CN113563412A - 一种自由流等电聚焦电泳缓冲体系 - Google Patents
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Abstract
本发明提供了一种自由流等电聚焦电泳缓冲体系,包括:缓冲体系组分,所述缓冲体系组分设有9个,9个组分包括1个阳极电解液、1个阳极占位缓冲液、5个分离缓冲液和1个阴极占位缓冲液、1个阴极极电解液,所述缓冲体系组分中不含甲基纤维素等大分子物质,pH组成为5‑10.5,等电点在200‑10000us之间。本缓冲体系在异构体制备完成后可采用超滤浓缩管进行回收,回收率可以达到90%以上;本缓冲体系不仅灵敏度较高可以达到0.03pH单位,而且可以明显提高异构体的回收率,降低异构体的后续纯化时间。
Description
技术领域
本发明属自由流等电聚焦电泳技术领域,具体涉及一种自由流等电聚焦电泳缓冲体系。
背景技术
电荷异构体分析是生物治疗蛋白质的一项监管要求。这些大的异质分子在生产过程中历经多种酶促翻译后修饰,例如糖基化和赖氨酸切断。此外,在纯化和储存过程中可发生多种化学修饰,例如氧化或脱氨。自由流电泳是制备蛋白质药物电荷异构体的最佳方法。
自由流电泳(Free Flow Electrophoresis,FFE)是半制备型分离技术,具有可连续分离、多种分离模式、无固定支持介质和分离条件温和等优势,特别适用于生物材料的分离纯化和制备。自由流等电聚焦电泳(IEF-FFE)利用连续流动的两性电解质溶液,在分离室内的电场方向形成线性pH梯度。样品随溶液纵向流动,并在电场作用下发生横向偏转,达到其等电点处不再横向移动,被强制聚焦在某位置,不受扩散和对流的影响,仅在溶液流动方向移动。具有不同等电点的蛋白质或肽等良性物质可通过IEF-FFE达到分离。由于电荷异构体的产生会影响药物的活性,因此现在制备电荷异构体主要是进行活性的分析,但是采用IEF-FFE制备电荷异构体后的缓冲液中存在甲基纤维素或羟甲基丙基纤维素等大分子,这些分子不仅影响异构体的浓缩回收和后续的活性分析,现在常用的纯化异构体的方法是进行亲和层析,由于酸性和碱性异构体含量较少,在亲和层析过程中回收过低而造成实验失败。
发明内容
本发明的目的在于提供一种自由流等电聚焦电泳缓冲体系,以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:一种自由流等电聚焦电泳缓冲体系,包括:缓冲体系组分,所述缓冲体系组分设有9个,9个组分包括1个阳极电解液、1个阳极占位缓冲液、5个分离缓冲液和1个阴极占位缓冲液、1个阴极极电解液,所述缓冲体系组分中不含甲基纤维素等大分子物质,pH组成为5-10.5,等电点在200-10000us之间。
优选的,所述阳极电解液为100mM硫酸,400mM吡啶乙醇。
优选的,所述阳极占位缓冲液为100mM磷酸,400mM吡啶乙醇,50mM乙二酸。
优选的,5个分离缓冲液分别设为分离缓冲液Ⅰ、分离缓冲液Ⅱ、分离缓冲液Ⅲ,分离缓冲液Ⅳ和分离缓冲液Ⅴ,所述分离缓冲液Ⅰ为20M HIBA,20mMES,20mM HEPES,1.5%pharmalyte8-10.5,1%pharmalyte 5-8,0.5%pharmalyte 2.5-5;所述分离缓冲液Ⅱ为100mM牛磺酸,2%pharmalyte8-10.5,1%pharmalyte 5-8;所述分离缓冲液Ⅲ为1.5%pharmalyte8-10.5,0.1%pharmalyte 5-8;所述分离缓冲液Ⅳ为2%pharmalyte8-10.5,0.1%pharmalyte 5-8,50mM乙醇胺;所述分离缓冲液Ⅴ为300mM KOH,100mM乙醇胺。
优选的,所述阴极占位缓冲液为150mM KOH,50mM乙醇胺,400mM EACA。
优选的,所述阴极电解液为100mM NaOH,400mM EACA。
本发明的技术效果和优点:本缓冲体系在异构体制备完成后可采用超滤浓缩管进行回收,回收率可以达到90%以上;本缓冲体系不仅灵敏度较高可以达到0.03pH单位,而且可以明显提高异构体的回收率,降低异构体的后续纯化时间。
附图说明
图1为实施例中的420nm光吸收图;
图2为实施例中的等电点标准品分布图;
图3为实施例中样本的CIEF结果图;
图4为实施例中的420nm光吸收的吸收峰图。
具体实施方式
实施例
采用该自由流等电聚焦电泳缓冲体系和样本进行异构体制备,并采用等电点标准品对异构体进行定位,等电点标准品中含有4个颜色不同的等电点物质,等电点物质对应的等电点为:6.4,7.5,8.5,10.1。该自由流等电聚焦电泳缓冲体系的组成如下表所示:
制备结果如下所示:图1为420nm光吸收图(等电点标准品中的等电点物质在420nm处均有光吸收,可以通过仪器直接检测420nm处光吸收进行检测),不同等电点的等电点物质均匀聚焦在不同的分流孔内;图2中可以看到不同颜色的等电点物质均匀分散在不同的分流孔中。从图上可以看到最后两个marker的pH是8.5和10.1,在1.6个pH单位分散了54个孔,孔之间的pH相差0.0296个pH单位,因此灵敏度在0.03个pH内。
图3为样本的CIEF结果,可以验证原始样本有四个电荷异构体,采用本缓冲体系均可以制备出来,最下面的线为原始样本,sample1-9为制备的电荷异构体的结果。图4为420nm光吸收的吸收峰图,图上的吸收峰和原始样本的四个异构体可以一一对应。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (6)
1.一种自由流等电聚焦电泳缓冲体系,包括:缓冲体系组分,其特征在于:所述缓冲体系组分设有9个,9个组分包括1个阳极电解液、1个阳极占位缓冲液、5个分离缓冲液和1个阴极占位缓冲液、1个阴极极电解液,所述缓冲体系组分中不含甲基纤维素等大分子物质,pH组成为5-10.5,等电点在200-10000us之间。
2.根据权利要求1所述的一种自由流等电聚焦电泳缓冲体系,其特征在于:所述阳极电解液为100mM硫酸,400mM吡啶乙醇。
3.根据权利要求1所述的一种自由流等电聚焦电泳缓冲体系,其特征在于:所述阳极占位缓冲液为100mM磷酸,400mM吡啶乙醇,50mM乙二酸。
