CN100548372C - α-1蛋白质抑制剂的制剂 - Google Patents
α-1蛋白质抑制剂的制剂 Download PDFInfo
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Abstract
本发明涉及用在pH低于6.0和低离子强度下溶液的阳离子色谱从生物流体包括血浆和血浆组分中提纯人α-1蛋白酶抑制剂(α-1 PI)的方法。阳离子色谱的优点在于：在这些条件下活性α-1 PI不与该阳离子柱结合而其它蛋白质包括变性的α-1 PI和白蛋白与该阳离子柱结合。结果，活性α-1 PI流过该色谱柱，而污染蛋白质留在柱上。α-1 PI的回收率高，且纯度的提高是惊人的。
Description
本申请是申请号为CN95108778.9母案的分案申请。该母案的申请日为1995年8月23日；发明名称为“用新的色谱分离条件纯化α-1蛋白酶抑制剂”。
技术领域
一般来讲，本发明涉及蛋白质的纯化方法，具体来讲，本发明涉及用阳离子交换在能使活性α-1 PI不与柱结合而污染蛋白质与柱结合的条件下纯化得自血浆部分的α-1蛋白酶抑制剂。
背景技术
α-1蛋白酶抑制剂(α-1 PI)是分子量为约55000道尔顿的糖蛋白。α-1 PI是蛋白酶例如胰蛋白酶、胰凝乳蛋白酶、胰弹性蛋白酶、皮肤胶原酶、血管紧张肽原酶、尿激酶和多形核淋巴细胞的蛋白酶的抑制剂。α-1 PI的现行治疗用途是抑制肺中的淋巴细胞弹性蛋白酶。该蛋白酶通过分解异种蛋白起作用。当α-1 PI存在的量不足以调节弹性蛋白酶的活性时，该弹性蛋白酶会破坏肺组织。此不平衡经过一定时间后会导致慢性肺组织损害和肺气肿。补给α-1 PI已成功地用于治疗这种肺气肿。
目前对α-1 PI的需求量超过了供给量。已将α-1 PI基团转移并在微生物、细胞株和绵羊中表达。但沿未制得令人满意的重组体产品。人血浆是治疗用α-1 PI的唯一被认可的来源。α-1 PI用于代替治疗并以正常基准长期给予病人。由于微量杂质能刺激病人的免疫反应，因此该产品的高纯度对成功的治疗是个关键。血浆-α-1 PI源是有限的，因此α-1 PI高收率的纯化方法是必要的。至今尚无可用的能获得高收率和高纯度的α-1 PI的实用的方法。
现已发表了多种由人血浆中提纯α-1 PI的方法。Bollen等人(美国专利4629567(1986))使用5个不同的色谱步骤从酵母、大肠杆菌和人血浆中提纯α-1 PI。这5个步骤包括DEAE离子交换、硫醇-二硫化物交换、肝素亲和、锋螯合色谱和氨基己基离子交换。未给出纯度和收率的数据。
Novika等人，Gematol.Transfuziol.34：46-50(1989)报道了制备血液制品和副产物的分离方法。他们采用了亲和、DEAE纤维素和凝胶过滤色谱。未给出纯度和收率数据。
Podiarene等人，Vopr.Med.Khim.35：96-99(1989)报道了用亲和层析以单克隆抗体，从人血浆中分离α-1 PI的一步法。α-1 PI活性增加61.1倍，收率为20％。
Burnouf等人，Vox.Sang.52，291-297(1987)以血浆上清液A(相当于Cohn部分II+III)为原料，使用DEAE色谱法和筛析色谱法制备α-1 PI，其纯度为80-90％(以SDS-PAGE)，纯度增大36倍。由上清液A的回收率为65-70％。
Hein等人，Eur.Respir.J.9：16s-20s(1990)提出了这样一个方法：用Cohn部分IV-1作原料，采取用聚乙二醇分级沉淀，随后在DEAE
上进行阴离子交换色谱。终产品的纯度为约60％，收率为45％。
Dubin等人，Prep.Biochem.20：63-70(1990)提出了两步色谱纯化法。先将α-1 PI、CI抑制剂、α-1抗胰凝乳蛋白酶和内α-1胰蛋白酶抑制剂(inter α-1
inhibitor)从Blue
中洗脱出来，然后将α-1 PI用凝胶过滤法纯化。未给出纯度和收率的数据。
Ballieux等人采用4-苯基丁胺新和层析、阳离子交换和最后的免疫亲和步骤从化脓性痰中提纯α-1 PI和蛋白酶3-复合体(Ballieux，B.E.等人，J.Immunol.Methods 159：63-70(1993).在该阳离子交换步骤中所用的缓冲液的pH为7.0。在所用的条件下，大多数痰蛋白质与树脂结合，而α-1 PI和蛋白酶-3能不结合通过。
Jordan等人，美国专利第4749783(1988)描述了在病毒灭活步骤后用亲和层析除去制剂中的无生物活性的蛋白质的方法。蛋白质的天然形式和变性形式之间进行分离的基础是天然蛋白质对亲和性树脂的生物活性，而不是天然的和变性的蛋白质之间的物理差异。
这些方法均未使用以强阳离子树脂在低
