TWI398453B - 層析媒介質 - Google Patents
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Description
本發明係關於新穎層析媒介質之製備及使用,該層析媒介質較佳為用於分離及純化諸如肽、蛋白質及抗體之各種生物分子之混合模式聚合層析媒介質。更特定言之,本發明揭示新穎層析媒介質,較佳為混合陰離子交換劑、混合陰離子-陽離子交換劑及疏水交換劑。藉由以聚乙烯亞胺及其官能化作用改質聚合物來製備該層析媒介質。已意外發現該等混合模式之聚合媒介質呈現增強之分離能力及蛋白質結合容量。
諸如由Pete Gagnon,"Purification Tools for Monoclonal Antibodies",Validated Biosystems,Inc.,(1996)已詳細記錄藉由離子交換層析媒介質分析蛋白質混合物。新近,蛋白質基藥物及疫苗之發展已增加了對於較大規模純化蛋白質混合物之需要。非常理想的是在該領域中可利用離子交換層析媒介質。
製備該等層析媒介質材料,尤其為含有第一胺及第二胺官能基之陰離子交換劑之一種方法係藉由以聚乙烯亞胺(PEI)塗覆多孔二氧化矽材料之內表面。例如,Alpert與Regnier,J.Chromatogr.185
,375-392(1979)中已揭示以聚乙烯亞胺塗覆二氧化矽粒子內表面,繼而經交聯固定且在Lawson等人,Anal.Biochem.133,85-93(1983)中已描述以彼方式製備之材料對於層析分離合成寡核苷酸之用途。
類似地,在JT Baker Chemical Co.之US 4,540,486中已揭示經PEI塗覆多孔二氧化矽粒子或藉由聚乙烯亞胺基丙基-三甲氧基矽烷之共價結合獲得之可控孔度玻璃粒子之製備。該相同專利揭示藉由PEI塗覆之二氧化矽與環狀羧酸酐反應形成具有混合弱鹼/弱酸官能基之層析樹脂。已顯示當該等材料用於層析管柱中時,其可分離細胞色素C、α1-酸糖蛋白、卵清蛋白及β-乳球蛋白(弱鹼媒介質)之混合物或卵清蛋白、細胞色素C、血色素及溶菌酶(混合弱鹼/弱酸媒介質)之混合物。在JT Baker Chemical Co.之US 4,606,825中亦已證實PEI衍生之多孔二氧化矽或其羧酸化變型在純化免疫球蛋白G中之功效。
如在JT Baker Chemical Co.之US 4,721,573中所揭示,上文所述之PEI塗覆二氧化矽之醯化形式可藉由引入磺酸基團而進一步轉化為混合弱酸/強酸官能基媒介質。以該磺化媒介質填充之管柱可供分離細胞色素C、血色素、溶菌酶及卵清蛋白之混合物以及分離融合瘤細胞培養基之蛋白質組份。
已與PEI共價結合之二氧化矽粒子亦可藉由與其中醯基可為線性烴鏈、苯基或經取代苯基之單醯氯或線性羧酸酐反應而轉化為疏水層析媒介質。如在JT Baker Chemical Co.之US 4,551,245中所揭示,以彼方式使用丁醯氯製備之二氧化矽基弱鹼/逆相層析媒介質分離細胞色素C、肌血球素、溶菌酶、卵清蛋白及α-胰凝乳蛋白酶原。
在Amersham之WO 98/58732中已揭示在聚乙烯醇(PVA)臂末端帶有離子交換基團之瓊脂糖珠粒。然而,與本發明不同,該PVA間隔臂對產物不貢獻任何離子交換容量,其本身亦不提供在混合模式產物中之一個官能基。
二氧化矽基層析填料之主要缺點之一為在高pH下其缺乏穩定性。由於以1N氫氧化鈉處理設備為常用之滅菌規範,因此對於涉及純化藥物之應用,此尤其為該種情況。
本發明提供在高pH下可最小化或避免該缺乏穩定性之層析媒介質。本發明亦提供混合模式之層析媒介質。已發現一種避免該不穩定性問題之可能方式為使用基於以PEI於聚合物表面上(意即,未經交聯)衍生之聚合載體之層析填料,其表面衍生之媒介質可藉由官能化試劑與聚乙烯亞胺之末端胺基於該聚合樹脂表面上之反應而進一步官能化。根據本發明,較佳可提供混合模式之媒介質,例如具有混合第一、第二及第三胺交換位點之媒介質、同時具有弱陰離子與弱陽離子交換位點之媒介質、具有弱陰離子、弱陽離子及強陽離子交換位點之媒介質、具有弱陰離子及疏水(逆相)交換位點之媒介質及具有弱陰離子與強陰離子交換位點之媒介質。此外,意外發現基於該聚乙烯亞胺衍生之聚合衍生物及其官能化衍生物之層析媒介質提供不同且獨特之分離特徵。在本發明之一態樣中,提供用於生物分離之聚合層析媒介質。由於目前該等層析媒介質為簡單離子交換劑,因此本發明在媒介質之製備方法及特徵上不同於目前之聚合層析媒介質。就製備方法、組份、用途及效能而言,本發明之媒介質亦不同於目前之二氧化矽基媒介質。令人驚奇地,根據本發明製備之聚合混合模式媒介質具有經增強之分離及結合容量。
