Name: capillary electrophoresis separation of monoclonal antibody isoforms using a neutral capillary
Uploaded: 2017-01-16T14:00:00.000+00:00
Duration: 14 min 53 s
Description: Watch this Scientific Journal Video about Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary at JoVE.com

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-Ab&#250;ndez, Jes&#250;s Padilla-Calder&#243;n, Jaime M. Uribe-Wiechers, N&#233;stor O. P&#233;rez, 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 &amp; Co. KGaA. This work was supported by CONACyT, Mexico, grant 230551.

1.溶液的制备<ol><li> 准备BGE解决方案。 <ol><li>制备0.05％组成的溶液100毫升（M / V）羟丙基甲基纤维素（HPMC），200mM的ε氨基正己酸（EACA）和30毫摩尔乙酸锂。 注:由于HPMC是一种粘弹性聚合物，倒出粉末放入玻璃烧杯中，加水80毫升，最后加入搅拌棒。继续添加其余试剂为常。处理醋酸锂的时候，因为它会导致眼睛不适戴安全防护眼镜。 </li><li>调节至pH值4.8±0.1，用50％（体积/体积）乙酸。使该溶液稳定5分钟，并根据需要进行调整。 </li><li>加水以弥补至100ml在容量瓶的最终体积。 </li><li>通过用0.2μm孔径的亲水性膜过滤。 </li><li>保存在2-8℃下7天。 </li></ol></li><li> 制备内标。 <ol><li>准备1毫升的一个的1％（M / V）组胺组成的溶液。 </li><li>通过用0.2μm孔径的亲水性膜过滤。 </li><li>保存在2-8℃下1天。 </li></ol></li></ol> 2.样品制备<ol><li>制备180微升用Tris缓冲液（50mM三（羟甲基）氨基甲烷，pH 8.0）中以1毫克/毫升的最终浓度稀释的mAb的样品。 </li><li>加入20μl的1％（M / V）组胺。 </li><li>混合，并在1000 xg离心离心5秒。 </li><li>放置一个微型瓶通用瓶内。 </li><li>样品输送到微瓶和盖子的普遍小瓶。为了避免引入任何气泡向上分配的样本。 </li><li>保存在2-8℃下1天。 </li><li>将通用瓶（含样品溶液微小瓶）在样品入口盘。 </li></ol> 3.准备CE系统。 <ol><li> CE系统的清洗。 注:次行为是过程每周至少一次，以避免从灰尘和碎屑积聚衍生电流泄漏。然而，根据不同的应用（ 例如 ，使用一个BGE的具有增加的粘度或高盐浓度，增加粘度的样品）电极和开口杆，可能需要更频繁地清洁，以防止交叉污染和样品残留。 <ol><li>关闭CE仪器的电源开关，打开前门。 </li><li>清洁样品盖的表面，样品保持系统（样品和缓冲液盘），墨盒盖，夹杆，接口模块和电极沾水擦拭无纺布。以重复该过程的乙醇蘸抹，使用前干燥。 </li><li>冲洗打开杆用清水彻底。清洁其表面用无纺布擦拭。与重复此过程乙醇浸湿擦拭，并在安装前干燥。 </li><li>清洁光缆小心的两端LY用蘸水的超细纤维布。重复该过程用乙醇湿布。 </li></ol></li><li> 盒组件。 <ol><li>从包装袋中取出一个新的中性毛细管（50微米内径）。 </li><li>大盘回落毛细管保护管到工作台的一端。解开，整顿和拉毛细管出管。 </li><li>粘一片带或纸到工作台和添加测量标记如下:长度到检测器（30厘米），毛细管窗口（0.3厘米）和长度，所述出口端（10厘米）（ 即 ，总40.2厘米长度30.0厘米有效长度）。 </li><li>对齐毛细管窗口以在参考纸0.2 cm度量标记。胶带固定毛细管和标记毛细管结束测量标志外2毫米。 </li><li>切在毛细管入口端在一个单一的直线运动用的裂解石的冲水边缘的保护帽（从窗口最远的一端）。<ol><li>浸入毛细管端插入，以避免到毛细管内涂层永久损坏充满水的封端普遍小瓶中。重复此过程的每个毛细管端被暴露于环境的时间超过1分钟的时间。 </li></ol></li><li>插入毛细管入口端插入暗盒的出口侧。 </li><li>推，并通过暗盒必要拉毛细管，直到毛细管窗口居中于盒窗口。 </li><li>插入孔插塞（100微米×200微米）到盒窗口。 注:确认当暴露于光的白色光源的白色光通过的窗口。如果通过通过的光具有褐色外观，根据需要调整。 </li><li>插入所述毛细管插入预形成的冷却剂管，用于为40.2厘米的总毛细管长度（预装油管螺母，垫圈和O形环在该顺序两端）。 </li><li>通过COOLAN推毛细管根据需要，直到毛细管出现在管的另一侧部T管材。 </li><li>将冷却剂管导入盒的出口侧的端部，并拧紧油管螺母。 </li><li>插入毛细管入口端插入暗盒的入口侧。 </li><li>推，并通过暗盒必要拉毛细管，直到毛细管出现在盒的入口侧。 </li><li>将冷却剂管导入盒的入口侧的端部，并拧紧油管螺母。 </li><li>切在在一个单一的直线移动所述毛细管出口端（从窗口最靠近端）在使用切割石材的冲水边缘的保护帽。 </li><li>将密封固定夹在毛细管两端并按下，使其完全到位。目视检查毛细管末端。如果不连，重复上述步骤。 </li><li>放置在工作台上的边缘毛细管长度的模板。 </li><li>将朝下AG墨盒ainst毛细管长度模板和对准毛细管模板上的参考线结束。如果毛细管测量标记（见3.2.4）不与基准线对齐，根据需要调整。 </li><li>靠住毛细管长度模板毛细管和使用的模板上的参考线下方的切割石头2mm左右齐平边缘切割毛细管的两端。 </li><li>目视检查毛细管用放大镜结束。如果毛细管末端不顺畅，使用软面切割石材的光泽他们。 </li><li>将光圈O型圈到使用O型圈插入工具孔塞孔。 </li><li>立即用插入到小瓶毛细管两端安装装满水到盒壳体和地点盒封万向小瓶入情况。对于短期和长期储存在保持2-8℃。 </li></ol></li><li> 缓冲盘的制备。 <ol><li>填写并放置封顶UNIVERS人小瓶在缓冲器入口和出口托盘（ 图1）。与样本的数目，根据在泳道2，3，4和5重复瓶位置进行分析，将一个车道每六个样品注射。 注:避免以防止电流泄漏润湿瓶盖。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/55082/55082fig1.jpg" /> 图1:位置和填写缓冲入口和出口托盘通用小瓶的水平。填充量:1/1 = 1400μL，3/4 = 1300μL和1/2 = 1000微升。缩写:W =水，盐酸= 0.1M盐酸。 <a href="http://ecsource.jove.com/files/ftp_upload/55082/55082fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="4"><li> CE系统组件装配。 <ol><li>安装UV检测器。 </li><li>安装打开杠杆按成接口块。 </li><li>安装光纤电缆。插入相应的结束夹栏，并按住两端，按顺时针方向旋转。另一端连接到UV检测器。 注意:小心使用光缆在此过程中，因为弯曲可能导致其断裂。 </li><li>地方样品和缓冲液盘插入样品保持系统和捕捉到的位置。 </li><li>将毛细管盒插入接口模块，并同时按下两端的夹杆，拧紧旋钮。 </li></ol></li></ol> 4.仪器方法建立<ol><li>电压，最大:使用以下参数和表1所述创建调理，运行和关闭的仪器方法30.0千伏;目前，最大:300.0μA;墨盒温度:20.0℃;样品储存温度:10.0℃; UV检测器的初始条件:波长:214纳米;数据速率:4赫兹;过滤器:正常;峰宽（分）:16-25;吸光度信号:直接。 注意:数据采集速率可以为了提高窄分离区的覆盖范围提高到25赫兹。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="8">调理方法时间程序</td></tr><tr><td> 时间（min） </td><td> 事件 </td><td> 值 </td><td> 为期 </td><td> 进瓶 </td><td> 出口小瓶 </td><td> 概要 </td><td> 注释 </td></tr><tr><td> - </td><td>冲洗 - 压力</td><td> 2068毫巴</td><td> 1.0分钟</td><td> BI:C1 </td><td> BO:C1 </td><td>前锋</td><td>盐酸0.1M的</td></tr><tr><td> - </td><td>冲洗 - 压力</td><td> 2068毫巴</td><td> 2.0分</td><td> BI:B1 </td><td> BO:C1 </td><td>前锋</td> &lt;TD&gt;水</td></tr><tr><td> - </td><td>冲洗 - 压力</td><td> 2068毫巴</td><td> 10.0分钟</td><td> BI:E1 </td><td> BO:C1 </td><td>前锋</td><td> BGE </td></tr><tr><td> 0.00 </td><td>独立 - 电压</td><td> 15.0 KV </td><td> 10.0分钟</td><td> BI:D1 </td><td> BO:D1 </td><td> 0.17敏斜坡，正极性</td><td>分离</td></tr><tr><td> 10.00 </td><td>冲洗 - 压力</td><td> 2758毫巴</td><td> 10.0分钟</td><td> BI:E1 </td><td> BO:C1 </td><td>前锋</td><td> BGE </td></tr><tr><td> 20.01 </td><td>等待</td><td> - </td><td> 0.0分</td><td> BI:A1 </td><td> BO:A1 </td><td> - </td><td>冲洗提示</td></tr><tr><td colspan="8">运行方法时间程序</td></tr><tr><td> 时间（min） </td><td> 事件 </td><td> 值 </td><td> 为期 </td><td> 进瓶 </td><tD&gt; 