.低盐浓度和适中的蛋白质浓度下的流过色谱作纯化步骤。出乎意料的是，在例如这些条件下，仅活性α-1 PI流过柱子。该方法从色谱桂的回收率可达到90％，在使用两去阳离子交换柱后纯度可达约95％。本发明提供以高纯度和高收率大规模地从人血浆中提纯α-1 PI的改进的方法。
“活性α-1 PI”或“天然的α-1 PI”是指在体外弹性蛋白酶测定中能显示抑制弹性蛋白酶活性的α-1 PI。
“非活性α-1 PI”或“变性的α-1 PI”是指在体外弹性蛋白酶评定中对弹性蛋白酶和活性无影响的α-1 PI。
“高度纯化的”指含20％以下的污染蛋白质。
“基本上不含非活性α-1 PI”指非活性α-1 PI和含量低于10％。
“基本上不含非活性病毒”指由于已经认可的病毒灭活步骤处理(例如巴氏灭菌或化学处理)活性病毒的含置降低。一般来讲这是指模型病毒的效价降低至少约41ogi。
所有的电导率测定均在25℃进行。
发明内容
本发明是由含蛋白质水溶液中提纯α-1 PI的方法，即在足以能确保活性α-1 PI不与介质(或离子交换树脂)结合的pH、离子强度和蛋白质浓度的条件下，在阳离子交换色谱介质上进行流通色谱。在优选的实施方案中，该方法包括以下步骤：
(1)将蛋白质溶液渗析或透析滤过至离子强度为约≤10mmho/cm；
(2)将溶液pH调至约≤6.0；
(3)将蛋白质溶液调至约≤10mg蛋白质/ml；
(4)使溶液通过阳离子交换色谱树脂；和
(5)收集色谱的流过部分作为纯化的α-1 PI。
附图说明
图1是说明一般用于提纯按照本发明的α-1 PI的Cohn分级分离法、得自这些流分的蛋白质和两个原料(Cohn组分IV-1和Cohn流出液II和III)的流程图。
图2是显示我们的α-1 PI纯化方法的优选的步骤的流程图。
图3是说明与在先技术相比本申请中所述的方法在纯度和比活性方面的改进的棒状图。
具体实施方式
本发明是由人血浆中提纯α-1 PI的改进的方法。本方法涉及在pH≤6.0下在低离子强度缓冲液中进行阳离子交换色谱。收集流过部分，该部分含纯化和α-1 PI。该阳离子交换色谱对α-1 PI是特异性的，实际上可用作直接由Cohn组分IV-1悬浮液的两步提纯法：一个阳离子交换柱接着第二只阳离子交换柱。
该方法是通用的，阳离子色谱足以能处理包括Cohn组分流出液II+III、Cohn组分IV-1糊状物(目前铖选的原料)和纯化的α-1 PI在内的各种原料，并仍能制备基本上纯的产品。
作为大规模制备药用产品的可供选择的方案，可以增加多个附加步骤包括病毒的灭活以使收率最佳化、增进病毒的安全性并保证调节的一致性。这些步骤可以包括以下步骤，但不限于这些：
(1)在弱离子交换树脂(DEAE)上进行初色谱；
(2)在强阴离交换树脂(QAE)上进行初色谱；
(3)用干热法或巴氏灭菌法将溶液中的病毒灭活；
(4)将病毒排阻过滤以除去可能的病毒污染物；
(5)化学处理例如溶剂净化处理以将病毒灭活；和
(6)沉淀步骤以在阳离子色谱前部分纯化原料。
pH通过改变蛋白质上的带电基团而在离子交换色谱中起关键作用。它能改变蛋白质与色谱树脂的结合行为，或者由未结合的转变成结合的，或者与此相反。优选用在本发明中的强阳离子树脂配体是与树脂珠直接或通过短碳基链相连的磺酸根
。该配体在1-14的pH范围内带有负电荷。若在给定的pH下蛋白质的净有效表面电荷是负的，则该蛋白质将无阻滞地流过该柱。一般来讲，若该溶液的pH在其pI以上，则该蛋白质具有负电荷。
在人血浆中的两个很特别的蛋白质是α-1 PI和白蛋白，其平均pI分别为4.8和5.3。这两种蛋白质在pH5.45均应带负电并应流过阳离子交换柱。出人意料的是，这些实验显示，在低盐浓度和pH5.45的条件下，白蛋白和变性的(或无活性的)α-1 PI与该树脂结合，而仅天然的(或活性的)α-1 PI流过该柱。对α-1 PI的这一观察结果提示在离子交换中，不仅蛋白质的p I而且其三级结构也是重要的。α-1PI的天然形式显然具有负性电荷表面，而变性形式的表面可能带有更多正电荷。因此，变性蛋白质意外地与阳离子树脂相结合。α-1 PI的天然形式在较高pH下要稳定得多。若考虑α-1 PI的稳定性和用离子交换色谱纯化α-1 PI，则pH5.45是实用的最低pH值。
离子交换树脂和蛋白质溶液之间的平衡受该溶液的离子强度、蛋白质浓度、pH和该树脂上的特定配体所影响。Yamamoto等人在Biote-chnol.Bioeng.25：1373-1391(1983)中提出了关于作为在低蛋白质浓度溶液的离子强度的函数的分布系数的半经验公式。该公式为：
K＝A(I)B+Kcrt
式中K代表分布系数，A和B代表经验常数，I代表溶液的离子强度，Kcrt代表在静电相互作用可被忽略的高离子强度下蛋白质的分布系数。在给定的离子强度下不同的蛋白质的分布系数不同。因此，不同的蛋白质在相同的条件下以不同的速度迁移。这适用于用该色谱法进行蛋白质的分离。在低盐浓度下，多数蛋白质的迁移速度由于在进样步骤中与树脂结合而减慢，天然的α-1 PI由于其独特的表面电荷性质通过该柱。若流过缓冲液的离子强度增大，则其它蛋白质与树脂的相互作用被改变，较大百分比例的蛋白质会流过该柱。因此，增加该溶液的离子强度会降低流过该柱的α-I PI的纯度。
