CN101262919A - 抗凝血酶iii的亲合色谱法 - Google Patents
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Abstract
本发明的目的是亲合色谱柱，它包含与固体载体连接的蛋白ATIII，其特征在于：蛋白ATIII是使用未改性的并富含活性种的低分子量肝素通过孵化进行预先激活，按照低于每ml水合树脂约2mg蛋白的比率，让蛋白ATIII以共价方式与树脂连接。
Description
本发明的目的是亲合色谱柱，它包含与固体载体连接的ATIII蛋白。
这些肝素，即动物源的硫酸化粘多糖混合物，是糖胺聚糖类的生物活性剂，它们具有特别有用的抗凝血性质。它们是由因其尺寸而非常异类的硫酸化多糖直链构成的。一些肝素的平均重量是约15000Da(来源：猪粘液)。
将肝素长多糖链截断成低分子量的更短链可以制备低分子量肝素(HBPM)和非常低分子量肝素(HTBPM)。因此，HBPM和HTBPM应该理解是其分子量分别为3000-6500Da和1500-3000Da的链。
抗凝血酶III(ATIII)(Chandra等人，1983，《(Proc.Natl.Acad.Sci.U.S.A.》，80：1845-1848)是一种特定的蛇根平，它对控制凝固的丝氨酸蛋白酶有低抑制活性。这种作用肝素存在下明显得到增强，该肝素连接并且激活ATIII。特别地，与肝素的连接引起在蛋白质中一组构象的变化，该变化与采纳非常有利于与靶丝氨酸蛋白酶相互作用的构象同时达到最高程度。ATIII是在激活构象中时，与肝素分子或启动构象变化的衍生分子的相互作用是明显得到增强。
肝素与ATIII的相互作用是由于特定的五糖序列。然而，仅仅三分之一多糖段具有能与ATIII的相互稳定作用的特定序列。因此，肝素与衍生物的制备在涉及对ATIII亲合性方面是异质的。重要的是能够考虑富集对ATIII亲合种的低聚糖群，这样一种富集能显著提高这个群的抗凝血活性。
在
等人(1976，FEBS Lett，66：90-93)中描述过分离肝素亲合部分和非亲合部分的ATIII亲合色谱法。曾发表了其它方法，但是再次采用
等人方法的基本点(Hopwood等人，1976，《FEBS Lett.》，69：51-54；Denton等人，1981，《Anal.Biol.》，118：388-391；Pixley& Danishefsky，1982，《Thromb.Res.》，26：129-133)。根据这种方法，在乙酰化肝素的存在下，将ATIII接枝在用CNBr活化的Sepharose B树脂上。使用肝素的目的是防止在与肝素键合位点处都接枝ATIII。
这种技术有两个主要制约因素。
首先，连接ATIII所使用的肝素是乙酰化肝素，以便避免有与乌洛托品残基NH2竞争的风险。然而肝素乙酰化作用表现在其亲合性严重降低，和因此其对键合位点的保护能力降低，从而能连接亲合种的ATIII分子数减少。
另一方面，从使用ATIII浓度来看(约7mg蛋白质/ml水合树脂)，纯化与ATIII亲合种时，在这些能与ATIII连接的多糖分子之间存在着严重的空间不便的风险。
本发明的目的是亲合色谱柱，它包含与固体载体连接的抗凝血酶蛋白III(ATIII)，其特征在于：
a.蛋白ATIII是野生蛋白或其变体，
b.使用未改性的并富含活性种的低分子量肝素(HBPM)通过孵化预先激活蛋白ATIII，
c.按照低于每ml水合树脂约2mg蛋白的比率，让蛋白ATIII以共价方式与树脂连接。
未改性的HBPM应该理解是在制备后未进行化学或酶改性的HBPM，特别地，它没有被乙酰化。富含活性种的HBPM应该理解是富含与ATIII亲合的多糖的HBPM。
根据本发明，树脂应该理解是在其上接枝ATIII的化学惰性大分子载体。这些载体特别地非限制性地包括琼脂糖珠、琼脂糖聚丙烯酰胺珠、多孔玻璃珠、聚乙烯或聚甲基丙烯酸酯珠，按照制造商指示或本技术领域的技术人员熟知的方法，其中特别是使用溴化氰与使用肼的接枝方法在该珠上接枝ATIII。当然也可以使用预活化树脂。
根据本发明非常特别有利的实施方式，使用的树脂是Sepharose，采用的接枝技术是使用溴化氰的接枝技术。
本发明的亲合色谱柱具有双重优点。