出口小瓶 </td><td> 概要 </td><td> 注释 </td></tr><tr><td> - </td><td>冲洗 - 压力</td><td> 2068毫巴</td><td> 1.0分钟</td><td> BI:C1 </td><td> BO:C1 </td><td>前进，输入/输出小瓶INC 6 </td><td>盐酸0.1M的</td></tr><tr><td> - </td><td>冲洗 - 压力</td><td> 2068毫巴</td><td> 2.0分</td><td> BI:B1 </td><td> BO:C1 </td><td>前进，输入/输出小瓶INC 6 </td><td>水</td></tr><tr><td> - </td><td>冲洗 - 压力</td><td> 2068毫巴</td><td> 4.0分</td><td> BI:E1 </td><td> BO:C1 </td><td>前进，输入/输出小瓶INC 6 </td><td> BGE </td></tr><tr><td> - </td><td>注射 - 压力</td><td> 34毫巴</td><td> 20.0秒</td><td> SI:A1 </td><td> BO:B1 </td><td>覆盖，前进</td><td>进样</td></tr><tr><td> - </td><td>等待</td><td> - </td><td> 0.4分</td><td> BI:A1 </td><td> BO:A1 </td><td>输入/输出小瓶INC 6 </td><td>清洗提示</td></tr><tr><td> 0 </td><td>独立 - 电压</td><td> 15.0 KV </td><td> 30.0分钟</td><td> BI:D1 </td><td> BO:D1 </td><td> 0.17敏斜坡，正极性，输入/输出小瓶INC 6 </td><td>样品分离</td></tr><tr><td> 0.5 </td><td>自动归零</td><td> - </td><td> - </td><td> - </td><td> - </td><td> - </td><td> - </td></tr><tr><td> 30.01 </td><td>停止数据</td><td> - </td><td> - </td><td> - </td><td> - </td><td> - </td><td> - </td></tr><tr><td> 30.02 </td><td>冲洗 - 压力</td><td> 2068毫巴</td><td> 2.0分</td><td> BI:B1 </td><td> BO:C1 </td><td>前进，输入/输出小瓶INC 6 </td><td>水</td></tr><tr><td> 32.03 </td><td>等待</td><td> - </td><td> 0.0分</td><td> BI:A1 </td><td> BO:A1 </td><td>输入/输出小瓶INC 6 </td><td>冲洗提示</td></tr><tr><td> 32.04 </td><td>结束</td><td> -</td><td> - </td><td> - </td><td> - </td><td> - </td><td>结束</td></tr><tr><td colspan="8">关机方法时间程序</td></tr><tr><td> 时间（min） </td><td> 事件 </td><td> 值 </td><td> 为期 </td><td> 进瓶 </td><td> 出口小瓶 </td><td> 概要 </td><td> 注释 </td></tr><tr><td> - </td><td>冲洗 - 压力</td><td> 2068毫巴</td><td> 1.0分钟</td><td> BI:E6 </td><td> BO:E6 </td><td>前锋</td><td>盐酸0.1M的</td></tr><tr><td> - </td><td>冲洗 - 压力</td><td> 2068毫巴</td><td> 6.0分</td><td> BI:F6 </td><td> BO:F6 </td><td>前锋</td><td>水</td></tr><tr><td> - </td><td>灯 - ​​关</td><td> - </td><td> - </td><td> - </td><td> - </td><td> - </td><td> - </td></tr><tr><td> - </td><td>等待</td><td> - </td><td> 0.0分</td><td> BI:A1 </td><td> BO:A1 </td><td> - </td><td>冲洗提示</td></tr></tbody></table> 表1:空调，运行和停机时间程序。 5.数据采集与处理<ol><li> 程序中的样本集。 <ol><li>程序使用调节仪器方法调理毛细管。当毛细管用于第一时间，节目四次重复;否则程序只有两个重复的调理方法。 </li><li>在正在运行的仪器方法的程序要分析的样本。 </li><li>编程为使用关断仪器方法存储毛细管，并重复第3.2.22所示的过程。 </li></ol></li><li>运行实验。 </li><li>导出电泳。 </li><li>计算迁移时间和基本的，主一个的百分含量第二酸性亚型使用电泳型材垂直落差线整合。 </li></ol>