盐浓度也能通过阳离子例如
与树脂上的氢离子进行可逆性交换而改变流出液的pH。增加盐浓度能使更多的
从树脂转移至流出液中，从而导致洗脱液pH降低。因此，当初始蛋白质溶液用平衡缓冲液超滤时，进样溶液的离子强度应等于或略高于平衡缓冲液的离子强度。于是pH在进样过程中应保持相同或仅略微降低，若在进样后将平衡缓冲液用作洗涤剂，则它可由于降低了离子强度而导致pH增大。因此，在该洗涤剂中可使用略高的离子强度缓冲剂以保持该pH。
如前所述，高蛋白质浓度也能影响蛋白质与树脂的结合平衡。当蛋白质浓度增高时，吸附等温线通表常表现出饱和曲线(Yamamoto等人，″Ion Exchange Chromatography of Protein 3″，1988，Chromat-ographic Science Series)。因此，当接近结合的饱和水平时，结合的杂质的实际量达最大。在达到最大后，通过该柱的杂质的相对百分数在样品的蛋白质浓度增加时会增大。因此，蛋白质浓度在低至足以在杂质的吸附曲线的线性范围内时最佳。
由于蛋白质往往能缓冲溶液的pH，因此蛋白质浓度还能影响流出液的pH。当将蛋白质通过与色谱介质结合从溶液中选择性去除时，该溶液的实际缓冲作用(即pH)被改变。对色谱法的影响是复杂的，因为pH变化将取决于被吸附到该柱上的蛋白质的相对百分数。吸附受柱的流通量、溶液的缓冲能力、蛋白质混合物的改变的性质和各种蛋白质的竞争性结合的影响。
我们的实验表明，在给阳离子交换柱进样时，用较高的蛋白质浓度给该阳离子交换柱进样，能导致流出液的pH逐渐增大。pH升高会导致α-1 PI的纯度降低。为使大规模纯化用的阳离子交换柱的载荷最大，可在杂质曲线和pH影响的可接受的范围内使用较高的浓度，尽管一般来讲较稀的蛋白质溶液是α-1 PI纯化色谱的较好的原料。
实施例1
优选的实施方案
在本发明的优选的实施方案中，用阴离子交换色谱从在加至强阳离子交换柱之前的Cohn组分IV-1(Fr.IV-1)悬浮液中部分提纯α-1 PI。根据比活性。Cohn组分IV-1悬浮液为约10％α-1 PI。IV-1悬浮液象其它血浆组分一样，含多种蛋白质：脂蛋白、免疫球蛋白、球蛋白、变性蛋白等。脂蛋白存在一个特殊问题。若它们与色谱树脂结合，则它们难以去除并能堵塞树脂孔，使整个树脂床的压力增大。而且，由于蛋白质残余累积在树脂上，结合能力丧失。为从Fr.IV-1悬浮液中除去脂蛋白和其它杂质，第一步采用DEAE离子交换色谱，而不用强阳离子色谱。DEAE荷载条件应为能使大部分脂蛋白通过该柱而不结合。DEAE离子交换色谱的α-1 PI洗脱条件基于获取α-1 PI的95-100％回收率来选择。
将IV-1悬浮液的电导率调至≥5mmho/cm，pH调至8.0。然后将该蛋白质溶液加在DEAE树脂上。用20mM磷酸氢二钠和95mM氯化钠在pH8.0洗脱α-1 PI。将DEAE流出液用20mM磷酸二氢钠和5mM氯化钠在pH6.5进行透析滤过。将经透析滤过的洗脱液调至pH5.45，然后将其加至强阳离子树脂上，α-1 PI流过该柱。将流出液调至pH7.0，并加入0.15M氯化钠。
若期望，可在此时将α-1 PI冷冻。对于病毒的灭活，将α-1 PI溶液融化，若有必要，在60mM组氨酸和5mM氯化钙中调至pH6.5，然后冻干，然后将冻干物于80℃加热72小时以将病毒灭活。然后将冻干物溶于净化水中，并加入37％(W/V)蔗糖和0.38M柠檬酸盐作稳定剂。加入0.3％磷酸三正丁酯(TNBP)和0.2％胆酸钠作为处理包膜病毒(enveloped viruses)的溶剂洗涤剂。于30℃培养3小时后，用透析滤过法除去TNBP和胆酸盐。
将已经病毒灭活处理的溶液用20mM磷酸二氢钠和5mM氯化钠在pH6.5进行透析滤过。将经透析滤过的溶液调至pH5.45，并加至第二强阳离子树脂中以除去任何剩余的污染物和由于病毒灭活步骤变性的α-1 PI。α-1 PI的天然形式流过该柱。将收集的流出液调至pH7.0，加入0.15M氯化钠，并使其通过0.15μM滤器作为补充的病毒灭活步骤。制得高纯度的基本上不含其它蛋白质的α-1 PI(＞95％)。DEAE色谱能除去脂蛋白，将α-1 PI的纯度增至20％。第一阳离子柱能制得约60-70％纯度的α-1 PI，同时回收率为约90％。第二阳离子柱能使终产品的纯度达到95％。将阳离子交换柱用1MNacl和1MNaOH洗涤以除去结合的蛋白质。将与两个阳离子交换柱结合的蛋白质依次用1M氯化钠和1M氯化钠和1M氢氧化钠除去。
实施例2