本發明涉及新穎聚合層析媒介質且較佳為混合模式之聚合層析媒介質之製備及使用。根據本發明,使用以聚乙烯亞胺衍生之聚合粒子,且較佳為該等以適當反應物進一步官能化之聚乙烯亞胺衍生之聚合粒子來製備聚合媒介質。
用於層析分離蛋白質之聚合材料較佳具有某些特性,諸如:1)微孔尺寸係足夠大以允許與樹脂粒子內外之蛋白質相同大小之分子快速擴散;2)蛋白質與非官能化聚合物間之相互作用將經減弱以避免"非特異性相互作用"且允許以高產量回收所需蛋白質;3)該等樹脂粒子將為剛性以避免在層析操作中所遇到之壓力下的壓縮及流速損失;4)該樹脂在所有分離過程中所遇到之條件下應為化學穩定的。
該等有待以聚乙烯亞胺進行表面衍生之聚合樹脂粒子可為任何合適之可以聚乙烯亞胺衍生之聚合樹脂粒子,且該等聚乙烯亞胺衍生之聚合物或其進一步官能化之衍生物可用作層析分離媒介質。根據本發明適合於以聚乙烯亞胺衍生之聚合樹脂粒子之實例包括(但不限於)纖維素、瓊脂糖、環氧化或鹵化聚苯乙烯、環氧化或鹵化聚丙烯酸酯或聚甲基丙烯酸酯及環氧化或鹵化聚二乙烯基苯。例如,在致孔劑存在下所製備之多孔聚(甲基)丙烯酸酯,基於具有多個可聚合雙鍵之(甲基)丙烯酸單體之高度交聯之樹脂,諸如二甲基丙烯酸乙二醇酯(Showa Denko之US 4,118,347)、三甲基丙烯酸異戊四醇酯(Toyo Soda之US 4,256,842)、三羥甲基丙烷三甲基丙烯酸酯(Rohm與Haas之US 4,582,860)或二甲基丙烯酸甘油酯(Mitsubishi之US 2,254,634),已顯示提供具有所要特性之材料。此外,將官能性單體添加至聚合混合物中產生可經形成共價鍵結合其它分子之具有官能基之最終產物。該等單體之實例為甲基丙烯酸甘油酯(-OH基)(Mitsubishi之US 2,254,634,1993)、甲基丙烯酸二甲胺基乙酯(第三胺)或甲基丙烯酸縮水甘油酯(Showa Denko之US 4,118,347、Toyo Soda之US 4,256,842、Rohm與Haas之US 4,582,860)。作為適合於與PEI表面衍生之聚合樹脂粒子反應之合適官能化試劑之實例為(例如):諸如戊二酸酐及丁二酸酐之環狀羧酸酐之酸酐、諸如順丁烯二酸酐之不飽和羧酸酐、諸如偏亞硫酸氫鈉之亞硫酸氫鹽之磺化劑、諸如丁醯氯及乙酸酐或丁酸酐之烷基氯或酸酐及諸如氯化(3-氯-2-羥丙基)三甲基鍍之含有第四銨官能基之烷基氯及該等官能化試劑之混合物。
在本發明之一實施例中,藉由聚乙烯亞胺與帶有環氧基或鹵基(諸如氯、溴或碘基)之聚合載體反應來製備具有混合第一及第二及第三胺位點之陰離子交換劑。該聚合載體可為任何合適之合成聚合物或天然聚合物樹脂,諸如聚(甲基)丙烯酸酯、纖維素、聚苯乙烯-二乙烯基苯及瓊脂糖。該等市售樹脂之實例為Tosoh Biosciences Toyopearl AF-環氧基650M、環氧基活化之瓊脂糖(Sepharose)6B。該等材料可經形成化學穩定之α-羥胺基與各種分子量之聚乙烯亞胺之末端胺基之一反應。根據以下實例1至5,在表1中概括以彼方式製備之材料之某些特性。
在第二實施例中,藉由使PEI官能化聚合珠粒與諸如以下實例6至10中之戊二酸酐或丁二酸酐之環狀羧酸酐反應來製備具有弱陰離子與弱陽離子交換位點之混合模式媒介質。
在第三實施例中,如實例11至13中所述,藉由使PEI衍生之聚合物與不飽和羧酸酐反應,繼而磺化來製備具有弱陰離子、弱陽離子及強陽離子位點之混合模式媒介質。
在第四實施例中,藉由使PEI衍生聚合物與烷基氯(實例14至18)或一元酸酐(實例19至24)反應來製備具有弱陰離子與疏水(逆相)位點之混合模式媒介質。使用甲苯或二噁烷作為溶劑在室溫下進行與丁醯氯之反應歷時2小時。使用三乙胺以清除作為反應副產物所產生之氫氯酸。
