使用中性毛细管单克隆抗体亚型的毛细管电泳分离
Summary
Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.
Abstract
Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.
Introduction
单克隆抗体（mAbs）是生物治疗性蛋白质与由于其充当针对多种慢性和变性疾病1容量越来越大的兴趣。像其他生物分子，单克隆抗体很容易在其生命周期的各个阶段要经历数理化修改（ 即从合成到最终产品）。这样的修饰包括，但不限于：脱酰胺，糖基化，氧化，环化，异构化，聚集和蛋白水解切割2。因此，需要能够解决固有亚型的分析技术来监测单抗异质性和稳定性，以便建立质量标准。
毛细管电泳（CE）是outinside充满背景电解质（BGE）一个狭窄的石英管（微米范围）进行高性能的分离技术。在一个电场的应用（高达30,000 V），带电荷的分子对朝带相反电荷（ 即电驱动的分离）的电极迁移。在CE使用高电压的允许快速分析和提高效率，这是优于传统的凝胶电泳。毛细管区带电泳（CZE）是在生物制药行业常规地用于产品质量评估3-9一个基于CE的技术。不像CE的其它模式（ 例如 ，毛细管凝胶电泳，毛细管等电聚焦）或基于色谱的方法，CZE可以不使用变性剂或固相接口来进行的，从而允许接近其天然状态10的mAb的内在非均质性的分析。单克隆抗体同种型的CZE分离覆盖有亲水性聚合物（中性毛细管）一个熔融石英毛细管的内部发生，并且是根据它们不同的电泳迁移率，这是由电荷，质量，尺寸和形状（或流体力学体积）11排除。单抗部分被检测到时，它们被动员并通过检测窗口，这是由紫外线（UV）吸收检测器在214nm 4感测到的。
成功实施此分析技术将取决于适当注意细节之前和在实验过程中。反之，就增加了成本和时间进行分析，最终导致恒定失败和挫折。
在这里，我们提出了一个一步一步的指导，由捷克通过解决方案和样品，CE系统，设置仪器方法，数据采集的制剂的制备的详细解释，进行单抗异质性的一个成功的分析，以及的处理。对于本教程的目的，重组完全人源抗肿瘤坏死因子α（抗TNFα）单克隆抗体作为蛋白质模式;但是，这个协议可以轻松定制审议短暂修改其他蛋白质的分析。一个dditionally，提出了若干建议，以减轻提出的潜在问题。鼓励读者将严格按照该协议，以接替将增加的可能性。
Protocol
Representative Results
Discussion
在本教程中，我们强调的正确实践的重要性，在进行捷克以增加的概率成功单克隆抗体的分析。然而，当CZE是在常规基础上使用的，出现不可避免12的问题。
为了达到最佳效果，遵循纳入整个协议的音符，因为它们将有助于克服和解决困难的步骤分析师是很重要的。一个主要考虑在给定的条件，以获得最佳分辨率是BGE解决方案的正确准备。这个过程需要具有以上缓冲?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Wiley for the granted permission to use the concepts of the following publication for this tutorial. Carlos E. Espinosa-de la Garza, Francisco C. Perdomo-Abúndez, Jesús Padilla-Calderón, Jaime M. Uribe-Wiechers, Néstor O. Pérez, Luis F. Flores-Ortiz, Emilio Medina-Rivero: Analysis of recombinant monoclonal antibodies by capillary zone electrophoresis. Electrophoresis. 2013. 34. 1133-1140. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. This work was supported by CONACyT, Mexico, grant 230551.
Materials
| Glacial acetic acid | Tecsiquim | AT0035-7 |
| ACS grade hydrochloric acid | J.T. Baker | 9535-05 |
| Histamine dihydrochloride | Fluka | 53300 |
| (Hydroxypropyl) methyl cellulose | Fluka | 09963 |
| Lithium acetate | Sigma-Aldrich | 517992 |
| 6-Aminocaproic acid | Sigma-Aldrich | A2504 |
| eCAP Tris Buffer, 50.0 mM, pH 8 | Beckman Coulter | 477427 |
| PA 800 Plus Pharmaceutical Analysis System | Beckman Coulter | A66528 |
| eCAP Neutral capillary | Beckman Coulter | 477441 |
| Vial, Micro, 200 µl | Beckman Coulter | 144709 |
| Universal Vial Caps | Beckman Coulter | A62250 |
| Universal Vials | Beckman Coulter | A62251 |
| Cable, Optics, UV/Vis | Beckman Coulter | 144093 |
| UV/Vis Detector Module | Beckman Coulter | 144733 |
| Cartridge Assembly Kit, Blank | Beckman Coulter | 144738 |
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