CN101171078A - 纤维蛋白溶酶原的亲和吸附剂 - Google Patents

纤维蛋白溶酶原的亲和吸附剂 Download PDF

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CN101171078A
CN101171078A CN200680016008.0A CN200680016008A CN101171078A CN 101171078 A CN101171078 A CN 101171078A CN 200680016008 A CN200680016008 A CN 200680016008A CN 101171078 A CN101171078 A CN 101171078A
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詹森·理查德·贝特利
詹姆士·克里斯托弗·皮尔森
克劳迪亚·希尔德加德·库恩
巴尔德夫·辛格·贝纳斯
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Astley Biological Separation Co ltd
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Abstract

为分开、除去、分离、纯化、表征、鉴别或量化纤维蛋白溶酶原或作为纤维蛋白溶酶原类似物的蛋白，使用亲和吸附剂，其是式(II)的化合物，其中一个X是N，另一个是N、C－Cl或C－CN；A是载体基体，任选地通过间隔基连接到三嗪环；Z是O、S或N－R，R是H、C1－6烷基、C1－6羟烷基、苯甲基或β－苯乙基；B是含有1～10个碳原子的任选地取代的烃键；D是H、OH或伯氨基、仲氨基、叔氨基、季铵、咪唑、胍基或脒基；或B－D是－CHCOOH－(CH2)3－4－NH2；q是2～6。