在本实施例中Cohn组分II+III流出液是原料。将其用20mM磷酸二氢钠和5mM氯化钠在pH6.5和5℃进行透析滤过以除去醇并降低离子强度。然后将该溶液调至pH5.45并加至预平衡的强阳离子交换柱中。收集流出液作为显著富集的α-1 PI部分。在该原料中的污染蛋白质被滞留在阳离子交换柱中。
在一次通过阳离子交换柱后。α-1 PI被纯化至基本上不含Cohn组分流出液II+III中的其它蛋白质。该流出液的纯度据SDS-PAGE为82％α-1 PI。比活性增大20倍，由每毫克总蛋白质0.03mg弹性蛋白酶抑制活性增至每毫克蛋白质0.59mg弹性蛋白酶抑制活性。
实施例3
本实施例使用部分纯化的可商购的α-1 PI(Prolastin，Miles，Inc.)作原料。将
用0.1MNaCl和0.02M磷酸钠在pH7.0缓冲。α-1 PI的浓度约为30mg/ml，蛋白质浓度为约60ml/ml。白蛋白通常为总蛋白质的12％。IgA的含量通常为约1mg/ml(总蛋白质的2.5％)。将
用5mM NaCl和20mM磷酸二氢钠透析滤过的降低离子强度。将溶液的pH降至5.45，蛋白质浓度降至5.3mg/ml，并使该溶液流过强阳离子交换柱。收集流出液作为纯化的α-1 PI在SDS-PAGE上仅显示出单体和二聚体α-1 PI，蛋白质分别为95.4％和4.6％。然后使该溶液于pH7.0在0.15M NaCl中稳定，并可被过滤或浓缩并冻干，得到最终的稳定的产品。
将结合的蛋白质流分洗脱，并在SDS-PAGE上进行电泳。用Coomassie Blue显色的蛋白质的44％是在α-1 PI的分子量范围内。该部分的弹性蛋白酶抑制作用测定表明无α-1 PI活性。这表明该带中的蛋白质是失活的α-1 PI。
对上述实施例的总结见下表。
以上描述了本发明，我们认为，蛋白质纯化领域的技术人员会对其做出变动。因此，以上实施例应仅被认为是对本发明的举例说明，本发明的范围应仅受附权利要求的限制。
Claims (2)
1.α-1蛋白酶抑制剂的制剂，基本上不含活性病毒，其中所述制剂含有少于10％的非活性α-1蛋白酶抑制剂，并且所述制剂的纯度大于90％，并且其活性为每mg总蛋白质至少0.9mg弹性蛋白酶抑制活性。
2.在药学上可接受的载体中的权利要求1的产品。
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| SE9600796D0 (sv) * | 1996-02-29 | 1996-02-29 | Pharmacia Biotech Ab | A method of preparing a liquid mixture |
| US5616693A (en) * | 1996-07-01 | 1997-04-01 | Alpha Therapeutic Corporation | Process for seperating alpha-1-proteinase inhibitor from COHN IV1 +1V4 paste |
| GB2318732A (en) | 1996-11-01 | 1998-05-06 | Johnson & Johnson Medical | Wound healing compositions containing alpha-1-antitrypsin |
| AT407114B (de) * | 1997-06-10 | 2000-12-27 | Immuno Ag | Alpha 1-antitrypsin-präparation sowie verfahren zu deren herstellung |
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| US6093804A (en) * | 1998-09-24 | 2000-07-25 | American National Red Cross | Method for purification of alpha-1 proteinase inhibitor |
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| DE1189674B (de) * | 1963-06-12 | 1965-03-25 | Behringwerke Ag | Verfahren zur Isolierung von alpha-Antitrypsin |
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| US5319072A (en) * | 1992-01-10 | 1994-06-07 | Alpha Therapeutic Corporation | Human antithrombin-III preparation |