在第五實施例中,如在以下實例20-22中所述,藉由PEI塗覆之樹脂與丁酸酐在1-甲氧基-2-丙醇中於60℃下反應3hr而獲得混合模式媒介質。
如在實例24(乙酸酐)或分別在實例20及23(混合酸酐)中所示,可藉由使用乙酸酐或連續添加丁酸酐及乙酸酐來製備混合模式弱鹼/逆相樹脂,其中疏水部分為甲基或甲基與丁基之組合。如此,可能藉由使用一種試劑或試劑之組合來改變且選擇適當之疏水性。
在第六實施例中,藉由使PEI衍生之聚合物與含有末端第四銨官能基之烷基氯反應來製備具有弱陰離子與強陰離子(第四銨)位點之混合模式媒介質。更特定言之,如以下實例25至30中所示,使PEI衍生之聚合物在水性氫氧化鈉中與氯化(3-氯-2-羥丙基)三甲基鍍在70-80℃下反應。
根據本發明,已意外發現該等聚合混合模式層析媒介質呈現增強之分離能力以及高容量與穩定性。舉例而言,如圖1中所示,聚合混合模式陰離子交換媒介質可分離溶菌酶、免疫球蛋白G、牛血清白蛋白、β-乳球蛋白A及β-乳球蛋白B,而PEI-二氧化矽基媒介質不可分離所有該等蛋白質。如圖2中所示,混合模式陽離子交換媒介質可有效分離BSA、IgG、細胞色素-C及溶菌酶,而混合模式之二氧化矽媒介質不可分離所有該等蛋白質。
將12 g平均粒徑為35微米直徑之含有甲基丙烯酸酯聚合物之環氧樹脂與250 ml二噁烷置於一配備有一漏斗、攪拌器、回流冷凝器及正壓氮氣入口之1 L圓底燒瓶中且攪拌30分鐘以使該樹脂膨脹。添加20 g聚乙烯亞胺(PEI,平均分子量600道爾頓(Dalton))且以額外150 ml二噁烷沖洗該漏斗。將該經攪拌之混合物回流隔夜。將混合物冷卻之後,將該混合物轉移至一吸濾瓶中,抽乾,以二噁烷洗滌一次、以甲醇洗滌三次且於60℃真空下乾燥。元素分析:57.8% C、7.4% H及5.6% N。
將5 g含有甲基丙烯酸酯聚合物之環氧樹脂與150 ml二噁烷置於一配備有一攪拌器及回流冷凝器之500 ml圓底燒瓶中。添加30 g PEI(平均分子量1200道爾頓)且將混合物回流隔夜。使其冷卻之後,將該混合物轉移至一吸濾瓶中,抽乾,以二噁烷洗滌兩次、以甲醇洗滌三次且乾燥。元素分析:55.7% C、7.8% H、5.5% N。
將5 g含有甲基丙烯酸酯聚合物之環氧樹脂與150 ml二噁烷與水之50/50混合物置於一配備有一攪拌器及回流冷凝器之500 ml圓底燒瓶中。添加30 g PEI(平均分子量1200道爾頓)且將混合物回流隔夜(89℃)。使其冷卻之後,將該混合物轉移至一吸濾瓶中,抽乾,以二噁烷洗滌兩次、以甲醇洗滌三次且乾燥。元素分析顯示最終產物含有:57.5% C、7.4% H、2.5% N。
將5 g含有甲基丙烯酸酯聚合物之環氧樹脂與100 ml 30重量%之10,000道爾頓平均分子量之PEI於二噁烷中之溶液置於一配備有一攪拌器、氮氣入口及回流冷凝器之250 ml圓底燒瓶中且將該經攪拌之混合物回流隔夜。使其冷卻之後,將該混合物轉移至一吸濾瓶中,抽乾,以60℃水洗滌三次、以甲醇洗滌三次且於60℃真空烘箱中乾燥隔夜。元素分析顯示最終產物含有:56.5% C、7.8% H、6.4% N。
將5 g含有甲基丙烯酸樹脂之環氧樹脂與100 ml 30重量%之10,000道爾頓平均分子量之PEI於水中之溶液置於一配備有一攪拌器及回流冷凝器之250 ml圓底燒瓶中且將該經攪拌之混合物回流17 1/2小時。使其冷卻之後,將該混合物轉移至一吸濾瓶中,抽乾,以水洗滌三次、以甲醇洗滌三次且於60℃真空烘箱中乾燥隔夜。元素分析顯示最終產物含有56.9% C、7.5% H及3.2% N。
如表1中之結果顯示,系統中存在之水對氮之併入具有不利影響,然而當各種分子量之PEI與樹脂在乾燥二噁烷中反應時獲得類似之氮含量(即使當時在實例1中PEI與聚合物之比率較低)。
如表1中之結果進一步顯示,在純二噁烷中所進行之反應相對於在二噁烷/水混合物或純水中之反應產生較高程度之官能化,如由終產物之氮含量所示。當將純二噁烷用作溶劑(表1之第一列)時,因為PEI之分子量自600增加至10,000道爾頓,所以未顯示出顯著較高水平之氮引入。
