CN101735991A - 用于制备非免疫原性聚合物性缀合物的无聚集体尿酸氧化酶 - Google Patents
用于制备非免疫原性聚合物性缀合物的无聚集体尿酸氧化酶 Download PDFInfo
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Abstract
本发明涉及用于制备非免疫原性聚合物性缀合物的无聚集体尿酸氧化酶。使基本上不含大聚集体的天然蛋白质或重组蛋白质、特别是猪尿酸氧化酶突变蛋白与足够少量数目链的聚合物缀合,可使其基本上无免疫原性,从而使所述蛋白的生物活性基本保留在缀合物中。所述缀合物非常适合治疗慢性疾病,因为与从含微量大聚集体的蛋白质制品制备的类似缀合物相比,它们不大可能诱导形成抗体和/或加速清除。
Description
本申请是申请日为2001年2月7日,申请号为01807750.1,发明名称为“用于制备非免疫原性聚合物性缀合物的无聚集体尿酸氧化酶”的发明专利申请的分案申请。
本发明的政府权益声明
本发明介绍的一部分研究是在美国-以色列两国工业研究发展基金会支持下进行的。因此,美国政府对本发明拥有一定权益。
发明背景
发明领域
本发明涉及纯化和化学修饰蛋白质,以延长其循环时间并降低其免疫原性。更准确地说,本发明涉及去除尿酸氧化酶(尿酸酶)八聚体以上的聚集体,然后使其与聚(乙二醇)或聚(环氧乙烷)缀合。这样基本上消除了尿酸酶免疫原性而不影响其尿酸分解活性。
相关技术介绍
本背景部分包含的陈述不代表承认为现有技术,相反,反映了本发明人进行本发明时对技术状况的主观评论和解释。这些解释可能包括本发明人个人迄今未公开的见解,见解本身不是现有技术的一部分。
尿酸氧化酶(尿酸酶;E.C.1.7.3.3.)是催化尿酸氧化成可溶性更强的产物尿囊素的酶,尿囊素是更容易排出的嘌呤代谢物。人体不能产生具有酶活性的尿酸酶,这是因为高等灵长类动物在进化期间尿酸酶基因获得性产生了几个突变。Wu,X等(1992)J Mol Evol 34:78-84。结果,在易感个体中,血中过高浓度的尿酸(高尿酸血症)和尿中过高浓度的尿酸(高尿酸尿)可引起疼痛性关节炎(痛风)、损伤性尿酸盐沉积(痛风石)以及肾衰竭。在一些患者个体中,有效药物如别嘌呤醇(一种尿酸合成抑制剂)产生治疗限制性副作用或不能充分减轻上述病情。Hande,KR等(1984)Am J Med 76:47-56;Fam,AG,(1990)Baillière′s Clin Rheumatol 4:177-192。注射尿酸酶至少可短暂减轻高尿酸血症及高尿酸尿。因为尿酸酶是人体外源性蛋白,所以即使第一次注射源自黄曲霉的未修饰尿酸酶蛋白,都导致百分之几的治疗患者产生过敏反应(Pui,C-H等(1997)Leukemia 11:1813-1816),而且免疫反应限制其长期或间断性治疗应用。Donadio,D等(1981)NouvPresse Méd 10:711-712;Leaustic,M等(1983)Rev Rhum Mal Osteoartic50:553-554。
美国专利申请序号09/370,084和已公布的国际申请号PCT/US99/17514公开保留了至少约75%非缀合尿酸酶尿酸分解活性并大大减少其免疫原性的聚(乙二醇)-尿酸氧化酶(PEG-尿酸酶)。在一种所述纯化尿酸酶中,每个亚单位平均与2-10条PEG共价连接,其中每个PEG分子的分子量可为约5kDa-100kDa。
已知蛋白质聚集增加其免疫原性。对这一点的认识使得研制在蛋白质用于制备疫苗或免疫动物以产生抗血清之前通过诸如热变性处理和戊二醛交联处理而目的在于使蛋白质聚集的方法。
此外已经知道,在治疗性蛋白质的临床应用期间,例如人γ球蛋白(Henney等(1968)N.Engl.J.Med.278:2244-2246)和人生长激素(Moore等(1980)J.Clin.Endocrinol.Metab.51:691-697,蛋白质非目的性聚集导致产生免疫或致敏。人干扰素-α聚集产生免疫原性已经在BALB/c小鼠得到证实(Braun等(1997)Pharm.Res.14:1472-1478),已经将酶联免疫吸附测定法(ELISA)发展用于治疗蛋白聚集性免疫原性测定(Braun等(1997)Pharm.Res.14:1394-1400)。
与蛋白质聚集对免疫原性的已知作用形成对照的是,尚没有聚集对与诸如PEG的聚(亚烷基二醇)缀合的蛋白质免疫原性产生影响的报告。需要基本上消除尿酸酶免疫原性而不影响其尿酸分解活性的聚(亚烷基二醇)-尿酸酶缀合物。本发明提供这样的组合物。
发明概述