一方面，使用为保护ATIII键合位点而未改性的HBPM能够固定呈激活构象的蛋白。在这种构象中，ATIII对亲合种的亲合性比没有预先激活的通过伯胺连接的ATIII强得多，选择性也高得多。
另一方面，低浓度的ATIII允许与这些亲合种相互作用而没有在它们之间的空间不便。当寻求分离大尺寸的种时，本发明的这种性质对于是特别有意义的。
为了使所有这些ATIII分子激活，优选使用饱和量的非改性的富含亲合种的HBPM。
根据本发明有利的实施方式，在HBPM中连接ATIII的位点量与ATIII分子的量的比是约5-约15。更确切地，本发明的目的是如前面定义的亲合色谱柱，其特征在于这个比是约10。
非常特别地，本发明的目的是亲合色谱柱，其特征在于使用
如HBPM，以保护ATIII键合位点。
如前面所解释的，本发明的另外优点是对在这种树脂上可接枝蛋白的浓度起作用时能使ATIII有更好的可达性。
根据另一个有利的实施方式，本发明的目的是亲合色谱柱，其特征在于ATIII/树脂比是每ml树脂约0.5-约1.5mg蛋白。例如，对于使用溴化氰(δ)激活的Sepharose B类树脂，这相应于每克干树脂约1.75mg-5.25mg蛋白(按照供应商的数据，1g干树脂在水合后得到约3.5ml树脂)。
于是，本发明亲合色谱柱的容量与选择性比现有技术中描述的这些柱高得多。
存在这样一些情况，是可希望从亲合和非亲合种混合物中纯化ATIII亲合种。ATIII亲合种应该理解是能特定地与ATIII连接的任何分子。例如，可期望将一组肝素低聚糖富集成亲合种。例如提高对ATIII的多克隆抗体溶液效价也可能是有意义的。
本发明的亲合色谱柱因此可以用于从混合物中纯化ATIII亲合种。因此，本发明另一个目的是在含有ATIII亲合和非亲合种的试样中纯化ATIII亲合种的方法，所述方法包括：
a.把所述试样加到对ATIII蛋白亲合的色谱柱中，所述的柱用适当盐缓冲液进行预先平衡；
b.使用适当的洗涤盐缓冲液洗涤所述柱的非特定保留种，以及
c.使用适当的洗脱盐缓冲液洗脱所述柱特定保留种。
特别地，根据一个非常特别有利的实施方式，采用本发明方法纯化的亲合种是构成这些肝素及其衍生物的低聚糖。
洗脱不同种之后，例如可以使用分光光度计测量不同馏分的吸光度。采用的波长应适合于纯化种的性质。例如，如果纯化一些低聚糖，则采用的波长应是232nm；如果纯化蛋白质，则波长应是280nm。
然后，洗脱馏分的组成可以根据本技术领域的技术人员熟知的方法进行分析。因此，如果分析的馏分含有低聚糖，本技术领域的技术人员应能采用在现有技术中描述的分析方法：例如非限制性地，毛细管电泳、MALDI-TOF质量分光光度法、高效液相色谱法。本技术领域的技术人员也能采用生物试验，如Xa因子抑制试验。
实施例
下面实施例说明本发明而非限制本发明。
附图的图例
图1：采用ATIII亲合色谱法的
分离色谱图。
图2：采用ATIII亲合色谱法的
分离色谱图。曲线1表示往柱1(保护
)注射HBPM，曲线2表示往柱1(保护
)注射缓冲液，曲线3表示往柱2(保护肝素)注射HBPM，曲线4表示往柱2(保护肝素)注射缓冲液。
实施例1：柱的制备和使用
1.柱的制备
在2ml水ppi中重新组成10mg ATIII，然后再溶于在18ml偶合缓冲液(0.2M NaHCO3，0.5M NaCl，pH＝8)中得到最后浓度0.5mg/ml(8.6μM)。往该溶液添加34.4mg的
其最后浓度是1.4mg/ml(215μM)。
该溶液按照比率1∶2与按照制造商的指示制备的用CNBr(δ)激活Sepharose B树脂进行混合，然后在冷条件下轻微搅拌一整夜。然后将整个溶液转移到封闭缓冲液(tampon de blocage)中(体积对体积)，并搅拌16小时。
把该树脂倒入配备2个活塞的恒温XK16柱(Amersham)中。倾析(décantation)后，这种树脂在封闭缓冲液与洗涤缓冲液之间交替洗涤4-5次。这种洗涤后，该柱用10M Tris-HCl，pH＝7.4、3M NaCl的缓冲液充分冲洗，以除去与ATIII络合的
分子。
最后，这个柱用10mM Tris-HCl，pH＝7.4、0.4M NaCl的缓冲液进行平衡
2.采用亲合性色谱法将亲合种与 
分离
往这个柱注射1mg/ml
