗體完整層級上鑑別修飾方法
對mAb進行在線IEX-MS分析。將一等份去醣基化mAb樣品(約50 µg)注射至YMC-BioPro SP-F強陽離子交換(SCX)管柱(100 × 4.6 mm)上用於質量量測,該管柱耦聯至Thermo Exactive Plus EMR質譜儀或Thermo Q Exactive plus質譜儀。使樣品分離並經20分鐘pH梯度用基於乙酸銨之緩衝液(緩衝液A:20 mM乙酸銨,pH 5.8;緩衝液B:200 mM乙酸銨,pH 7.6)溶析。在SCX管柱之後連接有分析型分離器(約200:1比率)以使分析流在質譜儀之前減小至約2 µL/min用於質量檢測。使來自分離器之高流轉向Waters ACQUITY光二極體陣列(PDA)檢測器以進行同步UV檢測(280 nm)。
結果
因此,檢測到酸性肩峰且歸於質量增加約176 Da之抗體變體。然而,由於醣化修飾之複雜化,在完整層級上之質量量測並不準確,該醣化修飾在相同酸性肩峰中溶析且質量接近(162 Da)。
實例 2 :在抗體亞域層級上檢測新酸性修飾方法
為提高酸性峰至主峰之解析度並改良質量量測精度,用FabRICATOR處理去醣基化mAb樣品,FabRICATOR係一種在兩個鉸鏈區二硫鍵之C末端裂解重鏈之酶。
結果
此處理產生一個Fab
2片段及經由非共價相互作用結合在一起之兩個相同之Fc/2片段(Fc*)。如上文所述,對一等份消化物進行在線IEX-MS分析。如預期,在Fab2及Fc* (圖1A-1P)之酸性峰中檢測到質量增加約176 Da之相同酸性變體。
實例 3 :新酸性修飾之鑑別及確認方法
為進一步鑑別胺基酸殘基之修飾位點並獲得此未知修飾之準確質量,自SCX管柱收集Fc*及Fab2片段之酸性部分,用於肽圖譜分析。首先在SpeedVac中乾燥收集之酸性部分,且然後藉由在50℃加熱30分鐘在20 μL含有5 mM二硫蘇糖醇(DTT)、8 M尿素及100 mM Tris-HCl (pH 7.5)之溶液中變性並還原。然後,藉由在室溫下在黑暗中培育30分鐘,用10 mM碘乙醯胺(IAA)使樣品烷基化。然後用175 μL 100 mM Tris-HCl (pH 7.5)稀釋經還原及烷基化之樣品,並用胰蛋白酶在37℃下以1:10 (w/w)之酶對受質比率消化4小時。藉由添加4 μL 10% FA停止消化。然後藉由RP-UPLC分離每個消化之蛋白質樣品之等份,隨後進行在線MS分析。在Thermo Q Exactive Plus MS系統上實施MS及MS/MS實驗,該系統具有在MS/MS實驗期間用於肽片段化之高能碰撞解離(HCD)。然後對照含有mAb序列及質量在170至180 Da之間之可變通配符修飾之數據庫搜索MS原始數據文件。
結果
結果顯示,此未知修飾展現+176.03 Da之單同位素質量且在mAb序列中之多個Lys殘基上以低含量出現(圖2)。基於準確之δ質量,假設此修飾具有與葡萄糖醛酸化相同之元素組成(C
6H
8O
6),然而,據報導,Ser及Thr經由O鍵在UDP-葡萄糖醛酸基轉移酶催化下發生了該修飾。
在37℃下將胰蛋白酶消化之酸性部分與50 mM NaBH
4一起培育1小時,然後用10%甲酸(FA)淬滅。然後對NaBH
4處理之樣品進行RP LC/MS分析。結果顯示,此修飾(+176 Da)可由NaBH
4還原(+178 Da),表明修飾中存在席夫鹼(Schiff base)結構(圖3A-3B)。
藉由將mAb樣品與100 mM葡萄糖醛酸在37℃一起培育24小時來實施強制葡萄糖醛酸化。隨後之胰蛋白酶消化及肽圖譜分析顯示,一系列葡萄糖醛酸化肽在強制修飾樣品中呈現高得多之豐度,顯示出與在未處理樣品中所發現相同之精確質量、MS2片段化圖樣及滯留時間(圖4A-4E、5A-5D、6A-6D及7A-7D)。