蛋白质与聚(亚烷基二醇)、特别是PEG缀合获得免疫原性降低且在血液中维持时间增加的缀合物。在试图制备基本上无免疫原性、基本上保留未修饰尿酸酶制剂全部尿酸分解活性的尿酸酶缀合物时,发现重复注射由包含大的尿酸酶聚集体的尿酸酶制备的PEG缀合物后,原料物质中的微量尿酸酶大聚集体在刺激抗体形成及加速从血液中清除方面都有惊人的效果,这两方面都是有害的。令人惊奇的是,本发明人发现,免疫原性增加和加速清除并不是由于存在比天然四聚体大的明确中等大小的尿酸酶亚单位聚集体,例如含八个亚单位的聚集体(八聚体)。在可通过紫外光吸光度(例如214nm或276nm)检测或通过折光率或其它蛋白质浓度测量检测的大多数尿酸酶制剂中存在足够高浓度的八聚体形式尿酸酶。然而,发现八聚体本身对免疫原性和加速PEG-尿酸酶缀合物清除的作用非常小,相比之下含量少得多而大得多的聚集体产生的作用大得多,所述大聚集体不能在所述检测条件下通过紫外吸光度检测到,但易于通过静态(Raleigh)或动态光散射检测到。因此发现,与PEG缀合之前去除所述微量的非常大的聚集体而以出人意料的程度降低所获得的PEG-尿酸酶缀合物的免疫原性和快速清除。
本发明的一个实施方案是基本上不含大于八聚体的聚集体的纯化尿酸氧化酶(尿酸酶)。优选所述尿酸酶为哺乳动物尿酸酶。更优选所述尿酸酶为猪肝尿酸酶、牛肝尿酸酶或羊肝尿酸酶。在此优选实施方案的一个方面,所述尿酸酶为重组尿酸酶。在此优选实施方案的另一方面,所述尿酸酶基本上具有猪肝、牛肝、羊肝或狒狒肝尿酸酶的序列。所述尿酸酶优选为嵌合体。优选所述尿酸酶为PKC尿酸酶。在此优选实施方案的另一方面,所述尿酸酶基本上具有狒狒肝脏尿酸酶序列,其中97位酪氨酸由组氨酸替代。所述优选尿酸酶包含一个氨基末端和一个羧基末端,其中所述尿酸酶在一个末端或两个末端截短。所述尿酸酶为真菌尿酸酶或微生物尿酸酶是有益的。优选所述真菌尿酸酶或微生物尿酸酶分离自黄曲霉(Aspergillusflavus)、球形节杆菌(Arthrobacter globiformis)、芽孢杆菌(Bacillus sp.)或产朊假丝酵母(Candida utilis),或者是基本上具有一种所述尿酸酶序列的重组尿酸酶。或者所述尿酸酶为无脊椎动物尿酸酶。优选所述无脊椎动物尿酸酶分离自果蝇(Drosophila melanogaster)或Drosophila pseudoobscura,或者是基本上具有一种所述尿酸酶序列的重组尿酸酶。在此优选实施方案的另一方面,所述尿酸酶为植物尿酸酶。优选所述植物尿酸酶分离自大豆(Glycine max)根瘤或者为基本上具有所述尿酸酶序列的重组尿酸酶。
此优选实施方案的一个方面中,上述尿酸酶与聚(乙二醇)或聚(环氧乙烷)在一定条件下缀合,使得所述缀合物尿酸酶基本上不含大于八聚体的聚集体。优选所述尿酸酶通过氨基甲酸乙酯(氨基甲酸酯)键、仲胺键或酰胺键与聚(乙二醇)或聚(环氧乙烷)缀合。在此优选实施方案的一个方面,所述聚(乙二醇)为单甲氧基聚(乙二醇)。在此优选实施方案的另一方面,所述聚(乙二醇)或聚(环氧乙烷)分子量为约5kDa-30kDa。优选所述聚(乙二醇)或聚(环氧乙烷)分子量为约10kDa-20kDa。所述聚(乙二醇)或聚(环氧乙烷)平均链数为每个尿酸酶亚单位约2-12条是有益的。更有益的是所述聚(乙二醇)或聚(环氧乙烷)平均链数为每个尿酸酶亚单位约6-10条链。最有益的是所述聚(乙二醇)或聚(环氧乙烷)平均链数为每个尿酸酶亚单位约7-9条。优选所述聚(乙二醇)或聚(环氧乙烷)为线型。或者所述聚(乙二醇)或聚(环氧乙烷)为支链型。
本发明还提供降低体液或组织中尿酸水平的药用组合物,它包括上述尿酸酶缀合物及药学上可接受的载体。优选通过冷冻干燥稳定所述组合物,复制时溶解获得适于胃肠外给予的溶液。
本发明的另一个实施方案是用于纯化免疫原性降低的尿酸酶的方法,它包括分离尿酸酶流分中大于八聚体的尿酸酶聚集体,并从所纯化的尿酸酶中去除所述聚集体的步骤。优选所述分离步骤包括检测至少一部分所述尿酸酶流分中大于八聚体的聚集体,并去除包含所述聚集体流分的步骤。所述检测步骤最好包括光散射检测步骤。
本发明还提供上述方法制备的分离尿酸酶。
附图简述
图1说明Pharmacia Biotech Mono Q(1×10cm)阴离子交换柱流分尿酸酶活性、总蛋白浓度以及盐浓度。通过在室温下监测100μM尿酸的200mM硼酸钠溶液pH 9.2的292nm吸光度降低,从而测量尿酸酶活性。根据大小排阻性HPLC分析中尿酸酶吸收峰曲线下面积确定总蛋白量。
图2说明在Pharmacia Superdex 200柱(1×30cm)上对包含突变R291K和T301S(PKS尿酸酶)的负荷样品(load)和选定流分进行的大小排阻性HPLC分析,所述流分得自对猪尿酸酶的制备型Mono Q色谱分析,图中所示数据为光散射检测器90°入射光检测结果(上部曲线)及276nm吸光度(下部曲线)检测获得的数据。未分级分离样品(负荷样品)和各种流分中四聚体、八聚体以及更高聚集形式尿酸酶的信号强度差异是显而易见的。所述负荷样品用Mono Q柱缓冲液稀释5倍,流分5稀释3倍,流分6稀释9倍。流分5和流分6混合形成“低盐合并物”。