在10mM Tris-HCl，pH＝7.4、0.4MNaCl缓冲液中的溶液。该柱用同样缓冲液充分洗涤，然后使用10mM Tris-HCl，pH＝7.4、3MNaCl缓冲液进行洗脱。
测量一些馏分在232nm的吸光度时，使用二极管阵列UV检测器(HP1100)监测亲合种的行为。如图1所表明的，这些亲合种特定地被保留在这个柱上，并只是在3M NaCl存在下被洗脱。
实施例2：与其它亲合色谱柱比较
1.容量
保护剂除外，采用同样的接枝方案制备两个柱：
·柱1：接枝时用40mg保护。
·柱2：接枝时用120mg肝素保护。
通过平均分子量的不同(比约为3.4)证明保护时使用量的不同。
为了测定柱容量，在10mM Tris-HCl pH＝7.4，0.2M NaCl的缓冲溶液中以流量＝0.5ml/min将国际申请WO 02/08295实施例7所描述的制备的400μg HBPM注入到每个柱中。使用3M NaCl进行洗脱。
通过测量这些馏分在232nm的吸光度时，使用二极管阵列UV检测器(HP 1100)监测不同多糖种的分离。
在两个柱上观察到分离，但是分离曲线是不同的(图2)。特别地，在柱2(保护：肝素)上，未保留种的峰被严重拖尾。此外，柱2(保护：肝素)的洗脱量比柱1(保护：)的洗脱量似乎更低。
为了定量地证明这个定性结果，使用HP
软件根据下式借助面积进行的积分计算出保留种的百分数：
％保留种＝洗脱峰面积/(未保留种的峰面积+保留种的峰面积)
已知在使用HBPM六糖馏分中亲合种百分数估计是22-24％，通过改变注射量，大致确定其容量。该容量这时相应于HBPM的最大注射量，其中洗脱时得到22-24％的百分数。
因此发现，柱1(保护：enoxaparine)的容量是115.8μg，而柱2(保护：肝素)的容量是64μg。
选择性
在本文中选择性定义为从非亲合多糖种分离亲合多糖种时用于分辨的树脂的容量。
为了进行这些分析，注射了其量低于预先测定的容量的HBPM。
a.柱1(保护：)，
多次注射427μg HBPM后将亲合与非亲合馏分合并，再采用CLHPCTA-SAX(MOURIER等人，2004，《Anal.Biochem.》，332：299-313)进行分析。得到的亲合馏分是纯的和完全的，它不含有非亲合种，并且它具有全部亲合六糖。非亲合馏分完全补足缺失的亲合种。
为了证实结构分析结果，测定了在柱1上(保护：
)分离的亲合和非亲合馏分抗-Xa活性。
亲合馏分的抗-Xa活性等于818±10Ul/mg；为了比较，六糖ΔUA-(1→4)α-GlcNAc(6S)-(1→4)β-GlcA-(1→4)α-GlcNS(NS，3，6S)-(1→4)β-1dA2S-(1→4)α-GlcNS(NS，6S)的活性是约650-700Ul/mg。相反地，还完全不能检测在这些非亲合馏分中抗-Xa活性。
因此，对这些馏分进行的生物学分析是完全符合这些结构分析的。
b.柱2(保护：肝素)
进行了对柱2(保护：肝素)的类似研究。为了优化这种树脂的性能，然而，这些注射量相对于预先测定容量更低(60μg)。
该亲合馏分与用柱1(保护：
)得到的也不是一样的纯度。事实上观察到存在非亲合六糖，特别是非常硫酸化六糖，如ΔUA2S-(1→4)α-GlcNS(NS，6S)-(1→4)β-ldA2S-(1→4)α-GlcNS(NS，6S)-(1→4)β-ldA2S-(1→4)α-GlcNS(NS，6S)或ΔUA2S-(1→4)α-GlcNS(NS，6S)-(1→4)β-ldA2S-(1→4)α-GlcNS(NS，6S)-(1→4)β-GlcA-(1→4)α-GlcNS(NS，6S)，其量不可忽略不计。
此外，在非亲合馏分中找到亲合种，特别是主要亲合种：ΔUA-(1→4)α-GlcNAc(6S)-(1-→4)β-GIcA-(1→4)α-GlcNS(NS，3，6S)-(1→4)β-ldA2S-(1→4)α-GlcNS(NS，6S)。
亲合与非亲合馏分的抗-Xa活性效价证实这些结构分析。事实上亲合馏分具有抗-Xa活性，该活性仅仅为565±45Ul/mg。另外，观察到在该非亲合馏分中不可忽略不计的残留活性，约15Ul/mg。这个活性是在这种结构分析时证明在非亲合馏分中残留亲合种的体现。
因此，显然柱1(
)的选择性高于柱2(肝素)的选择性。
Claims (7)
1.亲合色谱柱，它包含与固体载体连接的抗凝血酶蛋白III(ATIII)，其特征在于：