將30 g如實例1中所製備之PEI聚合物以其體積兩倍之100%乙醇洗滌且以與其相同體積之1-甲氧基-2-丙醇洗滌兩次以移除任何殘餘水分。隨後將該聚合物在450 ml 1-甲氧基-2-丙醇中形成漿料,轉移至一配備有頂置式攪拌器、回流冷凝器及正壓力氮氣入口之燒瓶中且加熱至60℃。添加19.43 g 95%之戊二酸酐溶液(基於PEI樹脂之氮含量為1當量)之後,將溫度維持在60+/-2℃下歷時21/2小時。隨後將反應混合物轉移至一吸濾瓶中,抽乾且將殘餘固體以100 ml 1-甲氧基-2-丙醇洗滌一次、以100 ml甲醇洗滌兩次且以100 ml儲備液(乙醇:水20:80 v/v,或100 Mm pH 4.5之含有2%苄醇之乙酸鈉)洗滌兩次。將產物儲存於分別用於特徵化及用於層析分離蛋白質之儲備液中。元素分析顯示最終產物含有:% C=55.1,% N=4.6。
將73.7 g如實例1中所製備之經PEI塗覆之聚合物、225 ml 1-甲氧基-2-丙醇及14.4 g丁二酸酐加熱至60+/-2℃且維持彼溫度21/2小時。隨後將該反應混合物轉移至一吸濾瓶中,抽乾且將殘餘固體以100 ml 1-甲氧基-2-丙醇洗滌一次、以100 ml甲醇洗滌兩次且以100 ml如實例5中所述之儲備緩衝液洗滌兩次。元素分析顯示最終產物含有:53.08% C、7.13% H及4.86% N。
如同實例7進行該反應,除丁二酸酐填料為17.43 g以外。元素分析顯示最終產物含有:53.18% C、7.97% H及5.18% N。
將11 g如實例1中所製備之經PEI塗覆之聚合物以其體積兩倍之100%乙醇洗滌且以與其相同體積之二噁烷洗滌兩次以移除任何殘餘水分。隨後將該樹脂在約200 ml二噁烷中形成漿料,轉移至一配備有一氮氣吸入管之三燒瓶中且加熱至50-60℃。一小時之後,添加7 g戊二酸酐且將溫度維持在60+/-2℃歷時2 1/2小時。隨後將該反應混合物轉移至一吸濾瓶中,抽乾且將殘餘固體以二噁烷洗滌三次、以甲醇洗滌三次且於真空下乾燥。元素分析顯示最終產物含有:56.64% C、7.54% H及4.36% N。
如同實例9進行該反應,除使用10 g根據實例1所製備之PEI聚合物與6.6 g丁二酸酐以外。元素分析顯示最終產物含有:54.46%C,7.96% H及4.6%N。
將86.5 g根據實例1所製備之PEI聚合物與865 ml甲氧基-2-丙醇置於一配備有一攪拌器及回流冷凝器之2公升圓底燒瓶中且攪拌30 min。添加23.7 g順丁烯二酸酐且將該混合物在60℃下攪拌2.5小時。使混合物冷卻之後,將該混合物轉移至一吸濾瓶中,抽乾,以甲氧基-2-丙醇洗滌一次、以水洗滌三次、以甲醇洗滌三次。元素分析顯示最終產物含有:% C 55.6,% N 4.9,% H 7.1,% S 0。
在190 g偏亞硫酸氫鈉存在下,在80+/-2℃下將86.5 g如實例11中所順丁烯二酸化之樹脂於900 ml 0.01 N氫氧化鈉中加熱6小時。使反應混合物冷卻之後,將該混合物轉移至一吸濾瓶中,抽乾,以甲氧基-2-丙醇洗滌一次、以水洗滌三次、以甲醇洗滌三次且儲存於儲備緩衝液中以供進一步使用。元素分析顯示最終產物含有:% C 49.8,% N 4.6,% H 6.8,% S 2.1。
如同實例12進行該反應,除該反應在80+/-2℃下進行20小時以外。元素分析顯示最終產物含有:% C 50.2,% N 4.5,% H 6.8,% S 2.1。
8小時反應(3.8% S,實例12)與20小時反應(3.40% S,實例13)之後所得樣品之硫含量比較顯示該反應在8小時後即完成。
將30 g根據實例1所製備之官能化PEI以120 ml 100%乙醇洗滌兩次且以120 ml甲苯洗滌兩次以移除殘餘水分。隨後將該材料在300 ml甲苯中形成漿料,添加9.29 g丁醯氯(基於該樹脂之氮含量為1當量)及9.24 g三乙胺且使該反應在25+/-2℃下進行2小時。隨後將該樹脂轉移至一吸濾瓶中且以300 ml甲苯洗滌、以300 ml甲醇洗滌、以300 ml去離子水洗滌兩次、以300 ml甲醇洗滌三次且以300 ml pH 5之儲備緩衝液(10 mM乙酸鈉,pH 4.5)洗滌兩次。元素分析顯示最終產物含有:53.7% C、7.5% H及5.6% N。
如同實例14進行該反應,除溶劑為450 m1二噁烷以外。元素分析顯示最終產物含有:54.7% C、8.0% H及5.5 % N。
