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      接枝聚合物配基的蛋白質(zhì)吸附層析

      2016-03-19 07:30:39余林玲孫彥
      化工學(xué)報 2016年1期
      關(guān)鍵詞:配基吸附層析

      余林玲,孫彥

      (天津大學(xué)化工學(xué)院生物化工系,天津 300072)

      ?

      接枝聚合物配基的蛋白質(zhì)吸附層析

      余林玲,孫彥

      (天津大學(xué)化工學(xué)院生物化工系,天津 300072)

      摘要:以離子交換、親和結(jié)合和疏水性吸附為主的蛋白質(zhì)吸附層析是藥用蛋白質(zhì)生產(chǎn)過程的核心技術(shù),開發(fā)新技術(shù)和提高蛋白質(zhì)吸附層析操作的分離效率(如選擇性和動態(tài)吸附容量等)是該領(lǐng)域的主要研究目標(biāo)。近年來聚合物配基接枝的層析介質(zhì)由于同時具有較高的吸附容量和傳質(zhì)速率,得到產(chǎn)學(xué)界的廣泛關(guān)注。本文針對聚合物配基接枝修飾的蛋白質(zhì)吸附層析介質(zhì)的配基化學(xué)特征、吸附和傳質(zhì)特性、層析分離應(yīng)用和設(shè)計等方面進(jìn)行評述。首先介紹不同種類的聚合物接枝介質(zhì),然后系統(tǒng)闡述聚合物配基化學(xué)特性對介質(zhì)吸附和傳質(zhì)性能的影響機(jī)制,并分析上述性質(zhì)對聚合物配基接枝層析介質(zhì)分離特性的影響機(jī)理和應(yīng)用,最后討論和展望了高效聚合物配基接枝介質(zhì)設(shè)計、開發(fā)和應(yīng)用的前景。

      關(guān)鍵詞:蛋白質(zhì);吸附;層析;聚合物接枝;配基

      2015-07-13收到初稿,2015-08-26收到修改稿。

      聯(lián)系人:孫彥。第一作者:余林玲(1987—),女,博士,講師。

      Received date: 2015-07-13.

      引 言

      隨著基因工程等生物技術(shù)的不斷發(fā)展,蛋白質(zhì)的表達(dá)水平大幅提升,使得胰島素、干擾素、生長因子、抗體等蛋白質(zhì)類生物藥物的廣泛使用成為可能。然而蛋白質(zhì)具有易失活變性、分離純化工藝流程單元操作多等特點(diǎn),醫(yī)藥產(chǎn)品又對純度要求高,對藥用蛋白質(zhì)的分離純化技術(shù)提出了極高的要求。以離子交換、親和結(jié)合和疏水性吸附為主的蛋白質(zhì)制備層析由于具有分離精度高、分離條件溫和、操作簡單且重復(fù)性高等特點(diǎn),已成為大規(guī)模生產(chǎn)藥用蛋白質(zhì)過程中最常用和使用頻率最高的一項分離純化手段[1-2]。

      為了提高蛋白質(zhì)制備層析的分離性能、實現(xiàn)高效化生產(chǎn),研究者們對蛋白質(zhì)吸附層析技術(shù)中的核心——層析介質(zhì)(吸附劑)不斷地探索研究,開發(fā)了表面薄殼層析介質(zhì)(pellicular particles)[3]、觸須型層析介質(zhì)(tentacle-type resins)[4-6]、流通層析介質(zhì)[flow-through media[7],又稱灌注層析介質(zhì)(perfusion media)[8-9],包括雙孔層析介質(zhì)(bidisperse porous media)[10-12]和凝膠殼層層析介質(zhì)(“gel in a shell”particles)[13-16]等]等層析介質(zhì)。觸須型層析介質(zhì)等是通過改造介質(zhì)基質(zhì)表面的功能性配基的物理化學(xué)性質(zhì),從而提高蛋白質(zhì)在介質(zhì)中的平衡吸附容量[17];表面薄殼層析介質(zhì)、流通層析介質(zhì)等則是通過改造介質(zhì)基質(zhì)的基本結(jié)構(gòu),改善流動相在介質(zhì)內(nèi)部的流動行為以及介質(zhì)的耐壓性,從而提高蛋白質(zhì)在介質(zhì)內(nèi)部的傳質(zhì)性能[7, 9, 14]。然而,對于層析技術(shù)而言,只有在上柱操作中的高流速下具有的較高動態(tài)吸附容量(dynamic binding capacity, DBC)才對蛋白質(zhì)的分離純化具有實際使用意義,而這則需要同時具有較高的平衡吸附容量和較快的傳質(zhì)速率才能實現(xiàn)。

      近年來,以葡聚糖接枝的瓊脂糖離子交換層析介質(zhì)為代表的聚合物配基修飾的層析介質(zhì)得到了產(chǎn)學(xué)界的廣泛關(guān)注。相較于傳統(tǒng)的短鏈配基修飾的層析介質(zhì)[功能性基團(tuán)通過間隔臂直接修飾在介質(zhì)基質(zhì)表面,圖1(a)]而言,聚合物鏈配基修飾的層析介質(zhì)[部分或全部的功能性基團(tuán)位于聚合物鏈上,功能化的聚合物鏈接枝于介質(zhì)基質(zhì)表面,圖1(b)~(e)]通常同時具有較高的平衡吸附容量和傳質(zhì)速率[18-23],因而也具有較高水平的DBC[24-26],極大地提升了層析操作的分離性能。由此,聚合物配基修飾的層析介質(zhì)的設(shè)計和高效吸附傳質(zhì)機(jī)理的解析和應(yīng)用也成為了蛋白質(zhì)層析中的研究熱點(diǎn)。

      圖1 蛋白質(zhì)層析介質(zhì)的基本結(jié)構(gòu)Fig. 1 Schematic diagram of surface structures of resins for protein chromatography