图3说明对图1Mono Q柱流分的大小排阻性分析,与图2相同,所示数据为光散射检测器检测90°入射光及通过276nm下的吸光度检测获得的数据。该图中所示流分用于形成“高盐合并物”,由所述高盐合并物制备PEG缀合物并注入BALB/c小鼠。所得的BALB/c小鼠血清活性及免疫反应见图5及图6。
图4说明未分级分离PKC尿酸酶和PKS尿酸酶的制备型MonoQ柱色谱的选择流分(图1)中的八聚体含量,八聚体含量通过276nm吸光度及90°光散射测定获得,根据图2及图3数据计算获得。
图5说明每次注射(每周一次,共6次)PKS尿酸酶的6×10-kDaPEG缀合物或Mono Q柱流分合并物后24小时取血清在37℃下温育4小时后尿酸酶活性UV分析(同图1)。
图6说明对PKS尿酸酶PEG缀合物的IgG抗体形成及对图1所示Mono Q柱流分合并物中的PEG缀合物的IgG抗体形成的ELISA分析,其中对BALB/c雌性小鼠每次注射(每周一次,共6次)0.2mg尿酸酶蛋白/20g体重后24小时取血清进行所述分析。对于每只小鼠,从第一次注射至第六次注射后24小时的取血分析获得的数据从左至右显示。检测条件描述于实施例6。各组中8只小鼠的数据按免疫反应递增顺序从左至右排列。
优选实施方案详述
以前的研究表明,与PEG缀合(PEG化)达到尿酸酶免疫原性和/或抗原性显著降低的同时,始终伴有尿酸分解活性大量丧失。本发明观察到,微量大于八聚体的尿酸氧化酶聚集体直接与PEG-尿酸酶缀合物免疫原性及诱导快速清除有关。这一发现最可能适用于尿酸酶以外的蛋白质,包括干扰素及生长因子。
生物药物的安全性、方便性、成本效益都受药物效价降低从的不利影响,结果是需要增加给药剂量。因此需要降低包括血液和尿液在内的体液中高尿酸水平的安全有效的替代方法。本发明提供生产用于合成PEG-尿酸酶、去除了大于八聚体的尿酸酶聚集体的尿酸酶的方法。所述PEG-尿酸酶保留了未修饰尿酸酶所有或几乎所有尿酸分解活性。本发明还提供基本上不含大于八聚体的聚集体的纯化尿酸酶。术语“基本上不含”是指包含不超过约2%、优选不超过约1%的大于八聚体的聚集体的纯化尿酸酶。
本发明提供纯化尿酸酶的方法,从而从纯化尿酸酶制剂中去除大于八聚体的尿酸酶。因为这些较大的聚集体具有高度免疫原性,所以纯化尿酸酶制剂不能含有所述聚集体。所述方法涉及通过光散射而不是通过280nm紫外吸光度或者通过光散射和280nm紫外吸光度监测色谱柱分离的流分,因为所述聚集体可能浓度太小而用紫外吸光度检测不到。然后将所述纯化尿酸酶与水溶性聚合物、优选聚(乙二醇)或聚(环氧乙烷)缀合,见共同未决的美国专利申请序号09/370,084介绍。
从主要由四聚体尿酸酶构成的制剂中去除聚集尿酸酶可通过本领域技术人员已知的任何方法实现,包括大小排阻性色谱法、离子交换色谱法、通过微孔膜超滤以及包括超速离心在内的离心。所述分离方法可包括各流分的分离和分析以及去除包含过量大聚集体的流分。所得尿酸酶制剂比未分级分离尿酸酶更适于合成基本上没有免疫原性的尿酸酶缀合物。对于长期用药,蛋白质的PEG缀合物如PEG-尿酸酶具有低免疫原性且重复给药后不引起药物从血液中进行性的更快速清除是重要的。
本发明还提供聚合物-尿酸酶缀合物的药用组合物。所述缀合物基本上无免疫原性,并保留未修饰尿酸酶的至少75%、优选85%、更优选95%或95%以上的尿酸分解活性。适于与水溶性聚合物缀合的尿酸酶包括从细菌、真菌、植物组织以及动物组织(包括脊椎动物和无脊椎动物)中分离的天然尿酸氧化酶,以及包括重组形式的尿酸酶,包括变异尿酸酶、杂交尿酸酶和/或酶性截短的活性尿酸酶变异体。适用于本发明的水溶性聚合物包括线型和支链型聚(乙二醇)或聚(环氧乙烷),通常都称为PEG。支链型PEG实例是美国专利5,643,575的主题。线型PEG的一个优选实例为单甲氧基PEG,通式结构为CH3O-(CH2CH2O)nH,其中n为约100-2,300。