4.根据权利要求1所述的一种自由流等电聚焦电泳缓冲体系,其特征在于:5个分离缓冲液分别设为分离缓冲液Ⅰ、分离缓冲液Ⅱ、分离缓冲液Ⅲ,分离缓冲液Ⅳ和分离缓冲液Ⅴ,所述分离缓冲液Ⅰ为20M HIBA,20mMES,20mM HEPES,1.5%pharmalyte8-10.5,1%pharmalyte 5-8,0.5%pharmalyte 2.5-5;所述分离缓冲液Ⅱ为100mM牛磺酸,2%pharmalyte8-10.5,1%pharmalyte 5-8;所述分离缓冲液Ⅲ为1.5%pharmalyte8-10.5,0.1%pharmalyte 5-8;所述分离缓冲液Ⅳ为2%pharmalyte8-10.5,0.1%pharmalyte 5-8,50mM乙醇胺;所述分离缓冲液Ⅴ为300mM KOH,100mM乙醇胺。
5.根据权利要求1所述的一种自由流等电聚焦电泳缓冲体系,其特征在于:所述阴极占位缓冲液为150mM KOH,50mM乙醇胺,400mM EACA。
6.根据权利要求1所述的一种自由流等电聚焦电泳缓冲体系,其特征在于:所述阴极电解液为100mM NaOH,400mM EACA。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100252435A1 (en) * | 2006-06-20 | 2010-10-07 | Gerhard Weber | Method and device for separation and depletion of certain proteins and particles using electrophoresis |
US20110005930A1 (en) * | 2007-06-20 | 2011-01-13 | Gerhard Weber | Ffe media and ffe methods comprising volatile separation media |
CN104056468A (zh) * | 2014-06-25 | 2014-09-24 | 上海交通大学 | 无需两性电解质的分离两性物质的电聚焦方法 |
CN104945466A (zh) * | 2014-03-28 | 2015-09-30 | 深圳华大基因科技有限公司 | 一种用于自由流电泳的缓冲液 |
CN105241728A (zh) * | 2015-09-28 | 2016-01-13 | 华南理工大学 | 一种人糖化血红蛋白的制备方法 |
CN106814122A (zh) * | 2015-11-30 | 2017-06-09 | 成都金凯生物技术有限公司 | 一种毛细管等电聚焦测定重组蛋白质等电点的改进方法 |
CN112986368A (zh) * | 2021-02-18 | 2021-06-18 | 华润昂德生物药业有限公司 | 聚乙二醇化重组人促红素电荷异质性的检测方法 |
-
2021
- 2021-06-23 CN CN202110696211.3A patent/CN113563412B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100252435A1 (en) * | 2006-06-20 | 2010-10-07 | Gerhard Weber | Method and device for separation and depletion of certain proteins and particles using electrophoresis |
US20110005930A1 (en) * | 2007-06-20 | 2011-01-13 | Gerhard Weber | Ffe media and ffe methods comprising volatile separation media |
CN104945466A (zh) * | 2014-03-28 | 2015-09-30 | 深圳华大基因科技有限公司 | 一种用于自由流电泳的缓冲液 |
CN104056468A (zh) * | 2014-06-25 | 2014-09-24 | 上海交通大学 | 无需两性电解质的分离两性物质的电聚焦方法 |
CN105241728A (zh) * | 2015-09-28 | 2016-01-13 | 华南理工大学 | 一种人糖化血红蛋白的制备方法 |
CN106814122A (zh) * | 2015-11-30 | 2017-06-09 | 成都金凯生物技术有限公司 | 一种毛细管等电聚焦测定重组蛋白质等电点的改进方法 |
CN112986368A (zh) * | 2021-02-18 | 2021-06-18 | 华润昂德生物药业有限公司 | 聚乙二醇化重组人促红素电荷异质性的检测方法 |
Non-Patent Citations (3)
Title |
---|
BRIAN D HOSKEN等: "isolation and Characterization of Monoclonal Antibody Charge Variants by Free Flow Isoelectric Focusing", 《ANAL CHEM》, vol. 88, no. 11, pages 5662 - 5669 * |
JOHAN MALMSTROM等: "Optimized Peptide Separation and Identification for Mass Spectrometry Based Proteomics via Free-Flow Electrophoresis", 《JOURNAL OF PROTEOME RESEARCH》, vol. 5, no. 9, pages 2241 - 2249 * |
YONGHUI WANG等: "Free flow electrophoresis coupled with liquid chromatography–mass spectrometry for a proteomic study of the human cell line (K562/CR3)", 《JOURNAL OF CHROMATOGRAPHY A》, vol. 1053, no. 1, pages 269 - 278, XP004601194, DOI: 10.1016/j.chroma.2004.08.164 * |
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