      本文針對蛋白質(zhì)層析中聚合物配基修飾介質(zhì)在近年來的研究進(jìn)展進(jìn)行評述,首先介紹了不同種類的聚合物配基接枝介質(zhì),然后系統(tǒng)闡述聚合物配基化學(xué)特征對介質(zhì)的吸附和傳質(zhì)性能的作用機(jī)制,并分析上述性質(zhì)對聚合物配基介質(zhì)分離特性的影響機(jī)理,以及基于該分離特性對介質(zhì)的應(yīng)用進(jìn)行舉例說明,最后討論聚合物配基的理性設(shè)計思路和方向,推動高效蛋白質(zhì)制備層析的理論和技術(shù)發(fā)展。

      1 聚合物配基化學(xué)

      1.1 葡聚糖接枝介質(zhì)

      葡聚糖是由D-葡萄糖通過a-1,6-糖苷鍵形成的高分子聚合物,基本結(jié)構(gòu)見圖2(a)。葡聚糖通常為分支很少(<5%)的線性直鏈狀分子[27],相對分子質(zhì)量通常從4×103到600×103不等。葡聚糖具有良好的親水性、高生物相容性和極低的非特異性吸附性能,是一種優(yōu)良的蛋白質(zhì)層析材料。葡聚糖可直接通過交聯(lián)作為層析基質(zhì)(例如Sephadex系列),1997年起GE healthcare公司將它作為層析基質(zhì)表面的修飾材料,接枝到瓊脂糖基質(zhì)表面,從而形成了葡聚糖接枝的瓊脂糖介質(zhì)(Sepharose XL、Streamline XL和Capto等系列)這類商品化的聚合物配基修飾介質(zhì)。

      葡聚糖分子[圖2(a)]內(nèi)部雖然有很多羥基,但是葡聚糖鏈自身分子尺寸較大、在溶液中呈無規(guī)則線團(tuán)狀[28],使得葡聚糖鏈內(nèi)部的羥基由于空間位阻較大,難以與介質(zhì)基質(zhì)表面反應(yīng)。因此在接枝反應(yīng)中,通常只有葡聚糖鏈兩端的羥基與介質(zhì)基質(zhì)表面發(fā)生化學(xué)反應(yīng),多形成單端較少位點(diǎn)固定的接枝鏈[見圖1(b)][18]。同時,由于相對分子質(zhì)量較大的葡聚糖鏈幾乎呈線狀,所以接枝后在介質(zhì)孔道內(nèi)伸展,可形成較厚的接枝層,同時葡聚糖鏈靈活性較強(qiáng),可在孔道內(nèi)自由擺動。但由于葡聚糖鏈自身沒有功能性基團(tuán),呈電中性,因此它在接枝到介質(zhì)基質(zhì)上后,還需要偶聯(lián)上功能性基團(tuán)(例如離子交換基團(tuán)磺酸基[29],混合模式基團(tuán)苯胺咪唑[20]、巰基甲基咪唑[30-31]等),才能吸附蛋白質(zhì)。這種修飾方法會使得功能性基團(tuán)隨機(jī)分布于介質(zhì)基質(zhì)表面和葡聚糖鏈上,如圖1(b) 所示,從而蛋白質(zhì)可吸附于介質(zhì)孔道基質(zhì)表面和靈活的葡聚糖鏈上。這種特殊的結(jié)構(gòu)使得葡聚糖接枝介質(zhì)表現(xiàn)出了對蛋白質(zhì)優(yōu)良的吸附和傳質(zhì)性能[25, 32-39],這將在之后的章節(jié)中詳細(xì)討論。

      圖2 聚合物鏈的化學(xué)結(jié)構(gòu)Fig. 2 Chemical structures of grafted polymers

      1.2 聚乙烯亞胺接枝介質(zhì)

      聚乙烯亞胺[poly(ethylenimine), PEI]是一種高分子陽離子聚電解質(zhì),通常為具有眾多分支結(jié)構(gòu)的長鏈,其基本結(jié)構(gòu)見圖2(b)。PEI相對分子質(zhì)量通常從400到750×103不等,分子中伯、仲、叔胺的理論比例為1:2:1[40],在較寬的pH范圍內(nèi)都能解離帶正電(在水溶液中所有氨基完全質(zhì)子化后的電荷密度為23.3 mequiv.·g-1[41])。PEI由于可以可逆性吸附帶負(fù)電的物質(zhì),同時又具有良好的生物相容性,因此被廣泛應(yīng)用于基因轉(zhuǎn)移(例如PEI-DNA復(fù)合物轉(zhuǎn)染等[42-43])和生物制品的分離純化(例如提取肝素[44]、移除細(xì)菌內(nèi)毒素[45]等)。近年來,研究者發(fā)現(xiàn)PEI有利于保持酶的活性和增加酶的穩(wěn)定性,因此PEI也常被用作離子交換配基偶聯(lián)于多種基質(zhì)表面,應(yīng)用于多種酶的固定化[46-47]和蛋白質(zhì)(包括酶蛋白)的離子交換層析[23, 48-50][圖1(c)]。此外,近期本課題組將PEI接枝的瓊脂糖介質(zhì)二次修飾(在PEI接枝鏈上修飾疏水基團(tuán)苯甲?;鵞51]、正丁基[52]等),將其應(yīng)用拓展于蛋白質(zhì)的混合模式層析分離中。