a.蛋白ATIII是野生蛋白或其变体，
b.使用未改性的并富含活性种的低分子量肝素(HBPM)通过孵化预先激活蛋白ATIII，
c.按照低于每ml水合树脂约2mg蛋白的比率，让蛋白ATIII以共价方式与树脂连接。
2.如权利要求1所述的柱，其特征在于在HBPM中存在的ATIII连接位点量与ATIII分子的量的比是约5-约15。
3.如权利要求2所述的柱，其特征在于这个比是约10。
4.如权利要求1-3中任一项权利要求所述的柱，其特征在于HBPM是
5.如权利要求1所述的柱，其特征在于ATIII/树脂比率是每ml树脂有0.5-1.5mg的蛋白。
6.在含有ATIII亲合和非亲合种的试样中纯化ATIII亲合种的方法，所述方法包括：
a.把所述试样加到根据权利要求1所述的亲合色谱柱中，所述的柱在适当的盐缓冲液中进行预先平衡；
b.使用适当的洗涤盐缓冲液洗涤所述柱的非特定保留种，以及
c.使用适当的洗脱盐缓冲液洗脱所述柱特定保留种。
7.根据权利要求6所述的ATIII亲合种的纯化方法，其特征在于所述种是低聚糖。
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| FR0507986 | 2005-07-27 | ||
| FR0507986A FR2889078B1 (fr) | 2005-07-27 | 2005-07-27 | Nouvelle methode de chromatographie d'affinite d'antithrombine iii |
| PCT/FR2006/001820 WO2007012754A1 (fr) | 2005-07-27 | 2006-07-26 | Methode de chromatographie d' affinite d' antithrombine iii |
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| CN114958963A (zh) * | 2022-07-29 | 2022-08-30 | 深圳传世生物医疗有限公司 | 一种作用于凝血酶的抗凝药物检测试剂盒及其应用 |
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| AR085961A1 (es) | 2011-04-11 | 2013-11-06 | Sanofi Sa | Polisacaridos que poseen dos sitios de union a la antitrombina iii, metodo para prepararlos y composiciones farmaceuticas que los contienen |
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| ZA814216B (en) * | 1980-06-27 | 1982-07-28 | Akzo Nv | Porous inorganic support material coated with an organic stationary phase for use in chromatography and process for its preparation |
| EP0048898B1 (en) * | 1980-09-30 | 1985-04-17 | Bayer Ag | Method for the production of an antithrombin-heparin complex and pharmaceutical compositions containing the complex |
| SU1708400A1 (ru) * | 1988-06-06 | 1992-01-30 | Таджикский государственный университет им.В.И.Ленина | Способ получени сорбента дл гемосорбции |
