CN101171076B - 因子ⅷ和冯威勒布兰特因子的亲和吸附剂 - Google Patents

因子ⅷ和冯威勒布兰特因子的亲和吸附剂 Download PDF

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CN101171076B
CN101171076B CN2006800159187A CN200680015918A CN101171076B CN 101171076 B CN101171076 B CN 101171076B CN 2006800159187 A CN2006800159187 A CN 2006800159187A CN 200680015918 A CN200680015918 A CN 200680015918A CN 101171076 B CN101171076 B CN 101171076B
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詹森·理查德·贝特利
巴尔德夫·辛格·贝纳斯
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Abstract

为分开、除去、分离、纯化、表征、鉴别或量化因子VIII、冯威勒布兰特因子或作为二者任一种的类似物的蛋白，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；Y是O、S或NR2；Z是O、S或N-R3；R2和R3各自是H、C1-6烷基、C1-6羟烷基、苯甲基或β-苯乙基；B和W各自是含有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为-CHCOOH-(CH2)3-4-NH2；以及R7是在中性pH下带有正电荷的基团。

Description

因子Ⅷ和冯威勒布兰特因子的亲和吸附剂
发明领域
本发明涉及化合物和它们用作亲和配体的用途。
背景技术
因子VIII(之前称为抗血友病因子)是在循环血浆中发现的～200ng/mL水平的糖蛋白。天然存在的成熟单链蛋白由于糖基化而具有265～270 kDa的不同分子量，但是由于在体内的等位变异、糖基化、诱发变异和片段化而在血浆中观察到170-280kDa较宽范围分子量的蛋白。许多重组因子VIII产物是可以得到的，并且在蛋白结构和分子量上变化很大。
血浆因子VIII具有额外的复杂因子，其以与冯威勒布兰特因子(vWF)紧密结合的形式存在，复杂的多聚蛋白也涉及凝块。vWF本身，单独地或与因子VIII结合，具有治疗应用。
在vWF/FVIII转化为纤维蛋白的过程中，因子VIII促进了复杂凝块级联达到顶点，并形成纤维蛋白凝块。因子VIII产生过程中的遗传异常导致A型血友病，主要表现为由于凝块形成的减少或缺乏而导致的重复性出血事件。A型血友病及其症状(比如不可控的出血)可以用从血浆得到的或重组的因子VIII处理。使用适当的给药法，预防性地应用因子VIII可以显著地降低出血事件的次数和严重程度，包括在外科手术过程中观察到的那些。
从血浆中纯化因子VIII/vWF或从重组源中纯化因子VIII是复杂的过程，通常包括许多色谱和/或沉淀和/或病毒灭活步骤。从血浆中纯化通常还包括初始的冷冻沉淀和重组步骤。这些复杂的纯化步骤和内在的蛋白不稳定性结合，导致不良的纯化产物收率。已经描述了免疫亲和色谱，但是除了其本身的昂贵性以外，所用的色谱材料也缺乏稳定性和可清洁性，导致重复利用非常受限。基于肽配体的介质作为免疫亲和介质具有类似的问题，没有被广泛接受。获得能够从许多源以迄今不可能的产率、纯度和成本有效性分离因子VIII或因子VIII/冯威勒布兰特因子的色谱材料将是所希望的。
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 S2006800159187D00021
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
Figure 2006800159187_0
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
Figure 2006800159187_1
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vWF/FVIII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X、Y、Z、n和A如上式I定义的；
R7是在中性pH下带有正电荷的基团；
W是任选的连接基；
V是如上对于Q所述的，但是作为替代方案可以为节结构；和