      PEI的結(jié)構(gòu)與1.1節(jié)中介紹的葡聚糖具有相似之處:兩者均為相對分子質(zhì)量較大的長鏈聚合物,具有良好的生物相容性,均在溶液中呈現(xiàn)無規(guī)則線團(tuán)狀。然而,兩者又具有不同之處:PEI分子分支較多且較長、呈樹枝狀長鏈[40, 53],通常以多位點(diǎn)連接于介質(zhì)基質(zhì)表面[49][圖1(c)];而葡聚糖分子分支較少且較短、呈直線性長鏈,通常由鏈末端的單位點(diǎn)連接于介質(zhì)基質(zhì)表面[18]。所以,接枝于介質(zhì)基質(zhì)表面的PEI鏈的靈活性(在孔道內(nèi)的可擺動程度)低于葡聚糖[23],而且PEI接枝層的伸展性(接枝層厚度的變化程度)也低于葡聚糖接枝層(PEI接枝層不易塌縮)[21]。同時,由于PEI在溶液中的伸展?fàn)顟B(tài)與PEI濃度直接相關(guān)(濃度越低,結(jié)構(gòu)越伸展),因此也影響了PEI接枝時的反應(yīng)位點(diǎn)數(shù)(結(jié)構(gòu)越伸展,反應(yīng)位點(diǎn)數(shù)越多)[41, 49, 54];而葡聚糖接枝時的反應(yīng)位點(diǎn)數(shù)與濃度基本無關(guān)(只與鏈末端單位點(diǎn)反應(yīng))。此外,由于PEI自身帶正電,PEI接枝介質(zhì)的離子交換基團(tuán)只分布于PEI接枝層,如圖1(c)所示,蛋白質(zhì)只吸附于PEI接枝鏈上,不同于葡聚糖接枝介質(zhì)中蛋白質(zhì)可同時存在于介質(zhì)基質(zhì)表面和葡聚糖鏈上。因此,PEI接枝介質(zhì)中,PEI接枝密度(離子交換容量)對蛋白質(zhì)吸附和傳質(zhì)行為的影響,直接體現(xiàn)了介質(zhì)中接枝層的作用,排除了葡聚糖接枝介質(zhì)中配基分布的影響。

      上述相似與不同之處也使得PEI接枝介質(zhì)不但具有接枝介質(zhì)的一般吸附性質(zhì)(高吸附容量和高傳質(zhì)速率)[23],也具有自身的吸附特性(存在臨界離子交換容量或臨界接枝密度[23, 55]和高耐鹽性[21, 51]等)。PEI接枝介質(zhì)對蛋白質(zhì)的吸附特性也將在之后的章節(jié)中詳細(xì)討論。

      1.3 聚(4-乙烯吡啶)接枝介質(zhì)

      聚(4-乙烯吡啶)[poly(4-vinylpyridine), P4VP]是一種線性長鏈高分子聚合物,其基本結(jié)構(gòu)如圖2(c)所示,相對分子質(zhì)量通常從4×103到160×103不等。由于吡啶基團(tuán)可與蛋白質(zhì)通過多種作用模式發(fā)生相互作用[56-57],且易修飾于多種基質(zhì)表面,P4VP又具有生物相容性好、價格低廉等特點(diǎn),所以P4VP被廣泛應(yīng)用于蛋白質(zhì)分離領(lǐng)域[58-59]。近期,本課題組考慮P4VP的主鏈為疏水性的烷基鏈、短側(cè)鏈為可解離帶正電的吡啶基團(tuán),且其單體結(jié)構(gòu)與商品化混合模式層析介質(zhì)MEP-HyperCel相似,也將P4VP作為聚合物鏈配基接枝于瓊脂糖基質(zhì)上,開發(fā)了一系列混合模式層析介質(zhì)[60]。

      P4VP和1.2節(jié)中介紹的PEI有相似之處:兩者均為相對分子質(zhì)量較大的長鏈聚合物,具有良好的生物相容性,均在溶液中呈現(xiàn)無規(guī)則線團(tuán)狀,可與介質(zhì)基質(zhì)表面多位點(diǎn)反應(yīng),且自身帶功能性基團(tuán),均可解離帶正電、接枝后介質(zhì)的離子交換基團(tuán)只分布于接枝鏈上。兩者的不同之處也很多:P4VP為無分支的線性長鏈,相對分子質(zhì)量相同時其鏈長度遠(yuǎn)大于PEI鏈,分子內(nèi)同時存在靜電作用和疏水作用,多位點(diǎn)修飾后會形成相互纏繞的接枝鏈和結(jié)構(gòu)較緊實的、較不伸展的接枝層,而固定于基質(zhì)表面的吡啶基團(tuán)上的胺基季胺化[58, 61],帶上較多正電荷,排斥P4VP長鏈,使接枝層保持了一定的伸展性,不至于完全塌縮[60][圖1(d)];而PEI呈樹枝狀長鏈,各個分支均帶正電相互排斥,修飾于介質(zhì)基質(zhì)表面后形成伸展的、不易塌縮的接枝層。此外,P4VP長鏈中各個自由吡啶基團(tuán)的可解離程度相似,離子交換基團(tuán)(帶電基團(tuán))均勻分布于P4VP長鏈中;而PEI中各個伯、仲、叔胺的解離性質(zhì)差別較大、分布不均,因此離子交換基團(tuán)在PEI鏈中分布不均 [40, 62-63]。

      P4VP接枝介質(zhì)的獨(dú)特的接枝層形態(tài)[圖1(d)]、接枝鏈的疏水和靜電性能以及功能性基團(tuán)分布[圖2(c)]使其具有不同于葡聚糖接枝介質(zhì)和PEI接枝介質(zhì)的吸附與傳質(zhì)特性,例如可在500 mmol·L-1NaCl條件下對γ-球蛋白的高效吸附和對γ-球蛋白和牛血清白蛋白(bovine serum albumin,BSA)的溫和洗脫等[60],這也將在之后的章節(jié)中詳細(xì)討論。

      1.4 團(tuán)簇型電荷修飾介質(zhì)

      團(tuán)簇型電荷修飾介質(zhì)(clustered-charge resin)是一類以寡聚氨基酸及其衍生物為功能性配基的介質(zhì),結(jié)構(gòu)如圖1(e)所示,可用于多種蛋白質(zhì)和核酸的分離純化[22, 64-67]。在團(tuán)簇型電荷修飾介質(zhì)中,通常由賴氨酸和精氨酸等堿性氨基酸五聚體的酰胺衍生物[64, 67]以及精胺[66][1,12-二氨基-4,9-二氮十二烷,結(jié)構(gòu)如圖2(d)所示]作為介質(zhì)的離子交換配基。此類團(tuán)簇型電荷配基的電荷密度較高、相對分子質(zhì)量較低,呈現(xiàn)一種納米級別的短鏈聚電荷團(tuán)簇。