本发明的一个实施方案为保留非缀合尿酸酶的至少约75%尿酸分解活性并且显著降低免疫原性的尿酸氧化酶(尿酸酶)缀合物。本发明此方面的尿酸酶可以是重组尿酸酶。不论是否重组尿酸酶还是非重组尿酸酶,所述尿酸酶可以是哺乳动物来源。在此实施方案的一个方面,所述尿酸酶可以是猪、牛或羊的肝脏尿酸酶。在此实施方案的另一方面。所述尿酸酶可以是嵌合体。所述嵌合体尿酸酶可包含猪肝脏和/或狒狒肝脏尿酸酶的组成部分。例如所述嵌合体尿酸酶可以是包含突变R291K和T301S的猪尿酸酶(PKS尿酸酶)。另一方面,所述尿酸酶可以是狒狒肝脏尿酸酶,其中97位的酪氨酸由组氨酸替代,所以所述尿酸酶比活可增加至少约60%。无论什么来源,本发明所述尿酸酶,也可以是截短形式,在氨基末端、或者在羧基末端、或者两个末端截断。同样所述尿酸酶可以是真菌尿酸酶或微生物尿酸酶。在本发明的一个实施方案中,所述真菌尿酸酶或微生物尿酸酶可以是源自黄曲霉(Aspergillus flavus)、球形节杆菌(Arthrobacter globiformis)、芽孢杆菌(Bacillus sp.)或产朊假丝酵母(Candida utilis)的天然或重组尿酸酶。或者,所述尿酸酶可以是无脊椎动物尿酸酶,例如源自果蝇(Drosophila melanogaster)或Drosophilapseudoobscura的天然或重组尿酸酶。本发明的尿酸酶也可以是植物尿酸酶,例如源自大豆(Glycine max)根瘤的天然或重组尿酸酶。所述PEG平均分子量可以是约5kDa-100kDa;优选所述PEG平均分子量约8kDa-60kDa;更优选所述PEG平均分子量约10kDa-40kDa,例如10-20kDa。共价连接的PEG链平均数可以是2-12条/尿酸酶亚单位;优选共价连接链平均数可以是6-10条/亚单位;更优选PEG链平均数可以是7-9条/亚单位。在本实施方案的一个方面,所述尿酸酶可以是四聚体。所述PEG链可以通过氨基甲酸乙酯(氨基甲酸酯)键、仲胺键和/或酰胺键与尿酸酶共价连接。当所述尿酸酶为重组形式的本文所述任何一种尿酸酶时,所述重组形式尿酸酶可以基本上具有天然形式尿酸酶序列。
一种优选哺乳动物尿酸酶为重组猪-狒狒嵌合体尿酸酶,由猪肝脏尿酸酶部分序列和狒狒肝脏尿酸酶部分序列组成,两部分均由Wu等(1989)首次测定。所述嵌合体尿酸酶的一个实例包含猪尿酸酶序列(SEQ ID NO:1)的前288个氨基酸及狒狒尿酸酶序列(SEQ ID NO:2)的最后16个氨基酸。Hershfield等,国际公布WO 00/08196,尿酸氧化酶,2000年2月17日公布。因为后一序列只在两个位点上不同于猪尿酸酶序列,赖氨酸(K)代替291位残基精氨酸,丝氨酸(S)代替301位残基苏氨酸,这种突变蛋白称为猪-K-S尿酸酶或PKS尿酸酶(SEQID NO:3)。PKS尿酸酶还具有一个赖氨酸残基,因此比猪或狒狒尿酸酶序列多一个潜在的PEG化位点。
亚克隆包括PKS尿酸酶在内的各种哺乳动物尿酸酶的cDNA,并用标准方法确定在大肠杆菌中表达的最佳条件。参见Erlich,HA,(Ed.)(1989)PCR Technology.Principles and Applications for DNAAmplification.New York:Stockton Press;Sambrook等(1989)MolecularCloning.A Laboratory Manual,第二版,Cold Spring Harbor,NY:ColdSpring Harbor Laboratory Press。提取并纯化所述重组尿酸酶,使用改进的标准测定法测定其稳定性及活性。参见Fridovich,I,(1965)JBiol Chem 240:2491-2494;Nishimura等(1979)及实施例1和5。
在本发明的一个实施方案中,可通过生物学上稳定的无毒性共价键将尿酸酶与较少数目的PEG链缀合。所述共价键可包括氨基甲酸乙酯(氨基甲酸酯)键、仲胺键以及酰胺键。适于这类缀合的各种活化PEG可购自Shearwater Polymers,Hunsville,AL.。
例如将尿酸酶在PEG的碳酸琥珀酰亚胺酯(SC)衍生物或碳酸对硝基苯酯(NPC)衍生物存在下温育可形成与尿酸酶连接的氨基甲酸乙酯键。SC-PEG可用美国专利5,612,460介绍的方法合成。NPC-PEG可按照Veronese,FM等(1985)Appl Biochem Biotechnol 11:141-152及美国专利5,286,637中介绍的方法通过PEG与氯甲酸对硝基苯酯反应而合成。可通过调节反应物的浓度保持类似的化学计量,使美国专利5,286,637介绍的方法适合于高分子量PEG。Büttner,W等,东德专利说明书DD 279 486A1介绍了合成NPC-PEG的替代方法。
使用PEG羧酸衍生物的N-羟基琥珀酰亚胺酯(ShearwaterPolymers)可获得与尿酸酶连接的酰胺键。可使用2,2,2-三氟乙烷磺酰PEG(tresyl PEG;Shearwater Polymer)或使用PEG醛(ShearwaterPolymers)和氰基硼氢化钠通过还原性烷化形成仲胺键。