<ol>
	<li>Bruno, V., Battaglia, G., Nicoletti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advent+of+monoclonal+antibodies+in+the+treatment+of+chronic+autoimmune+diseases.">The advent of monoclonal antibodies in the treatment of chronic autoimmune diseases.</a> Neurol. Sci. 31, 283-288 (2011).</li><li>Liu, H., Gaza-Bulseco, G., Faldu, D., Chumsae, C., Sun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+monoclonal+antibodies.">Heterogeneity of monoclonal antibodies.</a> J. Pharm. Sci. 97 (7), 2426-2447 (2008).</li><li>Creamer, J. S., Oborny, N. J., Lunte, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+analysis+of+therapeutic+proteins+by+capillary+and+microchip+electrophoresis.">Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis.</a> Anal. Methods. 6 (15), 5427-5449 (2014).</li><li>Fekete, S., Guillarme, D., Sandra, P., Sandra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatographic,+Electrophoretic,+and+Mass+Spectrometric+Methods+for+the+Analytical+Characterization+of+Protein+Biopharmaceuticals.">Chromatographic, Electrophoretic, and Mass Spectrometric Methods for the Analytical Characterization of Protein Biopharmaceuticals.</a> Anal. Chem. 88 (1), 480-507 (2016).</li><li>He, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+identity,+charge+variants,+and+disulfide+isomers+of+monoclonal+antibodies+with+capillary+zone+electrophoresis+in+an+uncoated+capillary+column.">Analysis of identity, charge variants, and disulfide isomers of monoclonal antibodies with capillary zone electrophoresis in an uncoated capillary column.</a> Anal. Chem. 82 (8), 3222-3230 (2010).</li><li>He, Y., Isele, C., Hu, W., Ruesch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+charge+variants+of+monoclonal+antibodies+with+capillary+zone+electrophoresis+in+dynamically+coated+fused-silica+capillary.">Rapid analysis of charge variants of monoclonal antibodies with capillary zone electrophoresis in dynamically coated fused-silica capillary.</a> J. Sep. Sci. 34 (5), 548-555 (2011).</li><li>Zhao, S. S., Chen, D. D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+capillary+electrophoresis+in+characterizing+recombinant+protein+therapeutics.">Applications of capillary electrophoresis in characterizing recombinant protein therapeutics.</a> Electrophoresis. 35 (1), 96-108 (2014).</li><li>&#352;t&#283;p&#225;nov&#225;, S., Ka&#353;i&#269;ka, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+impurities+and+counterions+of+pharmaceuticals+by+capillary+electromigration+methods.">Determination of impurities and counterions of pharmaceuticals by capillary electromigration methods.</a> J. Sep. Sci. 37 (15), 2039-2055 (2014).</li><li>&#352;t&#283;p&#225;nov&#225;, S., Ka&#353;i&#269;ka, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+applications+of+capillary+electromigration+methods+to+separation+and+analysis+of+proteins.">Recent applications of capillary electromigration methods to separation and analysis of proteins.</a> Anal. Chim. Acta. 933, 23-42 (2016).</li><li>Espinosa-de la Garza, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+recombinant+monoclonal+antibodies+by+capillary+zone+electrophoresis.">Analysis of recombinant monoclonal antibodies by capillary zone electrophoresis.</a> Electrophoresis. 34 (8), 1133-1140 (2013).</li><li>Staub, A., Guillarme, D., Schappler, J., Veuthey, J. L., Rudaz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intact+protein+analysis+in+the+biopharmaceutical+field.">Intact protein analysis in the biopharmaceutical field.</a> J. Pharm. Biomed. Anal. 55 (4), 810-822 (2011).</li><li>Altria, K. D., Altria, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troubleshooting.">Troubleshooting.</a> Methods in Molecular Biology, Vol 52. Capillary Electrophoresis Guidebook: Principles, Operation and Applications. , (1996).</li><li>Ma, S., Nashabeh, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein+therapeutics+by+capillary+electrophoresis.">Analysis of protein therapeutics by capillary electrophoresis.</a> Chromatographia. 53 (5), 75-89 (2001).</li><li>Jaccoulet, E., Smadja, C., Prognon, P., Taverna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+electrophoresis+for+rapid+identification+of+monoclonal+antibodies+for+routine+application+in+hospital.">Capillary electrophoresis for rapid identification of monoclonal antibodies for routine application in hospital.</a> Electrophoresis. 36 (17), 2050-2056 (2015).</li></ol>