      該聚電荷團(tuán)簇短鏈與前文介紹的PEI鏈、P4VP鏈也有一些相同之處:均具有良好的生物相容性,均可解離帶正電、接枝后介質(zhì)的離子交換基團(tuán)只分布于接枝鏈上。它們的不同之處在于:聚電荷團(tuán)簇短鏈中各個氨基的可解離程度相似且均較高[66],因此離子交換基團(tuán)均勻分布于該聚電荷團(tuán)簇短鏈中且密度較高[64];P4VP鏈中離子交換基團(tuán)雖分布均一但密度相對較低,而PEI鏈中的離子交換基團(tuán)分布不均且密度相對較低[40, 62-63]。而且,聚電荷團(tuán)簇短鏈相對分子質(zhì)量較低(僅3~5個重復(fù)單元),電荷密度較高,修飾于介質(zhì)基質(zhì)表面后形成的接枝層很薄,短鏈也幾乎不能在孔道內(nèi)自由擺動,這與葡聚糖和PEI接枝后的自由長鏈和較厚接枝層有著較大區(qū)別。

      這種局部電荷密度較高的團(tuán)簇結(jié)構(gòu)使得團(tuán)簇型電荷修飾介質(zhì)具有不同于傳統(tǒng)短鏈配基介質(zhì)和葡聚糖、PEI接枝介質(zhì)、P4VP接枝介質(zhì)等長鏈配基接枝介質(zhì)的吸附與傳質(zhì)特性[67],例如,對蛋白質(zhì)局部電荷密度變化的高敏感性[64],這也將在之后的章節(jié)中詳細(xì)闡述。

      2 吸附性能

      2.1 吸附平衡

      正如在前文中提到的,當(dāng)離子交換容量相近或略高時,葡聚糖接枝介質(zhì)、PEI接枝介質(zhì)這兩類長鏈聚合物配基修飾介質(zhì)以及團(tuán)簇型電荷修飾介質(zhì)這類短鏈聚合物配基對蛋白質(zhì)的平衡吸附容量均高于傳統(tǒng)短鏈配基介質(zhì)[18, 21, 23, 29, 39, 55, 64, 66, 68-70]。例如,

      商品化傳統(tǒng)短鏈配基修飾的瓊脂糖介質(zhì)SP Sepharose FF(介質(zhì)基質(zhì)為6%的交聯(lián)瓊脂糖Sepharose FF,其表面修飾磺酸基,配基密度200 mmol·L-1)在10 mmol·L-1的磷酸-磷酸氫二鈉溶液中(pH 6.5)對溶菌酶的平衡吸附容量僅為230 mg·ml-1,而同樣條件下的商品化葡聚糖接枝的瓊脂糖介質(zhì)SP Sepharose XL(介質(zhì)基質(zhì)為Sepharose FF,其表面接枝葡聚糖后再修飾磺酸基,配基密度220 mmol·L-1)高達(dá) 400 mg·ml-1[19]。類似的,PEI接枝的瓊脂糖介質(zhì)FF-PEI-L260也比其相對應(yīng)的商品化傳統(tǒng)短鏈配基修飾瓊脂糖介質(zhì)Q Sepharose FF對BSA的平衡吸附容量高20%以上[21];P4VP接枝的瓊脂糖介質(zhì)FF-P4VP-190,比其對應(yīng)的商品化傳統(tǒng)短鏈配基修飾瓊脂糖介質(zhì)MEP-HyperCel對γ-球蛋白和BSA的平衡吸附容量高20%以上[60];五聚賴氨酸接枝的瓊脂糖介質(zhì)(pentalysinamide aldehyde agarose)對Ca2+缺失α-乳清蛋白的吸附平衡容量可由傳統(tǒng)分散型賴氨酸配基修飾瓊脂糖介質(zhì)(lysinamide aldehyde agarose)的0.24 μmol·ml-1提升至0.48 μmol·ml-1[64]。

      2.1.1 長鏈聚合物配基修飾介質(zhì)的高吸附容量原理目前普遍認(rèn)為葡聚糖、PEI、P4VP等長鏈聚合物配基修飾介質(zhì)對多種蛋白質(zhì)表現(xiàn)出的高水平的平衡吸附容量是源于接枝于介質(zhì)基質(zhì)表面的長鏈聚合物可在介質(zhì)孔道內(nèi)自由擺動,并形成了伸展的接枝層[29, 36, 68]。因此,蛋白質(zhì)更易接近聚合物長鏈上的功能性基團(tuán)(例如帶電基團(tuán)),可靈活地與其發(fā)生相互作用,使蛋白質(zhì)的吸附位點(diǎn)從傳統(tǒng)短鏈配基修飾介質(zhì)的吸附平面[圖1(a)]拓展到整個接枝層的三維吸附空間[20, 23, 39][圖1(b)、(c)],提高了功能性基團(tuán)的利用率,從而有利于蛋白質(zhì)吸附,提升平衡吸附容量。P4VP這種線性長鏈聚合物配基,在修飾密度較高時,由于分子極長,即使鏈間和鏈內(nèi)纏結(jié)后,形成的接枝層還是較伸展,可以提供一定的三維吸附空間[圖1(d)],同時功能性基團(tuán)數(shù)量多、作用方式多樣,因此也十分有利于蛋白質(zhì)吸附,接枝介質(zhì)具有較高的平衡吸附容量[60]。

      (1)葡聚糖接枝介質(zhì)

      由于葡聚糖接枝的離子交換層析介質(zhì)中的葡聚糖接枝鏈分支少、接枝層較伸展、接枝層的電荷密度較低,因此在加鹽屏蔽電荷后,接枝層極易塌縮,并屏蔽吸附位點(diǎn),不利于蛋白質(zhì)吸附,導(dǎo)致介質(zhì)吸附容量隨鹽濃度提升而急劇降低,即對鹽濃度的敏感程度極高(高于傳統(tǒng)短鏈配基介質(zhì))[21, 29, 39]。同時,由于葡聚糖接枝鏈相對分子質(zhì)量較大、分支少、易塌縮,在修飾疏水性基團(tuán)[20]后,接枝鏈通過疏水相互作用纏繞結(jié)合,接枝層快速塌縮,無法提供三維吸附空間,會喪失聚合物配基接枝介質(zhì)的吸附優(yōu)勢。因此,葡聚糖接枝介質(zhì)不適宜拓展到疏水相互作用層析和混合模式層析中。