在含分子量为10kDa PEG的缀合物中,哺乳动物尿酸酶(如PKC尿酸酶,一种猪尿酸酶的突变蛋白;参见实施例5中的分析条件)每个亚单位连接PEG链的最大数目为约12条,同时保留未修饰尿酸酶至少75%的尿酸分解活性。后者PEG化程度相当于总氨基的约40%。本发明的一个实施方案中,每个尿酸酶亚单位连接的PEG平均链数为约2-12条。在一个优选实施方案中,每个尿酸酶亚单位连接的PEG平均链数为约6-10条。在一个更优选的实施方案中,每个尿酸酶亚单位共价连接的PEG平均链数为约7-9条。在另一个实施方案中,用于连接反应的PEG分子量为约5kDa-30kDa,优选约10kDa-20kDa。
若干因素可能影响用于与给定型尿酸酶连接的PEG最佳分子量和最佳链数选择。通常,与较少数目的高分子量PEG链相比,减少或消除免疫原性而不明显丧失尿酸分解活性可能需要尿酸酶与较多数目的低分子量PEG链连接。同样,每种不同形式的尿酸酶在PEG链的大小和数目上可能具有不同的最佳选择。可使用本文所述方法容易地确定PEG链的最佳数目和最佳PEG分子量。
当用纯化的尿酸酶四聚体和八聚体(含四个或八个约35kDa的亚单位)制备哺乳动物尿酸酶的PEG缀合物时,其在小鼠中的免疫原性显著降低,相反,含大聚集体的尿酸酶制剂的PEG缀合物(参见图6)具有中等免疫原性而未修饰尿酸酶的免疫原性非常高。
天然和重组尿酸酶纯化制剂除含有四聚体(140-kDa)和八聚体(280kDa)形式的尿酸酶外,通常包含尿酸酶非常大的聚集体混合物。四聚体或八聚体形式尿酸酶制剂的百分率通常在约20%-95%范围内(参见图2-4)。尽管有证据显示几种其它蛋白的未PEG化聚集体具有高度免疫原性(参见例如Moore,WV等(1980)J Clin Endocrinol Metab51:691-697),但是以前的PEG-尿酸酶研究没有介绍限制聚集体含量的任何偿试,提示未考虑PEG修饰聚集体的潜在免疫原性。根据本发明的观测结果,似乎可以看出所述聚集体存在于以前用于合成PEG-尿酸酶的酶制剂中。它们的存在可能会导致制备非免疫原性缀合物更困难。还可以看出以前所观察到的PEG化尿酸酶尿酸分解活性大量丧失与所连接的高数目低分子量PEG链有关。另一方面,至少对于某些尿酸酶,例如PKS尿酸酶(猪尿酸酶的突变蛋白)及源自嗜热芽孢杆菌的尿酸酶,本文所述尿酸酶纯化和PEG化方法容许每个尿酸酶亚单位与多达12条PEG链共价连接而保留75%以上的尿酸分解活性。
在本发明的另一个优选实施方案中,在得到的基本上不含聚集体的尿酸酶制剂与PEG缀合之前,在pH为约9-10.5、优选10.2的条件下,通过离子交换色谱法(图1-3)或大小排阻性色谱法可去除所述尿酸酶的几乎所有大的聚集体。可通过任何大小相关性分析技术监测制备型柱各流分中尿酸酶分子量,所述分析技术包括例如HPLC、常规大小-排阻性色谱法、离心、光散射、在非变性缓冲液中的毛细管电泳或凝胶电泳。对于使用大小-排阻性色谱法分离的没有聚集体的尿酸酶,可将只含140-kD和280-kDa形式尿酸酶的流分汇集在一起,用于与PEG聚合。对于使用离子交换色谱法分离的四聚体和八聚体尿酸酶,可对离子交换柱的各流分进行大小方面的分析,以确定含大量四聚体或八聚体形式而不含通过光散射检测到的大聚集体的流分。因此在所述纯化产物中,不需要的在聚集体可仅仅占总尿酸酶的约1%或更少。
本文所述结果表明,即使充分PEG化,大于八聚体的PKS尿酸酶形式可刺激小鼠快速清除所述尿酸酶(图5)及具有一定的免疫原性(图6)。相比之下,由基本上不含大的聚集体(可通过光散射检测)的尿酸酶制备的缀合物可以在1周周期重复注射至少6次而清除速率较慢(图5)且没有敏感型酶联免疫测定可检测到的抗体形成(图6)。使用高度纯化的四聚体或八聚体尿酸酶进一步将本发明中改进的PEG缀合物与从前介绍的PEG-尿酸酶制剂区别开来。相比之下,以前一些研究者使用的尿酸酶制剂中所含的大量大的聚集体可能使尿酸酶与大量数目的PEG链连接,以抑制其免疫原性。因此所得缀合物的酶活性大大降低。
本发明PEG-尿酸酶缀合物可用于降低哺乳动物、优选人的体液和组织中的尿酸水平,因此可用于治疗与包括痛风、痛风结节、肾功能不全、器官移植以及恶性肿瘤在内的病症伴有的尿酸水平升高。PEG-尿酸酶缀合物可通过无数给药途径中任何一种途径注射给予尿酸水平升高的哺乳动物,所述途径包括静脉内、皮下、真皮内、肌肉内以及腹膜内途径。另外也可雾化吸入PEG-尿酸酶缀合物。参见Patton,JS,(1996)Adv Drug Delivery Rev 19:3-36及美国专利5,458,135。本发明PEG-尿酸酶的有效剂量取决于尿酸水平及患者个体的大小。本发明此方面的一个实施方案中,以约10μg-1g剂量用药学上可接受的赋形剂或稀释剂给予PEG-尿酸酶。在一个优选实施方案中,给药剂量为约100μg-500mg。更优选所述缀合的尿酸酶给药剂量为约1mg-100mg,例如5mg、20mg或50mg。所述实施方案的给药剂量的质量是指所述缀合物中蛋白质量。