The authors Carlos E. Espinosa-de la Garza, Rodolfo D. Salazar-Flores, N&#233;stor O. P&#233;rez, Luis F. Flores-Ortiz and Emilio Medina-Rivero are employees of Probiomed S.A. de C.V., which is developing, manufacturing and marketing biosimilar products. All authors are involved in the development of biosimilar products for Probiomed.

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?...

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。

<table><tbody><tr><td>Glacial acetic acid</td><td>Tecsiquim</td><td>AT0035-7</td><td></td></tr><tr><td>ACS grade hydrochloric acid</td><td>J.T. Baker</td><td>9535-05</td><td></td></tr><tr><td>Histamine dihydrochloride</td><td>Fluka</td><td>53300</td><td></td></tr><tr><td>(Hydroxypropyl) methyl cellulose&amp;nbsp;</td><td>Fluka</td><td>09963</td><td></td></tr><tr><td>Lithium acetate</td><td>Sigma-Aldrich</td><td>517992</td><td></td></tr><tr><td>6-Aminocaproic acid</td><td>Sigma-Aldrich</td><td>A2504</td><td></td></tr><tr><td>eCAP Tris Buffer, 50.0 mM,&amp;nbsp; pH 8</td><td>Beckman Coulter</td><td>477427</td><td></td></tr><tr><td>PA 800 Plus Pharmaceutical Analysis System</td><td>Beckman Coulter</td><td>A66528</td><td></td></tr><tr><td>eCAP Neutral capillary&amp;nbsp;</td><td>Beckman Coulter</td><td>477441</td><td></td></tr><tr><td>Vial, Micro, 200 &amp;micro;l</td><td>Beckman Coulter</td><td>144709</td><td></td></tr><tr><td>Universal Vial Caps</td><td>Beckman Coulter</td><td>A62250</td><td></td></tr><tr><td>Universal Vials</td><td>Beckman Coulter</td><td>A62251</td><td></td></tr><tr><td>Cable, Optics, UV/Vis</td><td>Beckman Coulter</td><td>144093</td><td></td></tr><tr><td>UV/Vis Detector Module</td><td>Beckman Coulter</td><td>144733</td><td></td></tr><tr><td>Cartridge Assembly Kit, Blank</td><td>Beckman Coulter</td><td>144738</td><td></td></tr></tbody></table>