如同實例14進行該反應,除使用13.3 g丁醯氯與13.2 g三乙胺以外。元素分析顯示最終產物含有:53.6% C、7.9 % H及4.5 % N。
如同實例14進行該反應,除使用19 g丁醯氯與18 g三乙胺以外。元素分析顯示最終產物含有:55.4% C、7.8 % H及4.2 % N。
如同實例14進行該反應,除使用25.3 g丁醯氯與24 g三乙胺以外。元素分析顯示最終產物含有:55.0% C、7.9% H及4.5% N。
表3中概括使用甲苯作為溶劑所獲得之一些結果。
如表3中之結果所示,增加試劑量導致增加之官能化水平(如產物中氮含量之相應降低所示)。
使60 g如實例1中所製備且於200 ml 1-甲氧基-2-丙醇中膨脹之PEI官能化聚合物、600 ml 1-甲氧基-2-丙醇、56.3 g丁二酸酐(過量1.5莫耳)及36 g三乙胺在60 +/- 2℃下反應3小時。將反應混合物轉移至一吸濾瓶中,抽乾,以500 ml 1-甲氧基-2-丙醇洗滌一次,以500 ml甲醇洗滌一次,以500 ml去離子水洗滌兩次且以500 ml儲備緩衝液洗滌兩次。元素分析顯示最終產物含有:54.6% C、7.9% H及4.8% N。
使60 g如實例1中所製備且於200 ml 1-甲氧基-2-丙醇中膨脹之PEI官能化聚合物、600 ml 1-甲氧基-2-丙醇、6.2 g 丁二酸酐及4.0 g三乙胺在60 +/- 2℃下反應3小時。於彼時添加24.2 g乙酸酐且使該反應在相同溫度下再進行3小時。將反應混合物轉移至一吸濾瓶中,抽乾,以500 ml 1-甲氧基-2-丙醇洗滌一次,以500 ml甲醇洗滌一次、以500 ml去離子水洗滌兩次且以500 ml儲備緩衝液洗滌兩次。元素分析顯示最終產物含有:53.7% C、7.7% H及4.7% N。
如同實例19進行該反應,除使用6.26 g丁酸酐與4.0 g三乙胺以外。元素分析顯示最終產物含有:53.7% C、7.9% H及4.8% N。
如同實例19進行該反應,除使用1.52 g丁酸酐與1.0 g三乙胺以外。最終產物之元素分析為54.6 % C、8.3% H及5.0% N。
表4中概括一些結果。
表3與表4中之結果之比較顯示,使用顯著較低化學計量量之試劑以丁酸酐可得到類似於以丁醯氯所獲得之彼等取代水平。
如同實例20進行該反應,除使用1.52 g丁酸酐、1.0 g三乙胺與10.7 g乙酸酐以外。元素分析顯示最終產物含有:55.4 % C、7.9% H及4.9% N。
使60 g如實例1中所製備且於20 ml 1-甲氧基-2-丙醇中膨脹之PEI官能化聚合物、500 ml 1-甲氧基-2-丙醇及14.07 g乙酸酐在60 +/- 2℃下反應6小時。將反應混合物轉移至一吸濾瓶中,抽乾,以500 ml 1-甲氧基-2-丙醇洗滌一次、以500 ml 0.1 N NaOH洗滌兩次、以500 ml去離子水洗滌兩次且以500 ml儲備緩衝液洗滌兩次。元素分析顯示最終產物含有:53.8% C、7.5% H及5.3% N。
使80 g如實例1中所製備之PEI官能化聚合物與59 g氯化(3-氯-2-羥丙基)三甲基銨於500 ml 0.5 N氫氧化鈉中之60%溶液在80℃下反應8小時。將反應混合物轉移至一吸濾瓶中且以0.1 N氫氧化鈉洗滌兩次、以去離子水洗滌兩次且以儲備緩衝液洗滌一次。洗滌且乾燥之後,該樹脂具有以下元素分析:54.2% C、8.4% H及5.9% N。
如同實例25進行該反應,除氫氧化鈉溶液為0.05 N以外。洗滌且乾燥之後,該樹脂具有以下元素分析:51.9% C、7.6% H及5.3% N。
如同實例25進行該反應,除該反應持續16小時以外。洗滌且乾燥之後,該樹脂具有以下元素分析:53.4% C、8.4% H及5.7% N。
如同實例26進行該反應,除該反應持續16小時以外。洗滌且乾燥之後,該樹脂具有以下元素分析:52.8% C、8.1% H及5.7% N。
如同實例25進行該反應,除氯化(3-氯-2-羥丙基)三甲基銨之60%溶液與樹脂之比率為0.4而非0.73以外。洗滌且乾燥之後,該樹脂具有以下元素分析:53.72% C、7.32% H及6.04% N。
如同實例25進行該反應,除氯化(3-氯-2-羥丙基)三甲基銨之60%溶液與樹脂之比率為1.2而非0.73以外。洗滌且乾燥之後,該樹脂具有以下元素分析:53.72% C、7.32% H及6.04% N。