      (2)PEI接枝介質(zhì)

      由于PEI接枝鏈不但分支眾多、接枝層不如葡聚糖層伸展,而且PEI接枝層的電荷密度較高,在加鹽屏蔽電荷后,接枝層不易塌縮,因此PEI接枝介質(zhì)對鹽濃度的敏感程度較低(低于傳統(tǒng)短鏈配基介質(zhì)和葡聚糖接枝介質(zhì),在200~300 mmol·L-1NaCl時還能保持較高的吸附容量)[21]。同時,由于PEI鏈較葡聚糖鏈不易塌縮,在PEI接枝鏈上修飾疏水基團(tuán)苯甲?;鵞51]、正丁基[52]等形成的PEI接枝的混合模式層析介質(zhì),也可形成一定厚度的接枝層,以提供一定的三維吸附空間,有利于蛋白質(zhì)吸附,在較寬的鹽濃度范圍(0~2 mol·L-1NaCl)具有較高的吸附容量[51-52]。

      (3)P4VP接枝介質(zhì)

      P4VP接枝介質(zhì)則正如之前提到的,在修飾密度較高時,由于介質(zhì)中的功能性基團(tuán)數(shù)量多、與蛋白質(zhì)的作用方式多種,因此可在不同條件下以不同作用模式吸附蛋白質(zhì);同時較厚的接枝層結(jié)構(gòu)較緊實,在高鹽下也不易塌縮,可以提供一定的三維吸附空間,有利于較寬鹽濃度范圍(0~500 mmol·L-1NaCl)的蛋白質(zhì)吸附[60]。另外,P4VP接枝介質(zhì)和PEI接枝的混合模式層析介質(zhì)類似,其接枝層厚度與pH密切相關(guān)(pH越低,基團(tuán)解離程度越高,鏈越伸展,接枝層越厚),對蛋白質(zhì)可進(jìn)入的孔道和三維吸附空間影響極大,也造成了不同pH條件下蛋白質(zhì)的吸附差異性較大[51-52, 60],為混合模式層析分離提供良好基礎(chǔ),這將在下一節(jié)中詳細(xì)討論。

      2.1.2 團(tuán)簇型電荷修飾介質(zhì)的高吸附容量原理 然而,對于團(tuán)簇型電荷修飾介質(zhì)而言,由于其聚合物配基鏈較短,無法形成伸展的接枝層,因此幾乎不存在有利于蛋白質(zhì)吸附的三維吸附空間。但團(tuán)簇型電荷修飾介質(zhì)中具有多個納米級聚電荷團(tuán)簇[圖1(e)],該團(tuán)簇局部電荷密度高,內(nèi)部電荷分布均一,易于與多種蛋白質(zhì)中的用以蛋白-蛋白或者蛋白-核酸識別的保守區(qū)域(該區(qū)域通常也具有高電荷密度[71],例如α-乳清蛋白的兩個亞基交界處是特異性結(jié)合Ca2+離子域的保守區(qū)域,富含天冬氨酸,局部帶大量負(fù)電荷)的多位點(diǎn)發(fā)生相互作用,蛋白質(zhì)與吸附位點(diǎn)間的結(jié)合部位更多,具有更高的初始結(jié)合親和性[64, 67],從而強(qiáng)化了蛋白質(zhì)吸附行為,提高平衡吸附容量。但是,當(dāng)?shù)鞍踪|(zhì)的保守區(qū)域發(fā)生改變(例如α-乳清蛋白的保守區(qū)域被Ca2+飽和、細(xì)胞色素b5保守區(qū)域被突變但凈電荷不變)時,團(tuán)簇型電荷修飾介質(zhì)的平衡吸附容量急劇下降,表現(xiàn)出了對蛋白質(zhì)局部結(jié)構(gòu)變化的高度敏感性[64]。由于研究者們主要關(guān)注團(tuán)簇型電荷修飾介質(zhì)的吸附平衡容量及其對蛋白質(zhì)結(jié)構(gòu)的敏感性[22, 64-67],而對蛋白質(zhì)的傳質(zhì)速率研究較少,所以對團(tuán)簇型電荷接枝介質(zhì)就不再討論其吸附動力學(xué)。

      2.2 吸附動力學(xué)

      2.2.1 離子交換層析介質(zhì) 在具有較高平衡吸附容量的同時,蛋白質(zhì)在葡聚糖接枝介質(zhì)和PEI接枝介質(zhì)這兩類長鏈聚合物配基修飾的離子交換層析介質(zhì)中傳質(zhì)速率也遠(yuǎn)高于傳統(tǒng)短鏈配基介質(zhì)[18-21, 23, 29, 37-38, 55, 68, 70, 72-74]。在相同條件下,溶菌酶在商品化葡聚糖接枝介質(zhì)SP Sepharose XL中的有效孔擴(kuò)散速率(De)可達(dá)其在商品化傳統(tǒng)介質(zhì)SP Sepharose FF中的4.7倍[19]。溶菌酶在葡聚糖接枝的瓊脂糖介質(zhì)SP-T40-X-S6B中甚至高達(dá)自由溶液擴(kuò)散速率(D0)的10倍[18, 29],而蛋白質(zhì)在多孔介質(zhì)中的De值通常應(yīng)低于D0值。BSA在PEI接枝的瓊脂糖介質(zhì)FF-PEI-L740中的De值也達(dá)到了其在自由溶液中D0值的1.6倍[21]。