capillary electrophoresis separation of monoclonal antibody isoforms using a neutral capillary

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.

图2示出一个200毫EACA，30mM乙锂的典型电电流分布，pH值4.8 BGE与用Tris缓冲液（50mM，pH 8.0）中稀释的抗TNFα单克隆抗体样品。如可以观察到的，当前是整个分析稳定和可以为30至35微安值之间振荡。 图3示出一个空白样品的CZE电泳，其中所检测的峰对应于组胺内标。预计对组胺为具有3.7至4.1分的迁移时间。 图4显示抗TNFα单克隆抗体的CZE电?...

Watch this Scientific Journal Video about Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary at JoVE.com

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.

使用中性毛细管单克隆抗体亚型的毛细管电泳分离

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The ...

capillary-electrophoresis-separation-monoclonal-antibody-isoforms

Research

JoVE Journal

Biochemistry

12.6K 次观看.  Unidad de Investigación y Desarrollo, Probiomed S.A. de C.V.. Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.

视频: Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

毛细管电聚焦法对蛋白质及其亚型的高灵敏度和定量检测

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

科研

生物化学

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography.&#160;While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process.&#160;Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.).&#160;Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment.&#160;To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available.&#160;To demonstrate this protocol, representative results are presented in which chimeric&#160;murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber.&#160;Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins.&#160;Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

使用蛋白质膜吸收器和流线型工作流的奇美人 Fc 表达蛋白质的隔离和特征

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

生物工程

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

免疫与感染

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

使用自动微生物反应器系统从中国仓鼠卵巢细胞中纯化和分析单克隆抗体

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

全自动毛细 Electrophoretic-based 免疫分析方法的程序和关键优化策略

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

生物学

Capillary Electrophoresis (CE)

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Capillary electrophoresis (CE) is a separation technique that separates molecules in an electric field according to size and charge. CE is performed in a small glass tube called a capillary that is filled with an electrolyte solution. Analytes are separated due to differences in electrophoretic mobility, which varies with charge, solvent viscosity, and size. Traditional electrophoresis in gels is limited in the amount of voltage that can be applied because Joule heating effects will ruin the gel and the separation. Capillaries have a large surface area-to-volume ratio and thus dissipate heat better. Therefore, the voltages applied for a capillary electrophoresis experiment are quite large, often 10,000&#8211;20,000 V.
Capillary electrophoresis is useful for high-performance separations. Compared to liquid chromatography, CE separations are often faster and more efficient. However, capillary electrophoresis works best to separate charged molecules, which is not a limitation of liquid chromatography. CE has a greater peak capacity than high-performance liquid chromatography (HPLC), meaning the separations are more efficient and more peaks can be detected. The instrumentation can be very simple. However, HPLC is more versatile and many stationary and mobile phases have been developed for different types of molecules.

Capillary Electrophoresis: Principle, Procedure and Applications

毛细管电泳 (CE)

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Capillary electrophoresis (CE) is a separation technique that separates molecules in an ...

教育

JoVE Science Education

化学

分析化学

An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies

Source: Elgundi, Z., et al. <a href="https://app.jove.com/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for purifying monoclonal antibodies from the culture supernatant obtained from an antibody-producing cell culture. The process involves using a matrix of crosslinked agarose beads with conjugated Protein A, which immobilizes the antibodies in the supernatant on the beads, allowing for effective separation from contaminants. By applying an elution buffer with low pH, the protein A-antibody interaction is loosened, facilitating the collection of antibody-containing fractions.