使160 g根據實例1所製備之PEI官能化聚合物與111.8 g甲醛、400 ml乙腈及25.9 g氰基硼氫化鈉在40℃下反應5小時。反應之後,將該聚合物以480 ml乙腈洗滌、以480 ml去離子水洗滌且以480 ml甲醇洗滌3次且儲存於儲備緩衝液中以供進一步使用。(元素分析% C=55.2,% N=5.8)
將25 mg根據實例31所製備之聚合物與200 ml乙腈饋入一高壓釜中(安裝帶有Parr 4840溫度控制器之壓力反應器之Parr系列4500工作臺)。當在120 RPM下攪拌該聚合物時,饋入氯甲烷氣體且加壓至60 PSI。將該反應於80℃下持續5小時。反應之後,將產物以60 ml乙腈、120 ml 0.1 N氫氧化鈉、120 ml去離子水、240 ml儲備緩衝液洗滌且儲存於儲備緩衝液中。
將71 g根據實例1所製備之經PEI改質之聚合物與500 ml0.5 N氫氧化鈉混合且在80℃下以50-100 RPM攪拌時使其與49 g 3-(二甲胺基)丙基氯反應歷時8小時。反應之後,將產物以500 ml氫氧化鈉洗滌兩次、以500 ml去離子水洗滌兩次、以500 ml儲備緩衝液洗滌兩次且儲存於該緩衝液中以供進一步使用。元素分析-% C:55.4,% N:6.2。
將如實例1中所製備之層析媒介質與來自JT Baker(產品號7264,批次N16084)之PEI二氧化矽媒介質填充於一4.6 x 100 mm之層析管柱中。將200微升pH 6.2之1 mg/ml溶菌酶、2 mg/ml兔免疫球蛋白G、2 mg/ml牛血清白蛋白與各自2 mg/ml之β-乳球蛋白A及B於20 mM乙酸鈉緩衝液中之溶液注入該管柱中且使用1 ml/min之流速及30 min自100% pH 6.2之20 mM乙酸鈉緩衝液至100% pH 6.2之1.0 mM乙酸鈉緩衝液之梯度進行溶離。藉由一UV偵測器在280 nm下記錄蛋白質之溶離(圖1)。
將實例9中所製備之層析媒介質填充於一4.6 x 100 mm之層析管柱中。將200微升4 mg/ml BSA、2 mg/ml兔IgG、2 mg/ml細胞色素-c與2 mg/ml溶菌酶溶液於20 mM pH 6.2之乙酸鈉緩衝液中之溶液注入該管柱中,且使用1 ml/min之流速及40 min自100% pH 6.2之20 mM乙酸鈉緩衝液至pH 6.2之100%相同溶離劑(但每公升乙酸鈉含有1莫耳)之梯度進行溶離。藉由一UV偵測器在280 nm下記錄藉由該實例之媒介質及用於比較性二氧化矽基混合模式媒介質(JT Baker產品號7269)之蛋白質溶離(圖2)。
量測且比較根據實例9所製備之層析媒介質與二氧化矽基產物之容量。以該媒介質填充一管柱(4.6 mm x 50 mm)。以不同線性速度(cm/hr)將pH 5.6、6.2及6.9之IgG於20 mM乙酸鈉中之溶液(1 mg/ml)應用於該管柱。藉由監控280 nm下之UV來測定穿透率。測定該媒介質在管柱中之穿透容量為10%穿透(表7)。
將根據實例19所製備之層析媒介質與二氧化矽媒介質(J T Baker產品號7285)填充於一7.75 mm x 100 mm之管柱中。注入200 μl 2 mg/ml之細胞色素C、核糖核酸酶、溶菌酶及卵清蛋白於25 mM pH 7.0之磷酸鈉與1.7 M硫酸銨中之溶液。藉由100% 25 mM磷酸鈉與1.7 M硫酸銨至100% pH 7.0之25 mM磷酸鈉之60分鐘線性梯度將蛋白質自該管柱中溶離。藉由280 nm下之UV監控溶離曲線(圖3A及3B)。
將根據實例20所製備之層析媒介質填充於7.75 mm x 100 mm之管柱中。注入200 μl 2 mg/ml之細胞色素C、核糖核酸酶、溶菌酶及卵清蛋白於pH 7.0之25 mM磷酸鈉與1.7 M硫酸銨中之溶液。藉由100% 25 mM磷酸鈉與1.7 M硫酸銨至100% pH 7.0之25 mM磷酸鈉之60分鐘線性梯度將蛋白質自該管柱中溶離。藉由280 nm下之UV監控溶離曲線(圖4)。