| US5744457A (en) * | 1995-03-31 | 1998-04-28 | Hamilton Civic Hospitals Research Development Inc. | Compositions and methods for inhibiting thrombogenesis |
| CA2315143A1 (en) | 1997-10-23 | 1999-04-29 | Fujimori Kogyo Co., Ltd. | Method for refinement or method for elimination of thrombin substrate |
| JP2005013156A (ja) * | 2003-06-27 | 2005-01-20 | Fujimori Kogyo Co Ltd | 固定化物及びその再生方法 |
| FR2889078B1 (fr) | 2005-07-27 | 2007-11-02 | Aventis Pharma Sa | Nouvelle methode de chromatographie d'affinite d'antithrombine iii |
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| CN114958963A (zh) * | 2022-07-29 | 2022-08-30 | 深圳传世生物医疗有限公司 | 一种作用于凝血酶的抗凝药物检测试剂盒及其应用 |
| CN114958963B (zh) * | 2022-07-29 | 2023-09-05 | 深圳传世生物医疗有限公司 | 一种作用于凝血酶的抗凝药物检测试剂盒及其应用 |
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| IL188412A (en) | 2013-07-31 |
| US8608961B2 (en) | 2013-12-17 |
| FR2889078B1 (fr) | 2007-11-02 |
| KR20080031320A (ko) | 2008-04-08 |
| WO2007012754A1 (fr) | 2007-02-01 |
| PL1909938T3 (pl) | 2011-06-30 |
| PT1909938E (pt) | 2011-03-03 |
| JP4931923B2 (ja) | 2012-05-16 |
| EP1909938B1 (fr) | 2011-01-05 |
| DK1909938T3 (da) | 2011-04-26 |
| AU2006273918B2 (en) | 2011-04-07 |
| CA2616132A1 (fr) | 2007-02-01 |
| HK1124278A1 (en) | 2009-07-10 |
| IL188412A0 (en) | 2008-12-29 |
| CN101262919B (zh) | 2011-07-27 |
| JP2009503483A (ja) | 2009-01-29 |
| CY1118533T1 (el) | 2017-07-12 |
| SI1909938T1 (sl) | 2011-04-29 |
| KR101261662B1 (ko) | 2013-05-06 |
| US20080188650A1 (en) | 2008-08-07 |
| RU2008107317A (ru) | 2009-09-10 |
| AU2006273918A1 (en) | 2007-02-01 |
| ES2357220T3 (es) | 2011-04-20 |
| ATE494051T1 (de) | 2011-01-15 |
| RU2411982C2 (ru) | 2011-02-20 |
| BRPI0614669A2 (pt) | 2011-04-12 |
| DE602006019437D1 (de) | 2011-02-17 |
| FR2889078A1 (fr) | 2007-02-02 |
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