包含PEG-尿酸酶的药用制剂可通过常规技术制备,例如参见Gennaro,AR(Ed.)(1990)Remington′s Pharmaceutical Science,18版,Easton,PA:Mack Publishing Co.。用于制备注射溶液剂的合适赋形剂包括例如磷酸缓冲盐水、乳酸化Ringer′s溶液、水、多元醇以及甘油。胃肠外注射的药用组合物包括药学上可接受的无菌水溶液剂或非水溶液剂、分散剂、悬浮剂或乳化剂以及使用之前复制成注射无菌溶液或分散液的无菌粉末。所述制剂可包含附加组分如防腐剂、增溶剂、稳定剂、湿润剂、乳化剂、缓冲剂、抗氧化剂以及稀释剂。
PEG-尿酸酶还可以作为控释组合物用于植入患者体内以持续控制体液尿酸水平升高。例如聚乳酸、聚乙醇酸、再生胶原蛋白、聚L-赖氨酸、藻酸钠、凝胶化树胶、壳聚糖、琼脂糖、多层脂质体以及许多其它常规贮库型制剂包括可用生物活性组合物配制的可生物腐蚀或可生物降解物质。所述物质当植入或注射入体内时,渐渐分解,将所述活性物质释放到周围组织。例如一种包囊化PEG-尿酸酶的方法包括美国专利5,653,974中公开的方法。本发明特别考虑了可生物腐蚀、可生物降解以及其它贮库型制剂的应用。用于传递PEG-尿酸酶的输注泵和基质包埋系统的应用也在本发明范围内。PEG-尿酸酶还可有利地包封在胶粒或脂质体中。脂质体包囊技术为本领域熟知的技术。参见例如Lasic,D等(Eds.)(1995)Stealth Liposmes.Boca Raton,FL;CRC Press。
本发明PEG-尿酸酶药用组合物降低尿酸盐性肾衰高危患者对血液透析的需求,所述肾衰患者为例如器官移植接受者(参见Venkataseshan,VS等(1990)Nephron 56:317-321及具有某些恶性肿瘤患者。对于大量结晶尿酸盐累积(痛风石)的患者,所述药用组合物比现有治疗方法更迅速地改善患者生活质量。
以下实施例说明以上公开内容的各个方面,决不能解释为限制本发明。这些实施例说明通过活化PEG(如碳酸对硝基苯酯衍生物)与猪尿酸酶突变蛋白连接制备的PEG-尿酸酶。下述实施例指导本领域技术人员生产至少保留未修饰尿酸酶约75%尿酸分解活性且非常适于长期用药、基本上无免疫原性的尿酸酶缀合物。
实施例1
尿酸酶的制备型离子交换色谱
在快速蛋白质液相色谱(FPLC)仪(Amersham Pharmacia,Piscataway,NJ)上进行制备型离子交换色谱。除所述样品以较低流速装载以外,用pH 10.3的50mM碳酸钠、0.1M氯化钠(缓冲A)至pH 10.3的50mM碳酸钠、0.6M氯化钠(缓冲液B)的递度以0.5ml/分钟的流速洗脱Mono Q柱(1×10cm,Amersham Pharmacia)。该技术用来分级分离25mL PKS尿酸酶溶液(pH 10.3)。PKS尿酸酶由Bio-TechnologyGeneral Limited(Rehovot,Israel)获得。后者为重组猪尿酸酶,其中一个赖氨酸残基(K)和一个丝氨酸(S)残基分别代替了原猪尿酸酶序列中一个精氨酸残基和一个苏氨酸残基(Lee等(1988)Science 239:1288-1291;Wu等(1989)Proc.Natl.Acad.Sci.U.S.A.86:9412-9416)。加样后,用100mL缓冲液A洗涤该柱。在31mL 0-26%的线性递度缓冲液B结束时开始洗脱出尿酸酶峰。大多数尿酸酶通过7mL含26%缓冲剂B的缓冲液等度洗脱。用89mL 26%-100%线性递度缓冲液B洗脱回收尿酸酶的其余部分。收集4ml或6ml流分。检测#4-11等分流分的尿酸酶及总蛋白(图1),并通过实施例2所述大小排阻性高效液相色谱(HPLC)进行分析(图2及图3)。#5-10流分的其余部分如实施例3所述与PEG连接。基于实施例2的分析结果,如图1所示,#5-6流分的PEG缀合物合并为“低盐合并物”,#7-10流分的PEG缀合物合并为“高盐合并物”。
实施例2
通过光散射和紫外吸光度监测尿酸酶大小排阻性色谱
在室温下用Superdex 200柱(1×30cm,Amersham PharmaciaBiotech)对未分级分离PKS尿酸酶及得自实施例1PKS尿酸酶的制备型Mono Q色谱分析的选定流分进行大小排阻性HPLC。使用WyattTechnologies(Santa Barbara,CA)的MiniDawn检测器通过在90度对入射光的散射分析得自Thermo Separtions HPLC(Sunnyvale,CA)的吸收监测器(UV 2000)的洗脱液。