將根據實例22所製備之層析媒介質填充於7.75 mm x 100 mm之管柱中。注入200 μl 2 mg/ml之細胞色素C、核糖核酸酶、溶菌酶及卵清蛋白於pH 7.0之25 mM磷酸鈉與1.7 M硫酸銨中之溶液。藉由100% 25 mM磷酸鈉與1.7 M硫酸銨至100% pH 7.0之25 mM磷酸鈉之60分鐘線性梯度將蛋白質自該管柱中溶離。藉由280 nm下之UV監控溶離曲線(圖5)。
將根據實例23所製備之層析媒介質填充於7.75 mm×100 mm之管柱中。注入200 μl 2 mg/ml之細胞色素C、核糖核酸酶、溶菌酶及卵清蛋白於pH 7.0之25 mM磷酸鈉與1.7 M硫酸銨中之溶液。藉由100% 25 mM磷酸鈉與1.7 M硫酸銨至100% pH 7.0之25 mM磷酸鈉之60分鐘線性梯度將蛋白質自該管柱中溶離。藉由280 nm下之UV監控溶離曲線(圖6)。
實例41
將根據實例7所製備之聚合物媒介質填充於1.0 cm x 10 cm之管柱中。首先藉由通過10倍管柱體積之緩衝液A(0.05 M MES,pH 5.6)來平衡該管柱。平衡之後,注入0.5 ml含有兔球蛋白(0.5 mg/ml)及溶菌酶(0.25 mg/ml)之蛋白質溶液。藉由進行40分鐘100%緩衝液A至100%緩衝液B(緩衝液A中之1 M氯化鈉)之線性梯度將蛋白質自該管柱中溶離。第一次蛋白質分離進行之後,藉由以1 ml/min將500 ml 10 mM磷酸循環歷時48小時來洗滌該管柱。洗滌之後,再進行蛋白質分離以測定該管柱之酸穩定性(圖7)。由於在以磷酸洗滌該管柱之前及之後長達48小時期間IgG與溶菌酶之滯留時間無變化,因此該資料顯示該聚合混合模式媒介質在酸性條件下之極佳穩定性。
將根據實例7所製備之聚合物媒介質填充於1.0 cm x 10 cm之管柱中。首先藉由通過10倍管柱體積之緩衝液A(0.05 M MES,pH 5.6)來平衡該管柱。平衡之後,注入0.5 ml含有兔球蛋白(0.5 mg/ml)及溶菌酶(0.25 mg/ml)之蛋白質溶液。藉由進行40分鐘100%緩衝液A至100%緩衝液B(緩衝液A中之1 M氯化鈉)之線性梯度將蛋白質自該管柱中溶離。第一次蛋白質分離進行之後,藉由以1 ml/min將500 ml 0.1 M氫氧化鈉循環歷時24至48小時來洗滌該管柱。洗滌之後,再進行蛋白質分離以測定該管柱之鹼穩定性(圖8)。由於在以氫氧化鈉洗滌該管柱之前及之後長達48小時期間IgG與溶菌酶之滯留時間無變化,因此該資料顯示該聚合混合模式媒介質在鹼性條件下之極佳穩定性。
量測且比較根據實例1、25、26、27、28、29、30及32所製備之層析媒介質之容量。以該媒介質填充一管柱(4.6mm x 50 mm)。以1 ml/min之流速將pH 11之20 mM CAPS(3-[環己胺基]-1-丙磺酸)中之BSA溶液(1 mg/ml)應用於該管柱。藉由監控280 nm下之UV來測定穿透率。測定該媒介質在管柱中之穿透容量為10%穿透。在管柱上負載BSA之後,將該等管柱以CAPS緩衝液洗滌且隨後使用1 M氯化鈉溶離所吸收之BSA以計算飽和容量(表8)。
參考本發明之該等特定實施例本文已描述本發明。應瞭解,在不偏離本文所揭示之本發明之精神及範疇之情況下可進行改變、修改及變化。因此,意欲包含所有符合所附申請專利範圍之該等改變、修改及變化。
圖1係如由UV偵測器所記錄之使用根據實例1所製備之媒介質及根據程序實例34之PEI二氧化矽媒介質分離蛋白質之溶離曲線圖;圖2係如由UV偵測器所記錄之使用根據實例9所製備之媒介質及根據程序實例35之混合模式二氧化矽媒介質分離蛋白質之溶離曲線圖;圖3a及3b係如由UV偵測器所記錄之使用根據實例19所製備之媒介質及根據程序實例37之二氧化矽媒介質分離蛋白質之溶離曲線圖;圖4係如由UV偵測器所記錄之根據實例38之程序使用根據實例20所製備之媒介質分離蛋白質之溶離曲線圖;圖5係如由UV偵測器所記錄之根據實例39中之程序使用根據實例22所製備之媒介質分離蛋白質之溶離曲線圖;圖6係如由UV偵測器所記錄之根據實例40中之程序使用根據實例23所製備之媒介質分離蛋白質之溶離曲線圖;圖7係如根據實例41中之程序所測定之根據實例7所製備之媒介質之酸穩定性圖;且圖8係如根據實例42中之程序所測定之根據實例7所製備之媒介質之鹼穩定性圖。
Claims (8)
- 一種層析媒介質,其包含環氧化或鹵化聚合樹脂粒子,其係藉由與聚乙烯亞胺在該聚合物之表面上反應所衍生且其中該等環氧化或鹵化聚合粒子為環氧化或鹵化聚丙烯酸酯或聚甲基丙烯酸酯,其中以聚乙烯亞胺在該聚合物之表面上所衍生之該等聚合樹脂粒子係藉由一官能化試劑與該聚合樹脂表面上之聚乙烯亞胺之末端胺基反應來官能化,且其中該官能化試劑係選自由以下各物組成之群:酸酐、磺化劑、烷基氯及含有第四銨官能基之烷基氯及其混合物。
- 如請求項1之層析媒介質,其中該官能化試劑係選自由以下各物組成之群:環狀羧酸酐、不飽和羧酸酐、亞硫酸氫鹽、烷基氯、烷基酸酐、含有第四銨官能基之烷基氯及其混合物。
- 如請求項2之層析媒介質,其中該官能化試劑係選自由以下各物組成之群:戊二酸酐、丁二酸酐、順丁烯二酸酐、偏亞硫酸氫鈉、丁醯氯、乙酸酐、丁酸酐、氯化(3-氯-2-羥丙基)三甲基銨及其混合物。
- 如請求項1之層析媒介質,其中該媒介質係混合模式。
- 如請求項4之層析媒介質,其中該混合模式媒介質係選自由以下各物組成之群:具有混合第一、第二及第三胺交換位點之媒介質、同時具有弱陰離子與弱陽離子交換位點之媒介質、具有弱陰離子、弱陽離子及強陽離子交換位點之媒介質、具有弱陰離子及疏水(逆相)交換位點 之媒介質、及具有弱陰離子與強陰離子交換位點之媒介質。
- 一種用於層析之管柱,其係以如請求項1至5中之任一項之層析媒介質填充。
- 一種分離溶液組份之方法,其包含使該溶液通過如請求項6之層析管柱及溶離該溶液之組份。
- 如請求項7之方法,其中該溶液係含有蛋白質之溶液。
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MX2007008832A (es) | 2007-09-07 |
DK1841527T3 (da) | 2009-12-07 |
JP2008528966A (ja) | 2008-07-31 |
AU2005325711B2 (en) | 2010-08-05 |
CA2595735C (en) | 2015-08-11 |
WO2006081002A3 (en) | 2006-10-19 |
IL184761A0 (en) | 2007-12-03 |
IL184761A (en) | 2011-11-30 |
JP5094417B2 (ja) | 2012-12-12 |
WO2006081002A2 (en) | 2006-08-03 |
NZ556639A (en) | 2010-01-29 |
CA2595735A1 (en) | 2006-08-03 |
ES2332008T3 (es) | 2010-01-22 |
DE602005016972D1 (en) | 2009-11-12 |
KR20070102685A (ko) | 2007-10-19 |
KR101279305B1 (ko) | 2013-06-26 |
EP1841527B1 (en) | 2009-09-30 |
US20140194543A1 (en) | 2014-07-10 |
BRPI0519950A2 (pt) | 2009-03-10 |
AU2005325711A1 (en) | 2006-08-03 |
ATE444119T1 (de) | 2009-10-15 |
CN101146609A (zh) | 2008-03-19 |
MY144940A (en) | 2011-11-30 |
CN101146609B (zh) | 2012-03-14 |
PL1841527T3 (pl) | 2010-02-26 |
NO20074331L (no) | 2007-10-17 |
TW200637876A (en) | 2006-11-01 |
US20080203029A1 (en) | 2008-08-28 |
EP1841527A2 (en) | 2007-10-10 |
PT1841527E (pt) | 2009-11-20 |
BRPI0519950B1 (pt) | 2016-03-01 |
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