      研究者們對造成蛋白質(zhì)在上述兩類長鏈聚合物配基修飾的離子交換層析介質(zhì)中的De/D0>1現(xiàn)象的原理進(jìn)行了深入探究,目前較為廣泛接受的有如下幾種機(jī)理[75]。一類是,在上述長鏈聚合物配基修飾介質(zhì)中存在表面擴(kuò)散[包括“活性跳躍(activated jump)”[19, 32, 76]和“鏈傳遞(chain delivery effect)”[21, 23, 55]等機(jī)理],表面擴(kuò)散對傳質(zhì)的貢獻(xiàn)使得通過孔擴(kuò)散模型擬合得到的De值(集總的動力學(xué)參數(shù))偏大。另一類是,在上述介質(zhì)中存在電動效應(yīng)等非擴(kuò)散機(jī)理(包括電泳[20, 77]、電滲[77]、靜電耦合[29]等)促進(jìn)孔內(nèi)傳質(zhì),也使得擬合得到的De值偏大??偟膩碚f,研究者們均認(rèn)為在上述介質(zhì)中存在孔擴(kuò)散之外的傳質(zhì)機(jī)理對介質(zhì)內(nèi)部的蛋白質(zhì)傳遞過程做出較大貢獻(xiàn)。由于這些機(jī)理已在近期的綜述文章[75]中進(jìn)行了詳細(xì)的評述,所以在此不再贅述,只簡要地闡述其中的鏈傳遞機(jī)理。

      (1)鏈傳遞機(jī)理

      圖3 鏈傳遞機(jī)理[75]Fig. 3 Schematic drawing for mechanism of chain delivery effect[75](The blue circles represent adsorbed protein, and the dashed dark green lines as well as the faded circles represent the grafted polymers chains with adsorbed proteins after swing. The dashed pink boxes highlight the sites for the “relay”/delivery of protein. Other symbols in this figure are the same as in Fig.1)

      鏈傳遞機(jī)理可通俗地用“救火列隊作用(bucket brigade effect)”[35]描述,是一種特殊的表面擴(kuò)散,只存在于具有可自由擺動的聚合物鏈配基修飾介質(zhì)中[21, 23, 55, 70, 75],機(jī)理如圖3所示。本課題組在研究蛋白質(zhì)PEI接枝介質(zhì)中傳質(zhì)速率在臨界接枝密度(臨界離子交換容量)處快速增加[23, 55][圖4(a)]時,對這個機(jī)理進(jìn)行了詳細(xì)闡述。簡而言之,靈活的長鏈聚合物配基可在孔道內(nèi)自由擺動,當(dāng)入口處的聚合物長鏈上吸附蛋白質(zhì)后,聚合物長鏈會在朝向介質(zhì)中心的化學(xué)勢和由被吸附蛋白質(zhì)為媒介的鏈間作用驅(qū)動下,向孔道內(nèi)部擺動,將該蛋白質(zhì)運(yùn)送并轉(zhuǎn)移到鄰近的聚合物長鏈上(圖3),類似于在火災(zāi)時救火人員列隊傳遞接力水桶。由此,吸附相蛋白質(zhì)通過自身為媒介,通過聚合物長鏈的擺動,被動地在介質(zhì)表面的吸附位點(diǎn)上移動。

      圖4 蛋白質(zhì)在接枝介質(zhì)中的傳質(zhì)速率(De/D0值)隨聚合物鏈密度的變化Fig. 4 Effective diffusivities of BSA and γ-globulin on polymer-grafted resins with different grafting densities (a) PEI-grafted resins[23, 55]; (b) dextran-grafted resins[20]

      (2)鏈密度對鏈傳遞作用的影響

      由于PEI接枝介質(zhì)中吸附位點(diǎn)只存在于PEI接枝鏈上,所以當(dāng)接枝密度較低(低于臨界接枝密度)時,相鄰兩條鏈之間的距離太遠(yuǎn),無法發(fā)生鏈傳遞作用,蛋白質(zhì)在介質(zhì)中的傳質(zhì)以孔擴(kuò)散為主,傳質(zhì)較慢;而當(dāng)接枝密度較高(高于臨界接枝密度)時,相鄰兩條鏈之間的距離較近,鏈傳遞作用極大地促進(jìn)了吸附相蛋白質(zhì)傳質(zhì),蛋白質(zhì)在介質(zhì)中的傳質(zhì)速率大幅提升。因此,PEI接枝介質(zhì)中存在的臨界接枝密度,即為保證蛋白質(zhì)能在相鄰兩條鏈能發(fā)生鏈傳遞作用的距離。上述機(jī)理也是造成圖4(a)中PEI接枝介質(zhì)的De/D0值在臨界離子交換容量處快速增加的原因[23]。然而,由于葡聚糖接枝介質(zhì)中吸附位點(diǎn)既存在于聚合物接枝鏈上,又存在于介質(zhì)基質(zhì)表面的短鏈配基上,所以接枝鏈密度和配基密度(接枝鏈配基和短鏈配基總密度)都對鏈傳遞作用有影響。而在離子交換層析介質(zhì)的常用配基密度范圍(140~150 mmol·L-1),由于兩類配基間距離較近,聚合物接枝鏈與基質(zhì)表面的短鏈配基(或聚合物接枝鏈)間始終能發(fā)生鏈傳遞作用、促進(jìn)傳質(zhì),從而葡聚糖接枝介質(zhì)的De/D0值幾乎隨著接枝量線性增加[20][圖4(b)]。

      (3)鹽濃度對鏈傳遞作用的影響

      由于鏈傳遞作用是一種特殊的表面擴(kuò)散,因此增加鹽濃度減弱蛋白質(zhì)與吸附位點(diǎn)間的靜電相互作用,有利于鏈傳遞作用發(fā)生。同時,表面擴(kuò)散通量與吸附相蛋白質(zhì)密度正相關(guān),鏈傳遞作用對總傳質(zhì)速率的貢獻(xiàn)也與介質(zhì)對蛋白質(zhì)的吸附容量正相關(guān)。這兩方面的影響很好地解釋了葡聚糖接枝介質(zhì)和PEI接枝介質(zhì)的傳質(zhì)速率隨鹽濃度變化趨勢不同的現(xiàn)象(圖5)。葡聚糖接枝介質(zhì)的吸附容量對鹽濃度十分敏感,隨鹽濃度增加急劇下降[36, 39],因此,鏈傳遞作用的傳質(zhì)通量急劇下降,導(dǎo)致了總傳質(zhì)速率隨鹽濃度增加而下降的趨勢[19, 29]。PEI接枝介質(zhì)的吸附容量對鹽濃度變化不敏感,上述兩方面影響共同作用于鏈傳遞作用,所以總傳質(zhì)速率呈現(xiàn)隨鹽濃度增加先上升后下降的趨勢[21]。

      圖5 蛋白質(zhì)在接枝介質(zhì)中的傳質(zhì)速率(De/D0值)隨鹽濃度的變化(數(shù)據(jù)源于文獻(xiàn)[19, 21, 29])Fig. 5 Effective diffusivities of proteins on polymer-grafted resins at different salt concentrations (data from references[19, 21, 29])

      2.2.2 混合模式層析介質(zhì) 對于P4VP和疏水基二次修飾的PEI這兩類具有疏水性的長鏈聚合物配基修飾的混合模式層析介質(zhì)而言,其接枝鏈形態(tài)(靈活性和伸展性等)與接枝量、疏水基修飾密度、緩沖液鹽濃度和pH等密切相關(guān),這也使得其傳質(zhì)速率呈現(xiàn)更加復(fù)雜的趨勢[60]。其傳質(zhì)速率對緩沖液條件的依賴性也為這兩類介質(zhì)在層析柱上的操作靈活性和適用范圍廣泛性提供了良好的基礎(chǔ),這也將在下一節(jié)中詳細(xì)討論。

      3 蛋白質(zhì)層析

      由于上述幾種聚合物配基修飾的介質(zhì)均具有較高的吸附容量和傳質(zhì)速率,因此它們都常具有較高的DBC[24-25, 36, 60, 69-70, 72-74],被用于多種蛋白質(zhì)的分離純化工藝流程中[78-81]。例如,葡聚糖接枝的離子交換層析介質(zhì)Q Sepharose XL由于對BSA和單克隆抗體均具有較高的DBC,而這兩種蛋白質(zhì)的單體和聚集體又可以通過梯度洗脫分離,因此Q Sepharose XL可用于批量化分離BSA和單克隆抗體的單體和聚集體[82];類似的,Capto Q對12×103聚乙二醇修飾的BSA的DBC高達(dá)50 mg·ml-1(流速50 cm·h-1)和37 mg·ml-1(流速250 cm·h-1),實現(xiàn)了乙二醇化的BSA的高效分離[83];另外,Tugcu 等[79]利用市面上的多種商品化層析介質(zhì)設(shè)計了多種單克隆抗體純化方案,通過對比純化方案和優(yōu)化純化流程發(fā)現(xiàn),Q Sepharose XL由于DBC較高,其層析操作是單克隆抗體純化的最優(yōu)流程中不可或缺的重要環(huán)節(jié)。此外,這些聚合物配基修飾介質(zhì)的特殊的吸附性能造成其具有各自的層析分離特性,見表1。

      3.1 葡聚糖接枝的離子交換層析介質(zhì)

      如前所述,葡聚糖接枝的離子交換層析介質(zhì)的吸附容量和傳質(zhì)速率均隨著鹽濃度增加而急劇下降,這導(dǎo)致DBC具有極高的鹽濃度敏感性(在約150 mmol·L-1NaCl條件下就幾乎不吸附蛋白質(zhì))[29, 36-37, 84]。因此,該類介質(zhì)適宜于在低鹽條件下(0~100 mmol·L-1NaCl)的蛋白質(zhì)吸附;同樣,約150 mmol·L-1NaCl可作為洗脫條件,賦予了它較傳統(tǒng)短鏈配基介質(zhì)的低鹽高效洗脫優(yōu)勢[72]。所以,葡聚糖接枝的離子交換層析介質(zhì)通常用于0~150 mmol·L-1NaCl緩沖液條件下的蛋白質(zhì)高容量吸附與低鹽高效洗脫[85-86](表1)。

      3.2 PEI接枝的離子交換層析介質(zhì)

      相比之下,PEI接枝的離子交換層析介質(zhì)的DBC對鹽濃度的敏感程度則較低[24](遠(yuǎn)低于傳統(tǒng)短鏈配基介質(zhì)和葡聚糖接枝介質(zhì),這是由于其鏈內(nèi)靜電作用不易被屏蔽、接枝層不易塌縮所致,例如FF-PEI-L740在200 mmol·L-1NaCl時還能同時具有較高的吸附容量和傳質(zhì)速率[21]因而具有超過60 mg·ml-1的DBC[24])。所以,PEI接枝的離子交換層析介質(zhì)適用于較寬鹽濃度范圍(0~300 mmol·L-1NaCl)內(nèi)的蛋白質(zhì)吸附,其洗脫條件為約400 mmol·L-1NaCl[21, 24](表1)。同時,PEI接枝的離子交換層析介質(zhì)對流動相反離子種類也有較高的偏好性(不同的親和能力),例如FF-PEI-L680對反離子的偏好性隨SCN-

      3.3 PEI接枝的混合模式層析介質(zhì)

      表1 蛋白質(zhì)層析介質(zhì)的分離特性Table 1 Separation characteristics of resins for protein chromatography

      在接枝密度較低的PEI接枝的離子交換層析介質(zhì)的PEI鏈上,修飾疏水基團(tuán)苯甲?;鵞51]、正丁基[52]等,形成了PEI接枝的混合模式層析介質(zhì),也顯示了很好的蛋白質(zhì)層析分離應(yīng)用前景。例如,苯甲酰基修飾的PEI接枝的混合模式層析介質(zhì)B160-PEI330在0.5~2 mol·L-1NaCl條件下可保持恒定的吸附容量和傳質(zhì)速率,因此可提供較高的DBC,并表現(xiàn)了極好的耐鹽性;該介質(zhì)在pH 3條件下可以對BSA和γ-球蛋白完全洗脫,而在pH 4時BSA和γ-球蛋白的洗脫收率差異極大(8% v.s. 71%)[51]。上述耐鹽性和洗脫差異性可應(yīng)用于從含有白蛋白的混合物(例如血清)中分離抗體(表1)。但接枝密度較高的PEI接枝的混合模式層析介質(zhì),則由于其在低pH洗脫時的鏈內(nèi)靜電排斥作用太強(qiáng),形成接近剛性的接枝鏈,將孔道堵塞,阻礙蛋白質(zhì)從介質(zhì)內(nèi)部擴(kuò)散出來,造成洗脫回收率較低,因而不適用于混合模式層析分離[51]。