R8和R9为如对于R4、R5和R6定义的，但还包括环状结构，或R8和R9连接以形成这种环状结构。
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，通过参考引入本发明中。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vWF/FVIII。
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。
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，琼脂糖凝胶，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。
在本发明的化合物中，R7优选是式NR10R11的仲胺或例如更优选的叔胺，其中R10和R11各自独立地为C1-6烷基(比如乙基)，或NR8R9形成环，或R10和R11之一一起形成环，或与连接基W中的原子一起形成环。连接基W可以为烷基、芳基或芳烷基连接基，例如具有2～7，优选3～4个链原子，其本身可被取代，例如被OH基团取代。
另一个″臂″即Z-B-D的性质可以是较不关键的。
在本发明的一个优选实施方案中，通过结构III表示结合vWF/FVIII的吸附剂。
Figure S2006800159187D00041
在本发明的另一个优选实施方案中，结合vWF/FVIII的吸附剂由结构IV表示(在生理pH时质子化)。
Figure S2006800159187D00042
在本发明的一个最优选实施方案中，通过如下所示的结构V表示结合vWF/FVIII的吸附剂：
Figure S2006800159187D00051
本发明中记载的结合vWF/FVIII的配体和吸附剂可用于从复杂混合物纯化vWF/FVIII，所述复杂混合物包括但不限于人血浆和重组发酵上清液。这些应用在以下实施例4中说明，使用人血浆池进行色谱实验。
术语″vWF/FVIII″在本发明中是指vWF/FVIII本身以及具有vWF/FVIII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vWF/FVIII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。
二氯三嗪基-(R)-1-(3-甲氧基苯基)-乙胺的合成
将溶于丙酮(35mL)中的氰尿酰氯(5.0g)冷却到0℃，之后在冷却下用30分钟逐滴加入溶于丙酮(50ml)中的(R)-1-(3-甲氧基苯基)乙胺(4.1g)，以确保温度不超过5℃。然后慢慢地加入10M氢氧化钠(2.71mL)。45分钟之后，在搅拌下将反应混合物加入到冰水(20g)中。将油状产物萃取到二氯甲烷(150mL)中，用无水硫酸镁干燥，蒸发得到黄色油状产物(7.96g)。
实施例1  吸附剂V的合成
向氨基-Sepharose CL-4B(475g重，沉降在水中-16μmol/g取代)中加入二甲基甲酰胺(DMF)(475mL)。加入二异丙基乙胺(4.37mL)，搅拌混合物15分钟，之后将溶于DMF(250mL)中的二氯三嗪基-(R)-1-(3-甲氧基苯基)-乙胺(7.57g)加入到反应混合物中。搅拌该混合物3小时，之后用70％含水DMF(4×500mL)、50％含水DMF(2×500mL)和水(11×500mL)洗所述凝胶。在60℃将150g(沉降的)该材料在水(75mL)中浆化，并分批加到N，N-二乙基-1，3-丙二胺(3.8mL)在水(75mL)中的溶液中。加入之后，再次温热所述混合物到60℃，并在该温度下搅拌19小时。然后滤出凝胶并用水(12×150mL)洗，之后存储在20％含水乙醇防腐剂中。
实施例2  吸附剂III的合成
在60℃在水(75mL)中浆化来自实施例2的中间凝胶产物(150g沉降的)，并分批加入到2-(2-氨乙基)-1-甲基吡咯烷(3.5mL)在水(75mL)中的溶液中。加入之后，再次温热所述混合物到60℃，并在该温度下搅拌19小时。然后滤出凝胶并用水(12×150mL)洗，之后存储在20％含水乙醇防腐剂中。
实施例3  吸附剂IV的合成
将氨基-Sepharose CL-4B(100g重，沉降在水中-16μmol/g取代)悬浮在1M pH 7.0的磷酸钾(100mL)中，然后排水。然后向该凝胶中加入1M pH 7.0的磷酸钾(25mL)和RO水(250mL)。剧烈搅拌所述浆，同时加入丙酮(50mL)。在冰/盐浴中冷却30分钟之后，一次性加入在冷丙酮(25mL)中的氰尿酰氯(2.5g)。在0℃搅拌该混合物1小时，然后用50％含水丙酮(5×100mL)、RO水(5×100mL)、50％含水丙酮(5×100mL)和RO水(10×100mL)洗。然后在室温下，用含有2-氨基丁醇(1g)的50％含水DMF(100mL)浆化该沉降的凝胶4小时。过滤浆，然后用50％含水DMF(5×100mL)和RO水(10×100mL)洗。然后用含有4-氨基-a-二乙基氨基-o-甲酚二盐酸盐(4.2 g)的50％含水DMF(100mL)浆化所述沉降的凝胶，并用氢氧化钠(10M)调节至pH 9，并在60℃搅拌过夜。过滤浆，然后用50％含水DMF(5×100mL)和RO水(10×100mL)洗。将该凝胶在40℃在最终浓度0.5 M的NaOH/25％乙醇中培养3天，然后用0.5M NaOH/25％乙醇(5×100mL)洗，然后用RO水(10×100mL)洗。最后的清洗之后使得在重力下排水，加入1.0MNaOH(100mL)，并在40℃培养所述混合物3天。然后用0.5M NaOH(5×100mL)洗所述凝胶，然后用RO水(10×100mL)洗。所述凝胶用pH7.0的0.1 M PBS(3×100mL)洗后，再次用RO水(10×100mL)洗，之后在冷室中在4℃在20％v/v含水乙醇中存储。
实施例4  用吸附剂V对人血浆进行色谱分析的实施例
使用低压色谱系统(Kipp&Zonen平床图表记录器，Gilson minipuls3蠕动泵和Gilson UV检测器)，使用20mL柱容积的1.6cm直径XK16/20柱进行色谱实验。用5柱容积的20mM Tris.HCI、20mM柠檬酸钠、140mM氯化钠pH 7.5，以80cm/小时平衡所述柱。用20mMTris.HCl和100mM氯化钠(最终浓度)处理人源血浆。10μm过滤200mL经处理的血浆，然后以80cm/小时加入。加入后，用20mM Tris.HCl、20mM柠檬酸钠和140mM氯化钠pH 7.5洗至基线吸收度。然后用20mM Tris.HCl、20mM柠檬酸钠、3mM CaCl2、30％乙二醇和500mM氯化钠和0.01％Tween 80 pH 7.5洗脱所述柱。洗脱后，用8M脲pH 7.0对柱进行消毒。随后用0.5 M氢氧化钠清洗所述柱。通过浊度测定法、vWF ELISA和因子VIII显色活性试验分析加入的、未结合的和洗脱的级分，以确定回收率。还进行SDS PAGE以研究纯度。
回收率列于表1中。
表1
来自吸附剂V色谱分析的vWF/FVIII的回收率
加入 vWF  1632μg
FVIII  147 IU
(％回收)
未结合的 vWF  512μg 32
FVIII  29.2IU 20
(％回收)
加入 vWF  1632μg
FVIII  147 IU
洗脱 vWF  879μg 54
FVIII  79IU 54
通过显色活性试验(COATEST VIII：C/4，由Chromogenix提供)确定因子VIII结果。结果以国际单位(IU)表示，其中正常人血浆每mL含有1国际单位的因子VIII活性。
由ELISA确定vWF结果。

Claims (9)

1.亲和吸附剂用于分开、除去、分离、纯化、表征、鉴别或量化因子VIII、冯威勒布兰特因子的用途，其中所述亲和吸附剂是式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所述环；
Y是O、S或NR2；
Z是O、S或N-R3；
R2和R3各自是H、C1-6烷基、C1-6羟烷基、苯甲基或β-苯乙基；
B和W各自是含有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
R7是在中性pH下带有正电荷的叔胺基团。
2.权利要求1的用途，其中所述叔胺形成环的一部分。
3.权利要求1或2的用途，其中B或W包括芳基。
4.权利要求1或2的用途，其中所述吸附剂与血浆样品接触。
5.一种式III的化合物
Figure FSB00000092188200012
其中A是载体基体，任选地通过间隔基连接到所述环。
6.一种式IV的化合物
Figure FSB00000092188200021
其中A是载体基体，任选地通过间隔基连接到所述环。
7.一种式V的化合物
Figure FSB00000092188200022
其中A是载体基体，任选地通过间隔基连接到所述环。
8.一种化合物，其是根据权利要求5-7中任一项的化合物的前体，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。
9.根据权利要求1或2的用途，其中所述吸附剂是根据权利要求5-7中任一项的化合物。
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