一种用于纯化单克隆抗体的亲和层析技术

1. Antibody Purification by Affinity Chromatography Using an Automated Fast Protein Liquid Chromatography (FPLC) System
NOTE: The following procedure can ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Production and Purification

DFE-Based Affinity Chromatography: A Technique for Purification of 2F5 Monoclonal Antibodies from a Crude Sample Using High Ionic Strength-Based Elution Conditions

Source: Kn&#246;dler, M. et al., <a href="https://www.jove.com/v/59933">Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study</a>. J. Vis. Exp. (2019).
This video describes an affinity chromatography technique for purifying 2F5 monoclonal antibodies using the DsRed-2F5-Epitope or DFE ligands. The purified 2F5 monoclonal antibodies are essential drugs in immunotherapy.

基于 DFE 的亲和层析:一种使用基于高离子强度的洗脱条件从粗样品中纯化 2F5 单克隆抗体的技术

Source: Kn&#246;dler, M. et al., Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case ...

Biological Techniques

Chromatography Techniques

A Capillary Electrophoresis Immunoassay for Protein Biomarker Detection

Source: Sage, J. M., et al. <a href="https://app.jove.com/v/60638">Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates a capillary electrophoresis immunoassay protocol for detecting a protein biomarker associated with amyotrophic lateral sclerosis (ALS) from human blood platelets. Confirmation of the presence of the ALS biomarker protein is achieved through size-based separation of the platelet proteins followed by chemiluminescent detection.

用于蛋白质生物标志物检测的毛细管电泳免疫测定

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of buffers and ...

Characterization and Functional Analysis

Electrophoresis: Overview

Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric field. The core strength of electrophoresis lies in its ability to separate high-molecular-weight species in complex mixtures. It has found widespread use in biochemistry, molecular biology, and analytical chemistry, allowing the separation of compounds like amino acids, nucleotides, carbohydrates, and proteins with excellent resolution.
There are two main formats for electrophoretic separations: slab electrophoresis and capillary electrophoresis. Slab Electrophoresis uses a gel matrix, such as agarose or polyacrylamide, which acts as a sieve, allowing molecules to move through the pores. Capillary electrophoresis employs a narrow capillary tube filled with a buffer solution, providing high-resolution separation with rapid analysis time and minimal sample requirements. Although slab electrophoresis is widely used in biochemistry and biology to separate high-molecular-mass species, capillary electrophoresis is a more recent development with several advantages.
Capillary electrophoresis is a separation technique used to analyze complex mixtures of analytes based on their differential migration rates in an applied electric field. In this method, a sample is injected into a narrow capillary tube filled with a buffered solution, and an electric field is applied to facilitate separation. The migration rate of the analyte depends on its charge and size. The smaller, more highly charged ions migrate faster than the larger, less charged ones. In capillary electrophoresis, the migration of sample components is influenced by two types of mobility: electrophoretic mobility and electroosmotic mobility. Electrophoretic mobility is the solute&#39;s response to the applied electric field, with cations moving toward the negatively charged cathode, anions moving toward the positively charged anode, and neutral species remaining stationary. Electroosmotic mobility occurs when the buffer solution moves through the capillary in response to the electric field, typically toward the cathode.
Under normal conditions, capillary electrophoresis separates cations first, followed by neutral species, and finally, the anions. This separation is based on their respective electrophoretic mobilities and electroosmotic flow velocities. Notably, all separated species are eluted from the same end of the capillary, allowing their detection using quantitative detectors. The detector signals produce an electropherogram resembling a chromatogram but feature narrower peaks.
Capillary electrophoresis is a powerful tool for qualitative and quantitative analysis of complex mixtures. It provides information similar to that obtained from other separation techniques, such as gas chromatography (GC) or high-performance liquid chromatography (HPLC).

电泳:概述

Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric ...