CN104812491B - 阴离子交换-疏水性混合模式 - Google Patents

阴离子交换-疏水性混合模式 Download PDF

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CN104812491B
CN104812491B CN201380012891.6A CN201380012891A CN104812491B CN 104812491 B CN104812491 B CN 104812491B CN 201380012891 A CN201380012891 A CN 201380012891A CN 104812491 B CN104812491 B CN 104812491B
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J·廖
R·弗罗斯特
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Bio Rad Laboratories Inc
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Abstract

提供阴离子交换‑疏水混合模式配体及其使用方法。

Description

阴离子交换-疏水性混合模式
相关专利申请的交叉引用
本申请要求2012年3月8日提交的美国临时专利申请号61/608,418的优先权权益，其通过引用纳入本文用于所有目的。
发明背景
免疫球蛋白的液体源主要是哺乳动物体液或细胞培养收获物，来自液体源的免疫球蛋白提取物对于获得充分浓缩或纯化形式的免疫球蛋白来用于诊断和治疗用途以及一般实验室研究而言是重要的。类似地，纯化其它种类的蛋白质和来自生物样品的其它分子也是有价值的。
发明内容
提供连接至配体的色谱固体支持物(支持物(Support))。在一些实施方式中，所述支持物和配体具有下式：
支持物-(X)-N(R1,R2)-R3-L-Ar，其中，X是间隔子或不存在；R1和R2各自选自氢和包含1～6个碳的烷基；R3是包含1～6个碳的烷基或包含1～6个碳的环烷基；L是NR4、O或S；其中R4是氢或包含1～6个碳的烷基；且Ar是芳基。
在一些实施方式中，R1和R2是氢。
在一些实施方式中，R3是包含1～4个碳的烷基。
在一些实施方式中，R3是包含1、2、3、4、5或6个碳的烷基。
在一些实施方式中，所述芳基是苯基。在一些实施方式中，所述芳基是经取代的苯基。在一些实施方式中，所述经取代的苯基是烷基取代的苯基。
在一些实施方式中，R1和/或R2是包含1～6个碳的烷基且Ar是烷基取代的苯基。
在一些实施方式中，所述配体是N-苯基乙二胺或其盐。在一些实施方式中，所述配体是2-苯氧基乙胺或其盐。
在一些实施方式中，X(接头/间隔子)不存在。在一些实施方式中，X是间隔子。
还提供从样品纯化生物分子的方法。在一些实施方式中，所述方法包括：使该样品接触如上文或本文中他处所述的连接至配体的色谱固体支持物；和收集纯化的生物分子。
在一些实施方式中，所述生物分子是蛋白质。在一些实施方式中，所述蛋白质是抗体。
在一些实施方式中，所述样品包含单体抗体和聚集体，并且使单体抗体与所述聚集体分离，从而导致单体抗体的纯化。
在一些实施方式中，所述纯化的生物分子是单体抗体。在一些实施方式中，所述样品的单体抗体被固定在所述色谱固体支持物上，随后被洗脱。在一些实施方式中，所述样品的单体抗体流经所述色谱固体支持物并被收集。
定义
“抗体”是指免疫球蛋白、其复合(例如，融合)形式或其片段形式。该术语可包括但不限于：源自人或其它哺乳动物细胞系的IgA、IgD、IgE、IgG和IgM类的多克隆或单克隆抗体，包括天然形式或遗传修饰形式，如人源化、人、单链、嵌合、合成、重组、杂合、突变、移接和体外产生的抗体。“抗体”也可包括复合形式，其包括但不限于含有免疫球蛋白部分的融合蛋白。“抗体”还可包括抗体片段，如Fab、F(ab')2、Fv、scFv、Fd、dAb、Fc，无论它们是否保留抗原结合功能。
本文所用的术语“烷基”指直链或支链的，饱和的脂族基团，其具有指示数量的碳原子。例如，C1-C6烷基包括但不限于：甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、戊基、异戊基、己基等。其它烷基基团包括但不限于：庚基、辛基、壬基、癸基等。烷基可包括任何数量的碳，例如1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、2-3、2-4、2-5、2-6、3-4、3-5、3-6、4-5、4-6和5-6个。所述烷基基团通常是单价的，但可以是二价的，例如当烷基基团将两个部分连接到一起时。
本文所用的术语“环烷基”指饱和的或部分不饱和的单环、稠合二环或桥接多环组成物，其包含3～12个环原子，或指示数量的原子。单环包括，例如，环丙基、环丁基、环戊基、环己基和环辛基。二环和多环包括，例如，降莰烷、十氢化萘和金刚烷。例如，C3-8环烷基包括环丙基、环丁基、环戊基、环己基、环辛基和降莰烷。
本文中所用的术语“芳基”指单环或稠合二环、三环或更大的，芳族环组成物，其包含6～16个环碳原子。例如，芳基可以是苯基、苄基或萘基，优选苯基。“亚芳基”指源自芳基基团的二价基团。芳基基团可以是被单-，二-或三-取代有一个、两个或三个选自下组的基团：烷基、烷氧基、芳基、羟基、卤素、氰基、氨基、氨基-烷基、三氟甲基、亚烷基二氧基和氧基-C2-C3-亚烷基；其均任选地被进一步取代，例如如上文定义的那样；或1-或2-萘基；或1-或2-菲基。亚烷基二氧基是与苯基的两个邻近碳原子连接的二价取代基，例如亚甲基二氧基或亚乙基二氧基。氧基-C2-C3-亚烷基也是与苯基的两个邻近碳原子连接的二价取代基，例如，氧乙烯或氧丙烯。氧基-C2-C3-亚烷基-苯基的一个示例是2,3-二氢苯并呋喃-5-基。
经取代的苯基基团的示例有，例如，4-氯酚-1-基、3,4-二氯酚-1-基、4-甲氧基酚-1-基、4-甲基酚-1-基、4-氨基甲基酚-1-基、4-甲氧基乙基氨基甲基酚-1-基、4-羟基乙基氨基甲基酚-1-基、4-羟基乙基-(甲基)-氨基甲基酚-1-基、3-氨基甲基酚-1-基、4-N-乙酰基氨基甲基酚-1-基、4-氨基酚-1-基、3-氨基酚-1-基、2-氨基酚-1-基、4-苯基-酚-1-基、4-(咪唑-1-基)-酚-基、4-(咪唑-1-基甲基)-酚-1-基、4-(吗啉-1-基)-酚-1-基、4-(吗啉-1-基甲基)-酚-1-基、4-(2-甲氧基乙基氨基甲基)-酚-1-基和4-(吡咯烷-1-基甲基)-酚-1-基、4-(苯硫基)-酚-1-基、4-(3-苯硫基)-酚-1-基、4-(4-甲基哌嗪-1-基)-酚-1-基和4-(哌啶基)-苯基和4-(吡啶基)-苯基，其任选地在杂环中被取代。
本文中所用的术语“接头”或“间隔子”指将本发明的色谱配体连接至色谱基质的化学部分。可用于本发明的接头的长度可以是，例如，至多30个碳原子。用于将所述接头连接至本发明的化合物和生物分子的键的类型包括但不限于，酰胺、胺、酯、氨基甲酸酯、脲、硫醚、硫代氨基甲酸酯、硫代碳酸酯和硫脲。本领域技术人员会理解，其它类型的键也可用于本发明。如本文他处所述，所述配体和所述固体支持物基质之间的间隔子是任选的。
“生物分子制剂”和“生物样品”是指包含需纯化的生物来源的目标分子(“生物分子”)的任何组合物。在一些实施方式中，待纯化的目标分子是抗体或非抗体蛋白。
“结合-洗脱模式”是指色谱的操作方法，其中建立缓冲条件从而向配体(其任选地结合至固体支持物)添加样品时，目标分子以及，任选不需要的污染物结合至所述配体。随后可通过改变条件使目标从支持物洗脱来实现目标的分级分离。在一些实施方式中，污染物在目标洗脱之后仍保持结合。在一些实施方式中，污染物在目标洗脱之前流通或结合并在目标之前被洗脱。
“流通模式”是指色谱的操作方法，其中建立缓冲条件使待纯化的目标分子流通包含配体的色谱支持物，同时选择性地保留至少一些样品污染物，从而使它们被移除。
发明详述
I.介绍
本文描述一类色谱配体，其允许从样品有效纯化目标生物分子，并且发现这特别有利于从聚集目标生物分子纯化单体目标生物分子。特别地，实施例证明本文所述的配体在从抗体聚集体分离单体抗体上优于市售产品Capto AdhereTM(配体：N-苄基-N-甲基乙醇胺)。
II.色谱配体
本文所述的色谱配体在一些实施方式中可以由下式表示：
-N(R1,R2)-R3-L-Ar，或其盐
其中
R1和R2各自选自氢和烷基；
R3是烷基或环烷基；L是NR4、O或S；其中R4是氢或烷基；且
Ar是芳基。
N(R1,R2)的氮可以是仲胺、叔胺或季胺。该氮的带正电(盐)形式可在目标生物分子的结合中起作用(例如，在阴离子交换中)，因此，在纯化方法中，该配体常在该氮带正电的条件下使用，以供目标的至少部分结合至所述配体、清洗或洗脱。
在多个实施方式中，R1和R2是氢。实施例包括其中R1和R2是氢的若干实施方式，其显示优越的单体目标的纯化。
或者，在一些实施方式中，R1、R2或二者是烷基(例如，包括但不限于直链C1、C2、C3、C4或C5烷基)。在R1处包含甲基(C1)且Ar是苯基的实施方式在300mM NaCl条件下并不显著结合单克隆抗体。然而，不受任何特定理论或作用的限制，相信抗体结合至具有R1或R2烷基的配体能够通过利用所述配体的“Ar”位置处的烷基取代的芳基联合R1或R2烷基来得以改善。
R3可以是是烷基或环烷基。在一些实施方式中，R3是具有1、2、3、4、5或6个碳的直链烷基。例如，在苯基乙二胺和苯氧基乙胺中，R3是二-碳烷基。
部分"L"可以是氧(例如，醚形式)、硫(例如，硫醚形式)或NR4，例如NH或NR4是烷基的情况(例如，包括但不限于直链C1、C2、C3、C4或C5烷基)。
芳基基团一般是苯基。然而，在一些实施方式中，所述芳基是经取代的芳基，例如，被一个或多个烷基部分(例如，包括但不限于直链C1、C2、C3、C4或C5烷基)取代。在一些实施方式中，R1、R2或二者是烷基，且所述芳基是经取代的(例如，烷基取代的)芳基，例如烷基取代的苯基。
所述配体可连接至固体支持物(S)，任选地通过间隔子(X)连接，如下：
S-(X)-N(R1,R2)-R3-L-Ar，或其盐。
设想任何固体支持物用于连接至所述配体。所述固体支持物可以是，例如，多孔或无孔的，并且可以是如下形式，例如，基质、珠、颗粒、片基或其它构造，例如，膜或整料，即，单块、单团或单片材料。当用作基质时，颗粒可以是具有平滑表面或者具有粗糙或织构化表面的球或珠。许多(并且在一些情况下所有)的孔均是通孔(through-pore)，延伸穿过颗粒以用作足够大以允许流体动力学流过或快速扩散透过这些孔的通道。当以球或珠的形式时，在术语“直径”指的是颗粒的最长外部尺寸的情况下，中值颗粒直径在一些实施方式中是约25微米-约150微米。对满足本段描述的颗粒及其制备方法的公开见述于Hjertén等，美国专利号5,645,717，Liao等，美国专利号5,647,979，Liao等，美国专利号5,935,429，以及Liao等，美国专利号6,423,666。可以被聚合化以获得有用基质的单体的示例是乙酸乙烯酯、乙烯基丙基胺、丙烯酸、甲基丙烯酸酯、丙烯酸丁酯、丙烯酰胺、甲基丙烯酰胺、乙烯基吡咯烷酮(乙烯基吡咯烷二酮)，在一些情况下含有官能团。在许多实施方式中还采用交联剂，并且当存在交联剂时，在一些实施方式中会相对总单体构成约0.1-约0.7的摩尔比。交联剂的示例有：二羟基亚乙基二丙烯酰胺、二烯丙基酒石二酰胺、三烯丙基柠檬三酰胺、二丙烯酸乙二酯、双(丙烯酰)胱胺(bisacrylylcystamine)、N,N'-亚甲基双丙烯酰胺和哌嗪二丙烯酰胺。
如上所述，所述配体可直接(不通过间隔子)连接至固体支持物或通过接头连接。与所述固体支持物的连接将视所用具体固体支持物而定。在一些实施方式中，所述固体支持物包含二醇，二醇转化成醛，例如通过采用NaIO4的转化。所述配体的胺可通过还原性胺化反应连接至所述固体支持物上的醛，由此直接偶联所述配体至所述固体支持物。
在一些实施方式中，所述配体通过间隔子连接至所述固体支持物。所述间隔子可按照传统的共价偶联法引入。示例性的偶联化学品可涉及，例如，表氯醇、表溴醇、烯丙基-缩水甘油醚、二环氧化物如丁二醇二缩水甘油醚、卤素取代的脂族物质如二-氯-丙醇、二乙烯砜、羰二咪唑、醛如戊二醛、醌类、溴化氰、高碘酸盐如偏高碘酸钠、碳二亚胺、氯三嗪、磺酰氯如甲苯磺酰基氯和三氟乙基磺酰基氯(tresyl chlorides)、N-羟基琥珀酰亚胺、噁唑酮、马来酰亚胺、2-氟-1-甲基吡啶甲苯-4-磺酸盐(酯)、吡啶基二硫化物和酰肼。
所述固体支持物可用于任何常规配置，包括填充柱和流化或扩张床柱、整料或多孔膜，并且通过任何常规方法使用，包括分批模式加载、洗涤和洗脱，以及连续或流通(flow-through)模式。在一些实施方式中，柱的直径可以是1cm～1m，而其高度可以是1cm～30cm或更高。
III.方法
还提供从样品纯化一种或多种目标生物分子的方法，所述方法包括将所述样品施加至与固体支持物连接的配体，随后从所述固体支持物收集所述目标生物分子，由此从所述样品中的一种或多种成分纯化所述目标生物分子。如上所述，在一些实施方式中，所述目标生物分子是单体抗体，并且所述方法包括从所述样品中的聚集的抗体纯化所述单体抗体。
色谱配体可用于通过阴离子交换(即，所述配体带正电)/疏水混合模式色谱来纯化目标分子。可调节条件，从而能够以结合-洗脱模式或流通模式运行所述色谱。
可实施所述方法的蛋白质制备物可包括来自天然、合成或重组来源的未纯化的或部分纯化的抗体(例如，IgG)。未纯化的抗体制备物，例如，可来自多个来源，包括但不限于血浆、血清、腹水、乳品、植物提取物、细菌裂解物、酵母裂解物或经调理细胞培养基。部分纯化的蛋白质制备物可来自已经过至少一种色谱、沉淀、其它分级分离步骤或前述任何组合处理的未纯化制备物。一个或多个色谱步骤可采用任何方法，其包括但不限于尺寸排阻、亲和、阴离子交换、阳离子交换、蛋白A亲和、疏水相互作用、固化金属亲和色谱或羟基磷灰石色谱。一个或多个沉淀步骤可包括盐或PEG沉淀，或用有机酸、有机碱或其它试剂沉淀。其它分级分离步骤可包括但不限于：结晶、液体:液体分配或膜过滤。
本领域中会理解，用于所述混合模式色谱的加载、清洗和洗脱条件将视所用的具体色谱介质/配体而定。
在一些结合-洗脱模式实施方式中，加载(即，使抗体结合至基质)和任选的清洗，优选在高于7(例如，7～8，7～9等)的pH下进行。一些示例性的结合-洗脱条件是：
结合条件：100-300mM NaCl，pH 6.5-8.5于缓冲液中(例如，Tris或磷酸盐)；
洗脱条件：0-150mM NaCl，pH 4.0-6.0，采用乙酸钠缓冲液。
任选地，所述基质的清洗条件使得所述样品的一些成分从所述固体支持物移去，但目标生物分子保留固定在所述基质上。在一些实施方式中，所述目标生物分子后续通过降低与所述基质接触的溶液的盐浓度和/或降低pH来洗脱。
或者，所述样品可以流通模式施加，其中所述样品的一些成分被固定至所述基质，但目标生物分子流通(即，流动经过)所述固体支持物并被收集。一些示例性的流通条件是0-150mM NaCl，pH 4.0-8.0；可包括合适的缓冲剂，例如，MES、Bis-Tris、乙酸钠或柠檬酸盐-磷酸盐。
实施例
实施例1：包含N-苯基乙二胺的基质的生成