-
1994
- 1994-08-24 US US08/295,119 patent/US5610285A/en not_active Expired - Lifetime
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1995
- 1995-08-11 ES ES95112630T patent/ES2139794T3/es not_active Expired - Lifetime
- 1995-08-11 PT PT95112630T patent/PT698615E/pt unknown
- 1995-08-11 DK DK95112630T patent/DK0698615T3/da active
- 1995-08-11 DE DE69513751T patent/DE69513751T2/de not_active Expired - Lifetime
- 1995-08-11 AT AT95112630T patent/ATE187462T1/de active
- 1995-08-11 EP EP95112630A patent/EP0698615B1/en not_active Expired - Lifetime
- 1995-08-14 CA CA002692337A patent/CA2692337A1/en not_active Abandoned
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- 1995-08-21 JP JP23322895A patent/JP3770414B2/ja not_active Expired - Lifetime
- 1995-08-23 KR KR1019950026050A patent/KR100451266B1/ko not_active IP Right Cessation
- 1995-08-23 CN CNB2004100694447A patent/CN100548372C/zh not_active Expired - Fee Related
- 1995-08-23 CN CNB951087789A patent/CN1161371C/zh not_active Expired - Lifetime
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2000
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| Publication number | Publication date |
|---|---|
| EP0698615B1 (en) | 1999-12-08 |
| ES2139794T3 (es) | 2000-02-16 |
| DE69513751D1 (de) | 2000-01-13 |
| CN1572321A (zh) | 2005-02-02 |
| HK1072372A1 (en) | 2005-08-26 |
| GR3032728T3 (en) | 2000-06-30 |
| PT698615E (pt) | 2000-05-31 |
| KR100451266B1 (ko) | 2004-12-23 |
| DK0698615T3 (da) | 2000-05-15 |
| CA2692337A1 (en) | 1996-02-25 |
| AU700638B2 (en) | 1999-01-07 |
| CA2156007A1 (en) | 1996-02-25 |
| EP0698615A1 (en) | 1996-02-28 |
| DE69513751T2 (de) | 2000-04-27 |
| CN1161371C (zh) | 2004-08-11 |
| JP3770414B2 (ja) | 2006-04-26 |
| AU3011195A (en) | 1996-03-07 |
| JPH08176198A (ja) | 1996-07-09 |
| ATE187462T1 (de) | 1999-12-15 |
| US5610285A (en) | 1997-03-11 |
| CA2156007C (en) | 2010-03-09 |
| KR960007617A (ko) | 1996-03-22 |
| CN1125231A (zh) | 1996-06-26 |
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