Description

纤维蛋白溶酶原的亲和吸附剂
技术领域
本发明涉及化合物和它们用作亲和配体的用途。
背景技术
纤维蛋白溶酶原(可替代性地称为微纤溶酶、PLG或制管张素)是单链91kDa糖蛋白酶原，并且是纤维蛋白溶酶纤溶酶的前体。纤维蛋白溶酶原的天然形式由791个氨基酸组成，其中谷氨酸位于N-末端部分(Glu-纤维蛋白溶酶原)。纤维蛋白溶酶原具有五个被称为kringle的同源区域。这些区域对于位于tPA、uPA和凝血酶原的互补kringle是特异性的。这些kringle具有一个高亲和性和四个低亲和性赖氨酸结合位点，介导纤维蛋白溶酶原与纤维蛋白和α-2-抗纤维蛋白酶的相互作用。
纤维蛋白溶酶原通过导致纤维蛋白溶酶原的Arg560-Val561肽键水解的一系列的各种反应转化为纤溶酶，导致两个链通过二硫键保持共价键结合。胰蛋白酶样蛋白酶纤溶酶的主要作用是凝块的分解。
由编码DNA的同类或杂合突变产生的纤维蛋白溶酶原的不足表现在许多病理学中，包括木质化结膜炎和血栓形成。
纤维蛋白溶酶原以～100μg/mL的浓度存在于人血浆中。在很好地表征的亲和色谱法过程中，可以使用固定在固体载体上的赖氨酸从血浆中纯化纤维蛋白溶酶原。固定化赖氨酸色谱法也用于从收获的细胞培养物中初级捕获蛋白，比如组织纤溶酶原激活物(tPA)，一般用作凝块分解药。纤维蛋白溶酶原的可商业得到的树脂的结合能力通常为0.6～1.0mg蛋白/mL吸附剂。由这些材料洗脱的纤维蛋白溶酶原的纯度非常好，初级捕获的纯度通常＞95％。这对于使材料保持极好洗脱纯度的这些特征而显示出超过其目前可达到的结合能力来说将是有用的。
WO97/10887公开了用作亲和吸附剂的式I的基于三嗪的化合物：
Figure S2006800160080D00021
其中R1是H、烷基、羟烷基、环己基、NH2、苯基、萘基、1-苯基吡唑、吲唑、苯并噻唑、苯并唑或苯并咪唑，任意的这些芳基可以被烷基、烷氧基、酰氧基、酰氨基、氨基、NH2、OH、CO2H、磺酰基、氨基甲酰基、氨基磺酰基、烷基磺酰基和卤素中的一个或多个取代；
一个X是N，另一个是N、C-Cl或C-CN；
Y是O、S或NR2；
Z是O、S或NR3；
R2和R3各自是H、烷基、羟烷基、苯甲基或β-苯乙基；
Q是苯、萘、苯并噻唑、苯并唑、1-苯基吡唑、吲唑或苯并咪唑；
R4、R5和R6各自是H、OH、烷基、烷氧基、氨基、NH2、酰氧基、酰氨基、CO2H、磺酸、氨基甲酰基、氨基磺酰基、烷基磺酰基或卤素；
n是0～6；
p是0～20；和
A是载体基体，任选地通过间隔基连接到三嗪环。
式I的化合物公开为对于蛋白比如免疫球蛋白、胰岛素、因子VII或人生长激素具有亲和性。
相关结构的化合物公开在WO00/67900和WO03/097112中。它们对于内毒素具有亲和性。
发明内容
意外地，已经发现某些化合物可用于纤维蛋白溶酶原基于亲和性的分离，这些化合物中有许多是新的。这些化合物具有式II
其中，
A、X和Z如上式I中所定义；
B是含有1～10个碳原子的任选地取代的烃键；
D是H、OH或伯氨基、仲氨基、叔氨基、季铵、咪唑、胍基或脒基；或
B-D是-CHCOOH-(CH2)3-4-NH2；和
q是2～6。
另外，本发明的化合物包括相应的配体，其中A被官能团代替，直接地或间接地连接到三嗪环，其可以反应使得所述化合物固定在载体基体上。下文中，术语″配体″和″吸附剂″可互换地使用。
具体实施方式
WO97/10887、WO00/67900和WO03/097112公开了如何可以在固体载体上建立配体的组合库。它们的公开内容，包括实施方案的实施例和与本发明相同的步骤，通过引用并入本发明中。在用作为原料的人血浆池筛选这些组合库组期间，许多配体确定为能够选择性地结合和洗脱人纤维蛋白溶酶原。
可以通过本领域技术人员已知的方法制备用于本发明的式II的化合物。这种方法记载于上述3个PCT公开中；它们可以容易地适用于新化合物的制备。
在本发明的化合物中，q优选4。应知道分子的″臂″源自于赖氨酸。B优选包括芳环，例如苯环。
WO97/10887给出了A的实例，包括间隔基或连接基L，通过其三嗪环可连接到固体载体M。如WO97/10887所述，这种载体包括琼脂糖，二氧化硅，纤维素，右旋糖酐，淀粉，藻酸盐，角叉菜聚糖，合成聚合物，玻璃和金属氧化物。在反应以形成本发明的吸附剂以前可以活化这种材料。
L例如可以为-T-(-V1-V2)m-，其中
T是O、S或-NR7-；
m是0或1；
V1是任选地取代的2～20个C原子的烃基；和
V2是O、S、-COO-、-CONH-、-NHCO-、-PO3H-、-NH-亚芳基-SO2-CH2-CH2-或-NR8-；和
R7和R8各自独立地为H或C1-6烷基。
在本发明的一个优选实施方案中，通过结构III表示结合纤维蛋白溶酶原的吸附剂。
Figure S2006800160080D00041
在本发明的另一个优选实施方案中，通过结构IV表示结合纤维蛋白溶酶原的吸附剂。
在本发明的另一个优选实施方案中，通过结构V表示结合纤维蛋白溶酶原的吸附剂。
Figure S2006800160080D00051
本发明中记载的结合纤维蛋白溶酶原的配体和吸附剂可用于从各种流体和复杂混合物中亲和性分离和纯化纤维蛋白溶酶原，所述各种流体和复杂混合物包括但不限于人血浆、消除了一种或多种其它蛋白的人血浆和重组发酵上清液。这些应用在下实施例4中说明，其使用人血浆池通过色谱实验进行。
术语″纤维蛋白溶酶原″在本发明中是指纤维蛋白溶酶原本身以及具有纤维蛋白溶酶原功能特点的类似物，例如，就对于本发明中记载的给定化合物的亲和性而言。因此，所述分析物可以是作为纤维蛋白溶酶原功能片段的蛋白，或具有所有相同结合位点中的一个、多个的结构类似物，或融合蛋白。
以下实施例说明本发明。
实施例1  吸附剂III的合成
用RO水(670mL)、10M氢氧化钠(NaOH)(90mL)和表氯醇(127mL)将6％的交联Purabead琼脂糖凝胶(1000g沉降在反渗透(RO)水中)制成浆。搅拌所述浆2小时。取出用于分析的样品之后，过滤所述浆，然后用RO水(12×1L)洗。对于环氧基的分析表明每g沉降的凝胶衍生了17.3μmol的环氧基。
在加入RO水(800mL)和0.88比重的氨水溶液(200mL)以前，将凝胶中的水排出。搅拌该混合物并加热到40℃，然后在该温度搅拌16小时。取出用于分析的样品之后，过滤所述浆，然后用RO水(12×1000mL)洗。对于胺基的TNBS分析表明每g沉降的凝胶衍生了25.6μmol的胺基。
将沉降的氨化的凝胶(1000g)悬浮在1M pH7.0的磷酸钾(1L)中，然后排水。然后向该凝胶中加入1M pH7.0的磷酸钾(250mL)和RO水(250mL)。剧烈搅拌浆同时加入丙酮(500mL)。在冰/盐浴中冷却30分钟之后，一次性加入在冷丙酮(250mL)中的氰尿酰氯(25g)。在用50％含水丙酮(5×1L)、RO水(5×1L)、50％含水丙酮(5×1L)和RO水(10×1L)洗之前，在0℃搅拌该混合物1小时。在取出用于分析的样品以前，使得凝胶在重力下沉降。对于氯化物释放的分析表明每g沉降的凝胶衍生有16.8μmol取代的二氯三嗪。
在室温下，用含有盐酸赖氨酸(45g)的pH8.5的1M磷酸钾缓冲液(1L)浆化上述阶段的沉降的凝胶(1000g)16小时。过滤所述浆，然后用RO水(15×1L)洗。向在RO水(950mL)中浆化的950g(沉降的)凝胶中，加入乙醇胺(14.82g)。在60℃下搅拌该混合物过夜。取出用于分析的上清液样品之后，过滤所述浆，然后用RO水(12×1L)洗。对于氯化物释放的分析表明每g沉降的凝胶已经衍生了22.7μmol的乙醇胺。
在40℃下在最终浓度0.5M的NaOH中培养凝胶过夜，然后用0.5MNaOH(5×1L)洗。最后的清洗之后使得在重力下排水，加入0.5M NaOH(1L)，并在40℃下培养所述混合物过夜。然后用0.5M NaOH(5×1L)洗所述凝胶，然后用RO水(10×1L)洗。所述凝胶用pH7.0的0.1M PBS(3×1L)洗后，再次用RO水(10×1L)洗，之后在冷室中在4℃下存储在20％v/v含水乙醇中。
实施例2  吸附剂IV的合成
根据实施例1中所描述的方法制备二氯三嗪活化的琼脂糖。在60℃下使沉降的凝胶(41.5g，22μmol DCT/g沉降的)与盐酸赖氨酸(1.67g，10摩尔当量)在水(40mL)中的水溶液反应过夜，同时用10M NaOH保持pH为9.0。此时用50％含水二甲基甲酰胺(5×80mL)、RO水(5×80mL)、0.1M HCI(5×80mL)、30％异丙醇/0.2M NaOH(5×80mL)和RO水(5×80mL)洗所述凝胶，之后在冷室中在4℃下存储在20％v/v含水乙醇中。
实施例3  吸附剂V的合成
根据实施例1中所描述的方法制备二氯三嗪活化的琼脂糖。在5℃下使沉降的凝胶(43g，21.1μmol DCT/g沉降的)与2-(羟甲基)苯胺(0.56g，5摩尔当量)在50％DMF/水(40mL)中的溶液反应30分钟。在此时，用50％含水二甲基甲酰胺(5×80mL)和RO水(5×80mL)洗所述凝胶。在60℃下使所述凝胶与盐酸赖氨酸(1.71g，10摩尔当量)在水(40mL)中的水溶液反应过夜，同时用10M NaOH保持pH在9.0。此时用50％含水二甲基甲酰胺(5×80mL)、RO水(5×80mL)、0.1M HCI(5×80mL)、30％异丙醇/0.2M NaOH(5×80mL)和RO水(5×80mL)洗所述凝胶，之后在冷室中在4℃下存储在20％v/v含水乙醇中。
实施例4  使用人血浆用吸附剂III、IV和V进行色谱分析
使用Akta Explorer色谱系统，使用1.6厘米内径，10厘米床高度的柱色谱法柱进行色谱实验。用140mM氯化钠/20mM柠檬酸钠/20mM Tris pH7.5，以100cm/小时平衡所述柱。然后以50cm/小时加入人血浆(调节到20mM Tris/pH7.5，通过3、0.8、0.45和0.2μm过滤器过滤，1.3L)。加入后用140mM氯化钠/20mM柠檬酸钠/20mM TrispH7.5，以50cm/小时洗到基线吸收度。然后用140mM氯化钠/20mM柠檬酸钠/20mM Tris/500mMε-氨基己酸，pH7.5，以100cm/小时洗脱所述柱，并用0.5M氢氧化钠再生。通过浊度测定法分析加入的、未结合的和洗脱的级分，以确定结合能力。进行SDS PAGE以确定纯度。
从所有吸附剂洗脱的级分的纯度均＞85％。动态结合能力(在10％临界点)如表1所示。
表1
吸附剂 动态结合能力(mg/mL)
III 4.75
IV 4.0
V 17.0