图2-4所示结果表明尿酸酶亚单位的四聚体、八聚体以及更大聚集体的解析,以及在不同样品中检测到所述形式尿酸酶的不同信号比例。不同于与浓度成正比的吸收信号,光散射信号与浓度和光散射单位大小的乘积成正比。光散射检测器对非常少量的高度聚集尿酸酶的结果敏感度表明存在最大的聚集体,该聚集体在空隙容积或接近空隙容积(约7ml)时被洗脱出。
实施例3
PEG尿酸酶缀合物的合成
使用得自Shearwater Polymer(Huntsville,AL)的PEG的碳酸对硝基苯酯衍生物(NPC-PEG)使未分级分离的PKC尿酸酶(Bio-TechnologyGeneral Limited)及实施例1Mono Q柱流分尿酸酶与10-kDa PEG连接。若干报告(例如Veronese,FM等,(1985)Appl Biochem Biotechnol11:141-152;Kito,M等(1996)J Clin Biochem Nutr 21:101-111)中已介绍了使用氯甲酸苯酯由PEG制备NPC-PEG,包括本发明者在内的以前研究者已将NPC-PEG用于合成PEG-蛋白缀合物(例如Veronese等,见上文;Sherman,MR等,载于JM Harris等(Eds.)Poly(ethylene glycol)Chemistry and Biological Application(聚乙二醇化学及生物应用),ACSSymposium Series 680(第155-176页)Washington,DC:AmericanChemical Society)。根据Kunitani,M等(1991)J Chromatogr 588:125-137)介绍的方法,确定与每个尿酸酶亚单位连接的10-kDa PEG链数为6条。
实施例4
尿酸酶和PEG-尿酸酶的体内血清持续时间及免疫原性
在pH 7.4磷酸缓冲盐水(PBS)中,将按照实施例3方法制备的重组哺乳动物尿酸酶PEG缀合物调节为1mg蛋白/mL用于注射。冷冻样品并贮存以备分析或注射。样品温热至37℃持续达1小时,然后注射给予BALB/c雌性小鼠(8只/组)。各组小鼠在研究开始时平均体重为18-22g。
监测所有小鼠的体重并记录对实验注射产生的副反应或其它非健康表现。每次注射(每周注射一次,注射6次)后24小时,用氯胺酮麻醉小鼠,眶后取血100-200μL,当收集大量血液时放血处死小鼠。从于2-8℃凝固4-32小时的血液中制备血清。将血清贮存于-20℃。如实施例5所述分析血清测得尿酸分解活性,以及如实施例6所述分析血清中的抗尿酸酶抗体。
实施例5
注射PEG-尿酸酶的小鼠血清中PEG-尿酸酶的尿酸分解活性分析
在I.Fridovich方法(J Biol Chem.(1965)240:2491-2494)中使用微量板,用溶于pH 9.2的200mM硼酸钠的100μM尿酸作为底物进行基于紫外光吸收度(UV 分析)的活性分析,使用Molecular Devices(Sunnyvale,CA)的SpectraMAX 250微量板读取器于室温下对UV-透明底的96孔板(Costar,Corning,NY)监测292nm吸光度的下降,监测时间为15分钟。通过观察在10%-40%的底物被氧化期间所测吸光度的最大吸收坡度(每分钟毫吸光度单位)分析所述数据。此分析法获得的结果见图1和5。
根据注射后24和72小时获得血清的数据,第一次注射连接6条10-kDa PEG/亚单位(6×10kDa PEG PKS)的PKS尿酸酶的小鼠血清尿酸酶平均半衰期为29±4小时。
独立实验发现,在-20℃贮存期间注射PEG-尿酸酶小鼠血清中的可检测尿酸分解活性下降,在分析之前将血清于37℃温育4小时获得尿酸酶活性最大程度回收。图5表明,按照实施例3方法进行PEG化之前,如实施例通过Mono Q柱色谱法纯化PEG PKS尿酸酶时,每周重复性注射6×10-kDa PEG PKS尿酸酶后尿酸分解活性回收最多。注射由实施例1高盐脱出液合并物制备的缀合物后尿酸分解活性回收程度最高(见图1),所述脱出液合并物含有最少量的非常大的聚集体(见图3中7-10流分的光散射曲线)。用实施例1Mono Q柱的低盐脱出液合并物制备的缀合物获得中度回收,用未分级分离PKS尿酸酶制备的缀合物获得尿酸酶活性回收最差,所述未分级分离PKS尿酸酶含有最大量的非常大的聚集体(见图2)。在重复注射后,不论使用上述UV分析法或P.Fossati等(J.Clin Chem(1980)26:227-231)的改良比色法,以及不论是否在分析血清之前将其在37℃下温育,都观察到同样的血清相对活性回收顺序(高盐合并物>低盐合并物>未分级分离尿酸酶)。
实施例6
注射PEG-尿酸酶小鼠血清的酶联免疫吸附分析(ELISA)