      3.4 P4VP接枝的混合模式層析介質(zhì)

      P4VP接枝的混合模式層析介質(zhì)則由于具有特殊的接枝鏈形態(tài)以及接枝鏈形態(tài)(厚度和帶電性等)對pH的高度敏感性,可在pH 4~4.5實現(xiàn)對蛋白質(zhì)的完全洗脫[60],比現(xiàn)今報道過的大多數(shù)混合模式層析的洗脫條件都溫和[57, 88-89],這也顯示了P4VP接枝介質(zhì)在結(jié)構(gòu)敏感型蛋白質(zhì)的層析分離中的應(yīng)用前景(表1)。

      3.5 團(tuán)簇型電荷修飾介質(zhì)

      如2.1節(jié)中所述,團(tuán)簇型電荷接枝介質(zhì)對具有保守的高電荷密度區(qū)域的蛋白質(zhì)的吸附具有較高的吸附容量和選擇性,且吸附容量對蛋白質(zhì)結(jié)構(gòu)具有極高的敏感性[64, 87],對鹽濃度也極敏感[66],因此團(tuán)簇型電荷接枝介質(zhì)可用于此類蛋白質(zhì)的高分辨率分離(表1)。

      4 展 望

      通過上述對不同種類的聚合物配基的化學(xué)特征、吸附和傳質(zhì)特性、層析分離特性和應(yīng)用的系統(tǒng)闡述,總結(jié)了聚合物配基影響介質(zhì)的蛋白質(zhì)層析行為的作用機(jī)理。這些討論也為今后的聚合物配基的理性設(shè)計提供了研究思路和方向。例如,對于長鏈聚合物配基而言,靈活、伸展的聚合物長鏈配基更有利于蛋白質(zhì)的三維吸附和鏈傳遞作用,所以新型層析配基的理性設(shè)計應(yīng)重點(diǎn)開發(fā)相對分子質(zhì)量較大、分支較少、電荷密度較低的長鏈聚合物配基以提升吸附容量和傳質(zhì)速率。電荷密度較高的長鏈聚合物配基,雖不靈活,但耐鹽性高,所以適用于高鹽條件下的分離純化。具有疏水性的長鏈聚合物配基則需謹(jǐn)慎考慮其疏水性和帶電性的比例以及疏水性和伸展性的關(guān)系(聚合物鏈上疏水基團(tuán)增加一方面會增加蛋白質(zhì)吸附位點(diǎn)總數(shù),另一方面會造成鏈纏繞、接枝層塌縮、掩蔽吸附位點(diǎn)、減少可被蛋白質(zhì)利用的吸附位點(diǎn)),使這類聚合物配基適用于蛋白質(zhì)混合模式吸附層析。而對于團(tuán)簇型短鏈配基而言,電荷密度越高、電荷分布越均一的團(tuán)簇型電荷配基更有利于對具有保守的高電荷密度區(qū)域的蛋白質(zhì)的高分辨率吸附。此外,蛋白質(zhì)層析的聚合物鏈配基研究還有很多不足,例如葡聚糖接枝介質(zhì)中功能性基團(tuán)的分布(葡聚糖接枝鏈配基和基質(zhì)表面短鏈配基的分布)對吸附和傳質(zhì)的影響仍不清楚;團(tuán)簇型短鏈配基的相對分子質(zhì)量和帶電基團(tuán)在短鏈中的分布(團(tuán)簇短鏈自身的電荷密度)對吸附和傳質(zhì)的影響,團(tuán)簇型短鏈配基能否拓展到混合模式吸附層析,團(tuán)簇型短鏈配基能否應(yīng)用于更廣泛的蛋白質(zhì)高分辨率分離等問題仍需研究。最后,利用分子模擬方法研究和解析表面聚合物配基結(jié)合蛋白質(zhì)的分子傳遞(鏈傳遞行為),將為聚合物配基設(shè)計提供理論基礎(chǔ),進(jìn)一步推動蛋白質(zhì)吸附層析技術(shù)的發(fā)展。

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      Foundation item: supported by the National Natural Science Foundation of China (21236005).

      Adsorptive protein chromatography with grafted polymeric ligands

      YU Linling, SUN Yan
      (Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China)

      Abstract:Adsorptive protein chromatography, mostly based on ion exchange, affinity binding and hydrophobic interactions, is the key technology in the large-scale production of therapeutic proteins. The development of novel technology as well as improvement of chromatographic separation efficiency, such as dynamic binding capacity and selectivity, is generally the main objective of fundamental studies on protein chromatography. Recently, polymeric ligand-modified matrices have been developed and widely studied due to the findings that some of them not only possess high equilibrium adsorption capacity but also high uptake rate. This review is devoted to an overview of the polymeric ligands for protein chromatography. Different kinds of polymeric ligands are first introduced. This is followed by the effects and functional mechanisms of the polymeric ligand chemistry on protein adsorption equilibria, uptake kinetics as well as separation performance of the ligand-modified matrices. Applications of the matrices based on the mechanisms are then illustrated to offer insight into the design of new polymeric ligands. Finally, a perspective for further development and fundamental studies on polymeric ligand-based protein chromatography is discussed.

      Key words:protein; adsorption; chromatography; polymer grafting; ligand

      Corresponding author:Prof. SUN Yan, ysun@tju.edu.cn

      基金項目:國家自然科學(xué)基金項目(21236005)。

      中圖分類號:TQ 033

      文獻(xiàn)標(biāo)志碼:A

      文章編號:0438—1157(2016)01—0140—12

      DOI:10.11949/j.issn.0438-1157.20151120

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