Claims (7)

1.亲和吸附剂用于分开、除去、分离、纯化、表征、鉴别或量化纤维蛋白溶酶原或作为纤维蛋白溶酶原类似物的蛋白的用途，其中所述亲和吸附剂是式II的化合物：
Figure S2006800160080C00011
其中一个X是N，另一个X是N、C-Cl或C-CN；
A是载体基体，任选地通过间隔基连接到所述三嗪环；
Z是O、S或N-R，R是H、C1-6烷基、C1-6羟烷基、苯甲基或β-苯乙基；
B是含有1～10个碳原子的任选地取代的烃键；
D是H、OH或伯氨基、仲氨基、叔氨基、季铵、咪唑、胍基或脒基；或
B-D是-CHCOOH-(CH2)3-4-NH2；和
q是2～6。
2.权利要求1的用途，其中所述吸附剂具有式III。
3.权利要求1的用途，其中所述吸附剂具有式IV。
4.权利要求1的用途，其中所述吸附剂具有式V。
5.前述权利要求任一项的用途，其中所述纤维蛋白溶酶原在血浆样品中。
6.一种新型化合物，其具有前述权利要求任一项中所定义的式II。
7.一种化合物，其是根据权利要求6的化合物的前体，其中A被官能团代替，直接地或间接地连接到所述三嗪环，这允许所述前体固定在固体载体上。
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