圖1A-1F為顯示示例性mAb之Fab
2電荷變體分析之層析圖。圖1G-1P為顯示示例性mAb之Fc電荷變體分析之層析圖。
圖2為顯示酸性Fc、鹼性Fc及酸性Fab
2分離之層析圖。
圖3A顯示未用NaBH
4處理之樣品之MS2片段化圖樣。圖3B顯示用NaBH
4處理之樣品之MS2片段化圖樣。
圖4A為使用WCX管柱之層析圖。箭頭指示強制葡萄糖醛酸處理。圖4B為顯示Fab
2對照之結果之圖。圖4C為顯示用葡萄糖醛酸處理之Fab
2之結果之圖。圖4D為顯示Fc對照之結果之圖。圖4E為顯示用葡萄糖醛酸處理之Fc之結果之圖。
圖5A為天然樣品之層析圖,其顯示預先存在之葡萄糖醛酸化。圖5B為用葡萄糖醛酸處理之樣品之層析圖,其顯示強制葡萄糖醛酸化。圖5C顯示預先存在之葡萄糖醛酸化之M2片段化圖樣之結果。圖5D顯示用葡萄糖醛酸處理之樣品之M2片段化圖樣之結果。
圖6A為第二樣品之層析圖。6B為用葡萄糖醛酸處理之第二樣品之層析圖,其顯示強制葡萄糖醛酸化。圖6C顯示預先存在之葡萄糖醛酸化之M2片段化圖樣之結果。圖6D顯示用葡萄糖醛酸處理之樣品之M2片段化圖樣之結果。
圖7A為第三樣品之層析圖。7B為用葡萄糖醛酸處理之第三樣品之層析圖,其顯示強制葡萄糖醛酸化。圖7C顯示預先存在之葡萄糖醛酸化之M2片段化圖樣之結果。圖7D顯示用葡萄糖醛酸處理之樣品之M2片段化圖樣之結果。
Claims (18)
- 一種包含治療性蛋白質之蛋白質藥物產品,其中少於10%之該治療性蛋白質之胺基酸經葡萄糖醛酸化。
- 如申請專利範圍第1項之蛋白質藥物產品,其中少於5%之該治療性蛋白質之胺基酸經葡萄糖醛酸化。
- 如申請專利範圍第1或2項之蛋白質藥物產品,其中該等胺基酸為離胺酸胺基酸。
- 如申請專利範圍第1至3項中任一項之蛋白質藥物產品,其中該治療性蛋白質為抗體或融合蛋白。
- 如申請專利範圍第4項之蛋白質藥物產品,其中該抗體為單株抗體或其抗原結合片段。
- 一種包含治療性蛋白質之蛋白質藥物產品,其中少於3%之該治療性蛋白質之離胺酸胺基酸經葡萄糖醛酸化。
- 一種提高蛋白質藥物產品之純度之方法,其包括: 分析該蛋白質藥物產品以鑑別該蛋白質藥物產品之一或多個胺基酸之一級胺之葡萄糖醛酸化;及 自該蛋白質藥物產品去除葡萄糖醛酸化蛋白質,以產生經純化之蛋白質藥物產品。
- 如申請專利範圍第7項之方法,其中該等葡萄糖醛酸化蛋白質係藉由高效液相層析技術去除。
- 如申請專利範圍第8項之方法,其中該等層析技術係選自由尺寸排阻層析、離子交換層析、親和層析及其組合組成之群。
- 如申請專利範圍第9項之方法,其中該層析技術與質譜分析耦聯。
- 如申請專利範圍第7至10項中任一項之方法,其中該等胺基酸為離胺酸胺基酸。
- 如申請專利範圍第7至11項中任一項之方法,其中該治療性蛋白質為抗體或融合蛋白。
- 如申請專利範圍第12項之方法,其中該抗體為單株抗體或其抗原結合片段。
- 一種用於鑑別轉譯後修飾之蛋白質藥物產品之方法,其包括: 分析自哺乳動物細胞培養物獲得之蛋白質藥物產品之葡萄糖醛酸化,其中該蛋白質藥物產品中存在葡萄糖醛酸化係表明該蛋白質藥物產品包含轉譯後修飾。
- 如申請專利範圍第14項之方法,其中該哺乳動物細胞培養物包含中國倉鼠卵巢細胞。
- 如申請專利範圍第14或15項之方法,其中該葡萄糖醛酸化係在該蛋白質藥物產品之離胺酸胺基酸上。
- 如申請專利範圍第14至16項中任一項之方法,其中該蛋白質藥物產品為抗體或融合蛋白。
- 如申請專利範圍第17項之方法,其中該抗體為單株抗體或其抗原結合片段。
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