以球状珠的形式采用UNOsphereTM二醇(20mL)，一种3-烯丙氧基-1,2-丙二醇和乙烯基吡咯烷酮的共聚物，用N,N'-亚甲基双丙烯酰胺交联并且二醇密度为200-300μmol/mL。使这些珠悬浮在20mL的0.1M乙酸钠或水中。添加高碘酸钠至浓度在50-100mM范围内，并且所得混合物在室温(大约70°F(21℃))下孵育3-24小时。反应导致二醇基团转化为醛基(150-250μmol/mL范围内)。将所得的醛官能化树脂转移到20-mL柱，在柱中用100mL的水洗涤该树脂。
然后，使20毫升的UNOsphere醛树脂悬浮于包含0.6g的N-苯基乙二胺的pH7.0的20ml的0.20M磷酸钠中。这之后，使混合物在室温下孵育15分钟(振荡，200rpm)，然后添加200mg NaBH3CN并使该反应持续3-20小时。经测定，该反应中N-苯基乙二胺的浓度应在25-200mM。在反应最后，将树脂转移至20ml柱，用3CV水清洗后用1-2CV的0.1N HCl清洗，然后用5CV水清洗。所述N-苯基乙二胺配体密度在25-100μmol/ml范围内。
实施例2：包含N-苯基乙二胺的基质的应用：pH 8.5-4.5梯度
将含N-苯基乙二胺配体的树脂(如上所述生成)填充进入7mm(i.d.)x 5.5cm柱，并用包含300mM NaCl,pH 8.5的20mM Tris-HCl缓冲液平衡。将500μl的6.0mg/ml的单克隆IgG抗体溶液(包含5-10％的聚集的抗体)以2ml/分钟的流速施加至该柱。所述抗体在平衡缓冲液至20mM的乙酸钠洗脱缓冲液(含150mM NaCl，pH 4.5)的10ml梯度中洗脱，然后用洗脱缓冲液进行30ml等度洗脱。收集的抗体洗脱分级分离物用尺寸排阻高效液相色谱(HPLC-SEC)分析，以确定洗脱分级分离物中聚集的抗体的含量。在该抗体洗脱分级分离物中没有检测到抗体聚集体。
采用Capto AdhereTM(配体：N-苄基-N-甲基乙醇胺)柱进行相似实验。在来自CaptoAdhereTM柱的所有分级分离物中发现聚集的抗体物质。
实施例3：包含N-苯基乙二胺的基质的应用：pH 7.0-4.5梯度
采用含20mM磷酸钠缓冲剂(含300mM NaCl)的pH 7.0平衡缓冲液进行与上述实验类似的实验。在单克隆抗体分级分离物中没有检测到聚集的抗体。
采用Capto AdhereTM(配体：N-苄基-N-甲基乙醇胺)柱进行相似实验。在来自CaptoAdhereTM柱的分级分离物中发现聚集的抗体物质。
实施例4：包含2-苯氧基乙胺的基质的生成
采用实施例1中提供的反应条件生成包含配体2-苯氧基乙胺的基质，用2-苯氧基乙胺替代N-苯基乙二胺。2-苯氧基乙胺配体的密度是约49μmol/ml。
实施例5：包含2-苯氧基乙胺的基质的应用：pH 7.0-4.5
以类似于实施例2中所述方法，采用包含配体2-苯氧基乙胺的基质来纯化抗体。
将含2-苯氧基乙胺配体的树脂填充进入7mm(i.d.)x 5.5cm柱，并用20mM磷酸钠缓冲液(含300mM NaCl,pH 7.0)平衡。将500μl的6.0mg/ml的单克隆IgG抗体溶液(包含5-10％的聚集的抗体)以2ml/分钟的流速施加至该柱。所述抗体在平衡缓冲液至20mM乙酸钠洗脱缓冲液(含150mM NaCl，pH 4.5)的10ml梯度中洗脱，然后用洗脱缓冲液进行30ml等度洗脱。收集的抗体洗脱分级分离物用尺寸排阻高效液相色谱(HPLC-SEC)分析，以确定洗脱分级分离物中聚集的抗体的含量。在抗体洗脱分级分离物中没有检测到抗体聚集体。
实施例6：包含2-苯氧基乙胺的基质的应用：pH 4.5流通模式
以类似于实施例2描述的方法，采用包含配体2-苯氧基乙胺的基质来纯化抗体，不同之处在于，采用pH 4.5的平衡缓冲液将该抗体样品施加至所述柱。
将含2-苯氧基乙胺配体的树脂填充进入7mm(i.d.)x 5.5cm柱，并用20mM乙酸钠(含75mM NaCl,pH 4.5)平衡。将750μl的2.0mg/ml的单克隆IgG抗体溶液(包含15-20％的聚集的抗体)以2ml/分钟的流速施加至该柱。抗体流通该柱。所述柱在平衡缓冲液至20mM乙酸钠洗脱缓冲液(pH 4.5)的10ml梯度中清洗，然后用洗脱缓冲液进行10ml等度洗脱。收集的流通分级分离物中的抗体用尺寸排阻高效液相色谱(HPLC-SEC)分析，以确定聚集的抗体的含量。在抗体流通分级分离物中没有检测到抗体聚集体。
在所附的权利要求书中，术语“一个”或“一种”是用来表示“一个(种)或多个(种)”。在述及步骤或要素时，其前导术语“包含”及其变化形式例如“包括”和“含有”旨在表示其它步骤或要素的增加是可任选且不被排除的。本说明书中引用的所有专利、专利申请和其它公开的参考材料都通过引用全文结合入本文中。

Claims (28)

1.一种连接有配体的色谱固体支持物，所述连接有配体的色谱固体支持物具有下式：
支持物-(X)-N+(R1,R2)-R3-L-Ar，或其盐
其中
X是间隔子或不存在；
R1和R2各自选自氢和包含1～6个碳的烷基；
R3是包含1～6个碳的烷基或包含1～6个碳的环烷基；L是NR4、O或S；其中R4是氢或包含1～6个碳的烷基；并且
Ar是芳基，所述芳基是含6～16个环碳原子的芳族环组成物。
2.如权利要求1所述的色谱固体支持物，其特征在于，所述配体是2-苯氧基乙胺或其盐的质子化形式。
3.如权利要求1所述的色谱固体支持物，其特征在于，R1和R2是氢。
4.如权利要求1或3所述的色谱固体支持物，其特征在于，R3是包含1～4个碳的烷基。
5.如权利要求1或3所述的色谱固体支持物，其特征在于，R3是包含2个碳的烷基。
6.如权利要求1所述的色谱固体支持物，其特征在于，所述芳基是苯基。
7.如权利要求1所述的色谱固体支持物，其特征在于，所述芳基是经取代的苯基。
8.如权利要求7所述的色谱固体支持物，其特征在于，所述经取代的苯基是烷基取代的苯基。
9.如权利要求1所述的色谱固体支持物，其特征在于，R1和/或R2是包含1～6个碳的烷基，且Ar是烷基取代的苯基。
10.如权利要求1所述的色谱固体支持物，其特征在于，所述配体是N-苯基乙二胺或其盐的质子化形式。
11.如权利要求1所述的色谱固体支持物，其特征在于，X不存在。
12.如权利要求1所述的色谱固体支持物，其特征在于，X是间隔子。
13.一种从样品纯化生物分子的方法，所述方法包括：
使所述样品与权利要求1～12中任一项所述的连接有配体的色谱固体支持物接触；和
收集纯化的生物分子。
14.如权利要求13所述的方法，其特征在于，所述生物分子是蛋白质。
15.如权利要求14所述的方法，其特征在于，所述蛋白质是抗体。
16.如权利要求13所述的方法，其特征在于，所述样品包含单体抗体和聚集体，并且使单体抗体与所述聚集体分离，由此导致单体抗体的纯化。
17.如权利要求13所述的方法，其特征在于，所述纯化的生物分子是单体抗体。
18.如权利要求17所述的方法，其特征在于，所述样品的单体抗体被固定在所述色谱固体支持物上，随后被洗脱。
19.如权利要求17所述的方法，其特征在于，所述样品的单体抗体流经所述色谱固体支持物并被收集。
20.如权利要求3所述的色谱固体支持物，其特征在于，所述芳基是苯基。
21.如权利要求4所述的色谱固体支持物，其特征在于，所述芳基是苯基。
22.如权利要求5所述的色谱固体支持物，其特征在于，所述芳基是苯基。
23.如权利要求3所述的色谱固体支持物，其特征在于，所述芳基是经取代的苯基。
24.如权利要求4所述的色谱固体支持物，其特征在于，所述芳基是经取代的苯基。
25.如权利要求5所述的色谱固体支持物，其特征在于，所述芳基是经取代的苯基。
26.如权利要求1所述的色谱固体支持物，其特征在于，L是NR4。
27.如权利要求1所述的色谱固体支持物，其特征在于，L是O。
28.如权利要求1所述的色谱固体支持物，其特征在于，L是S。
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