用结合于96孔Immulon 2板(Dynex Technologies,VWR Scientific,San Francisco,CA)的猪尿酸酶进行非竞争性ELISA分析。第一抗血清得自注射尿酸酶或注射按照实施例3方法制备的6×10-kDa PEG缀合物的小鼠。如B.Porstmann等所述(J Clin.Chem.Clin.Biochem.(1981)19:435-440),第二抗体为偶联辣根过氧化物酶的山羊抗小鼠IgG(Calbiochem-Novabiochem #401 253,La Jolla,CA),底物为邻苯二胺二盐酸盐(Sigma P-9187,St.Louis,MO)。
图6说明非竞争性ELISA分析的结果。该结果证明用实施例1Mono Q柱的高盐洗出液按照实施例3方法合成的6×10-kDa PEGPKS尿酸酶(见图1)在连续6周每周接受注射的8只小鼠中没有任何一只产生可检测的免疫反应。有几只注射按照实施例3方法由未分级分离PKS尿酸酶制备的缀合物的小鼠表现低度但可检测的免疫反应。免疫反应发生率最高为注射按照实施例3方法从实施例1Mono Q柱的低盐脱出液合并物制备的缀合物的小鼠。
没有如实施例2所述的用于大小排阻性HPLC分析的光散射检测器的优势,最大聚集体形式而不是八聚体形式尿酸酶的存在与重复注射后BALB/c小鼠PEG-尿酸酶缀合物回收进行性下降有关(见实施例5观察结果(图5)),而免疫原性提高(见实施例6观察结果(图6)),这样的结果不明显。所述结果对于用作生产临床应用的PEG-尿酸酶的原料的尿酸酶规格具有重要意义。
虽然为了清楚理解本发明,借助于阐述和实施例在一定详细程度上介绍了上述发明,但是根据本发明指导本领域普通技术人员显而易见的是:在不背离本文介绍和要求保护的主题及范围内对本发明进行的某些改变和改进。
序列表
<110>Sherman,Merry R.
Saifer,Mark G.P.
Williams,L.David
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Claims (18)
1.一种通过减少大于八聚体的聚集体的量而纯化保留了尿酸分解活性的尿酸氧化酶(尿酸酶)的方法,所述方法包括:
(a)分级分离所述尿酸酶;
(b)检测含尿酸酶的流分中大于八聚体的聚集体;并且
(c)去除含所述大于八聚体的聚集体的流分。
2.权利要求1的方法,其中所述分级分离是用选自离子交换色谱法、大小排阻性色谱法和超滤的方法完成的。
3.权利要求1的方法,其中所述检测包括测量光散射。
4.纯化的用权利要求1的方法制备的尿酸氧化酶(尿酸酶)。
5.一种制备尿酸酶缀合物的方法,所述方法包括:
(a)将含有四聚体尿酸酶、八聚体尿酸酶和大于八聚体的尿酸酶聚集体的尿酸酶溶液加样到至少一个分离柱;
(b)从所述柱回收包含分离的四聚体和八聚体尿酸酶的一个或多个流分,其中所述一个或多个流分基本上不包含大于八聚体的尿酸酶聚集体;
(c)将所述尿酸酶与PEG缀合。
6.权利要求5的方法,其中所述尿酸酶溶液在pH为10.2的条件下加样至所述柱。
7.权利要求5的方法,其中所述分离柱选自离子交换柱和大小排阻柱。
8.权利要求5的方法,其进一步包括分析所述流分以确定至少一种选自以下的特性:存在所述四聚体和八聚体尿酸酶,以及不存在大于八聚体的聚集体。
9.权利要求8的方法,其中所述分析包括至少一种选自以下的分析:色谱、离心、光散射和电泳。
10.权利要求9的方法,其中所述色谱是高效液相色谱。
11.分离的用权利要求5的方法制备的尿酸氧化酶(尿酸酶)缀合物。
12.与聚乙二醇(PEG)缀合的纯化的尿酸酶和药学上可接受的载体在制备用于降低哺乳动物体液或组织中尿酸水平的药物中的用途,其中所述纯化的尿酸酶包含四聚体和八聚体形式的尿酸酶,并且所述尿酸酶基本上不包含大于八聚体形式的尿酸酶聚集体。
13.权利要求12的用途,其中所述哺乳动物是人。
14.权利要求12的用途,其中升高的尿酸水平与选自痛风、痛风结节、肾功能不全、器官移植以及恶性肿瘤的病症有关。
15.一种降低体液或组织尿酸水平的药用组合物,它包含与聚乙二醇(PEG)缀合的纯化的尿酸酶和药学上可接受的载体,其中所述纯化的尿酸酶包含四聚体和八聚体形式的尿酸酶,并且所述尿酸酶基本上不包含大于八聚体形式的尿酸酶聚集体。
16.权利要求15的药用组合物,其中所述组合物通过冷冻干燥实现稳定,复制时溶解。
17.权利要求16的药用组合物,其中所述组合物能够被复制以提供适于胃肠外给予的溶液。
18.一种包含与聚乙二醇(PEG)缀合的纯化的尿酸酶的药用组合物,其中每个尿酸酶亚单位与平均2至10条PEG链共价连接,其中PEG的平均分子量为约10kDa-30kDa,其中所述纯化的尿酸酶包含四聚体和八聚体形式的尿酸酶,并且所述尿酸酶基本上不包含大于八聚体形式的尿酸酶聚集体。
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