Name: laboratory scale production and purification of a therapeutic antibody
Uploaded: 2017-01-24T13:00:00.000+00:00
Duration: 9 min 54 s
Description: Watch this Scientific Journal Video about Laboratory Scale Production and Purification of a Therapeutic Antibody at JoVE.com

The research was supported by the University of Sydney. pVITRO1-Trastuzumab-IgG1/&#954; was a gift from Andrew Beavil (Addgene plasmid # 61883). We thank Tihomir S. Dodev for useful discussions regarding pVITRO1-Trastuzumab-IgG1/&#954;.

注:合适的哺乳动物表达载体，必须使用该协议。这里，使用含有两个表达盒的单个构建体（ 即重链和轻链表达由单独的启动子驱动）。曲妥单抗重和轻链先前克隆到载体中。这个载体是从安德鲁Beavil的礼物，通过一个不以营利为目的质粒库13获得。 1.恢复和载体DNA的规模化发展注:载体DNA收到大肠杆菌 XL-1蓝色株软琼脂穿刺培养;载体携带潮霉素抗性。 <ol><li>将无菌接种刺伤或循环进入穿刺培养，然后连胜75微克准备了卢里亚BERTANI（LB）琼脂平板上的软琼脂/ ml潮霉素的单菌落。孵育在37℃的板18-24小时。 </li><li>接种单个菌落到5ml了不起肉汤（TB）的含有75微克/毫升潮霉素。 INCU大怒的文化，在37℃，18〜24小时，225转摇晃。 </li><li>使用过夜培养轻轻用200μl80％甘油混和并冷冻在-80℃下混合800微升培养以制备载体DNA的甘油。 <ol><li>将100μl过夜培养物加至100毫升结核病含75微克/一个挡板摇瓶中（ 即 1/1000稀释）ml潮霉素。孵育在37℃下培养18至24小时，以225 rpm振摇。 </li></ol></li><li>过夜培养后，提取和纯化根据与以下异常的midi / MAXI制备试剂盒的制造商的说明将DNA;期间与水（pH 7.0-8.5）中的最后一步，洗脱或重悬的DNA。 </li><li>检查浓度与由吸光度读数的DNA纯度在260和280nm，然后储存的DNA在-20℃。 注:可选的（强烈推荐）:DNA序列使用载体特异性引物，以确认身份。 </li></ol> 2。 HEK-293细胞的稳定转染<ol><li>生长并保持根据在补充有在锥形瓶中的0.1％的非离子表面活性剂的无血清培养基的标准协议HEK-293细胞（悬浮细胞）。保持在2×10 5个细胞/ ml传代培养每第四天。培养细胞在37℃，5％的CO 2和120rpm的旋转。 注:细胞应已在培养不少于4天，不超过4周前，转染。生长在含有血清的培养基单层的HEK-293细胞也可以用于此过程。 </li><li>在转染前一天，种子的HEK-293细胞以3×10 5个细胞/在12孔平板的孔中毫升2-毫升Dulbecco氏改良的Eagle培养基（DMEM）中的补充有10％热灭活的胎牛血清（ FBS）。在转染日，检查细胞达到80-90％汇合</li><li>稀释转染媒体DNA和聚乙烯亚胺（PEI）分别然后混合在一起。<ol><li>稀释300微升转染媒体每孔1.25微克载体DNA（15微克的12口井）。在室温下孵育5分钟。 </li><li>稀释的1mg / ml的PEI溶液2.5微升（30μl的12口井， 即 1:PEI的2比的DNA）在300微升转染培养基。在室温下孵育5分钟。 </li><li>稀释的DNA添加至稀释的PEI，轻轻混合并在室温下孵育15分钟。 </li></ol></li><li>加入50μl的DNA / PEI混合物滴加至板的各孔中。轻轻摇动平板分发然后转染混合物放置板在37℃，5％CO 2的24小时。 </li><li>加50微克/ ml潮霉素，每孔B和返回板37℃，5％CO 2的10天。 注意:通过第10天，未转染的细胞将死亡和拆卸从板的表面上，而稳定地转染的细胞将是可行的，并附着到板上。 </li><li>在水井更换介质1/4体积的无血清培养基和3/4体积的DMEM补充有10％FBS（ 注:0.5 ml无血清培养基+ 1.5的10ml DMEM = 7.5％的最终血清浓度），并孵育4天。 </li><li>用补充有10％FBS（ 即 1毫升无血清培养基+ 1的10ml DMEM = 5％的最终血清浓度）1/2体积的无血清培养基和1/2体积的DMEM替换在孔中的媒体并孵育4天。 </li><li>用补充有10％FBS（ 即 1.5毫升无血清培养基+ 0.5的10ml DMEM = 2.5％的最终血清浓度）3/4体积的无血清培养基和1/4体积的DMEM替换在孔中的媒体并孵育4天。 </li><li>用2ml补充有0.1％的非离子表面活性剂（即0％的最终血清浓度）的无血清培养基的更换在孔中的媒体并孵育4天。 注:保持无血清适应过程中50μg/ ml的潮霉素B的选择性细胞上的压力。适应于无血清培养基的细胞从孔表面分离，并且可以第一组ñ停牌。参见步骤2.1培养条件与50微克的额外补充/ ml潮霉素B. </li><li>使用移液管，轻轻在12孔板和池细胞悬浮培养物混合到一个单独的管中。 <ol><li>使用血细胞计数器，枚举汇集细胞和种子在30毫升无血清培养基中一个锥形烧瓶中，在2×10 5个细胞/ ml。培养细胞在37℃，5％的CO 2和120rpm的旋转。亚文化，每第四天扩大。 </li></ol></li><li>继续扩大培养规模到所需细胞密度为1×10 7个细胞，以获得10小瓶/小瓶在液氮冷冻保存。冻结细胞在含有10％二甲基亚砜（DMSO）的无血清培养基。 注:空调含有抗体媒体可以在每个传代培养收获确认蛋白质生产或用于优化纯化条件。收获条件培养基和维护传代细胞，离心在文化300 XG 5分钟，然后用0.22微米的过滤器过滤消毒上清液。过滤的上清液可保持在4℃下1-2周或-20℃下长期储存。 </li></ol> 3.大规模抗体生产批量从长满文化注:抗体的生产可以从步骤2.11，其中现有的单元格扩展到基于批处理长满培养体积上所需的细胞密度是设置应遵循的。否则，已经从冷冻解冻细胞开始在该阶段，一旦扩大到合适的细胞密度和体积。各种培养补充剂可用于优化抗体生产;利用蛋白胨，增加抗体产量是表现在这个协议。 <ol><li>种子细胞在两个Erlenmeyer烧瓶的100ml无血清培养基2×10 5个细胞/ ml（以比较从非补充和营养补充培养抗体的产量）。文化在37 ℃，5％CO 2的</sUB&gt;和120转。 </li><li> 24小时后，加入胰蛋白胨为0.5％的终浓度向营养补充培养。均衡与无血清培养基未补充的文化音量。 </li><li>从第8天，进行使用血球监测细胞生存力每日培养物的细胞计数。收获培养上清一次细胞存活率小于80％。 <ol><li>收获上清液通过培养物离心以3000 xg离心15分钟，然后通过0.22微米的过滤器过滤，消毒上清液（含有抗体）。存储上清在4℃（短期），或在-20°C（长期）冻结。 </li></ol></li></ol> 4.抗体纯化通过亲和层析法使用自动化快速蛋白质液相色谱（FPLC）系统注:以下步骤一般可以适用于大多数的自动化系统。纯化可在室温下或在4℃下（如果FPLC系统被保持在阴凉室）中进行。可以进行一系列的侦察测试，以找出最佳纯化条件包括适当的列矩阵，结合缓冲液，洗脱缓冲液和pH值，以确保从条件培养基纯化的抗体的最大恢复（参见结果部分）。最优条件取决于被纯化的抗体或蛋白质。纯化了一个自动化FPLC系统上进行。纯化用5ml的蛋白A柱在室温下进行。 <ol><li>使用超纯水制备下列缓冲液和调整到推荐pH值，然后通过0.22微米的过滤器进行过滤。 <ol><li> 0.14 M氯化钠，0.0027米氯化钾，0.01M 的 Na 2 HPO 4和0.001摩尔KH 2 PO 4:通过混合下列制备1升磷酸盐缓冲盐水（PBS）pH 7.4的（结合缓冲液）的。过滤缓冲区之前必要时进行调整pH值。 </li><li>制成500毫升之0.1M甘氨酸 - 盐酸pH值2.7（洗脱缓冲液）的。过滤的buff前调节pH值呃。 </li><li>准备50ml的摩尔Tris pH值9.0（和缓冲液）的。 </li></ol></li><li>确保计算机是由系统电源和通讯连接。设备应在软件"系统控制"模块可见。确保UV单元被设定在280nm波长。可选的:如果连接，也可以使用校准pH计。 </li><li>沉浸A，B和采样泵的入口管超纯水冲洗系统。清除泵用注射器是否有空气管道或空气的系统中的一个怀疑。通过系统控制器或经由自动化的方法，通过手工操作用水冲洗该系统。观察到压力，导电性，UV280跟踪和pH保持一致，并且该列中的压力限制不超过根据制造商的规范。 注:异常可能表明空气或堵塞应该在继续之前加以解决系统。 </li><li>在系统控制器模块，从1开始的流速毫升/分钟通过手动在任何负载泵A（绕过样品环）或注射（通过样品环）的位置，开始连接列。在列位置入口断开系统管道和删除连接到柱入口塞子。 <ol><li>允许水从系统管滴流在塔的顶部则系统管道连接到柱上。具有柱的入口和与水滴入口配件溢流确保无气泡的连接。 </li></ol></li><li>连接柱子出口的下游出口，并确认所有的配件都紧紧系住。与列现在连接至系统，如在列规范概述不超过最大压力极限和流量限制。 </li><li>用的水5倍柱体积（CV）的洗涤塔。如果有必要，继续洗柱，直到UV280跟踪已趋于稳定。 </li><li>沉浸A的入口管结合缓冲液，B在洗脱缓冲液和结合缓冲器T采样泵Ø平衡系统在正确的缓冲区。使用PumpWash通过手动操作缓冲填充进气管。 </li><li>在软件的"方法编辑器"模块中，使用该方法的向导设置为，目的是要使用的柱的亲和色谱法。 注:自动化系统FPLC配方法预先填充推荐的设置（ 即流量和压力限制），并运行基于选定净化柱（制造商，矩阵和大小）的步骤。 <ol><li>使用蛋白质A亲和层析以下运行步骤: <ol><li>平衡系统用5CV结合缓冲液，并收集到流出液废物容器。 </li><li>负载样品上柱（体积要加载手动指定的方法），并收集流过到一个单独的容器中。 </li><li>洗系统用5CV的结合缓冲液，并收集流过到一个单独的容器中。 </li><li>用5CV isocrat柱洗脱IC分离使用洗脱缓冲液，并收集纯化抗体样品由馏分收集器部分。 </li><li>平衡系统用5CV结合缓冲液，并收集到流出液废物容器。 </li></ol></li></ol></li><li>一旦该方法已经设置，指定的条件培养基的体积要施加到列和保存方法。 注:在该方法中指定的体积应比实际体积，以避免样品运行期间引入空气少5-10毫升。在这个协议中指定的卷是90-120毫升。 </li><li>浸没样品泵管到含有经调节的培养基的容器中。 </li><li>准备一个单独的容器通过指定的出口管收集样品流出液。 注:收集在运行或柱的结合能力过程中发生错误的情况下的流过液是很重要的超过需要的流过液被重新应用到列或外净化epeated。 </li><li>在馏分收集器准备收集管。加入100微升每毫升体积分数和缓冲的。在FPLC系统将自动洗脱馏分进入收集管中。 </li><li>在软件的"系统控制"模块，打开方法来运行。 注:该方法运行在一系列的包括检查的方法中，馏分收集器安装程序的变量和定义的结果的文件名和存储位置的页面启动。 </li><li>点击START开始运行。运行可以在"系统控制"模块中进行监控。 </li><li>在净化运行完成后，检查所生成的色谱系统软件的"评估"模块中。 </li><li>将所有含蛋白质的分数到一个单独的管，缓冲交换和使用离心过滤装置与切断30 kDa的分子量在PBS集中。根据制造商的执行离心步骤说明。 </li><li>使用根据制造商的说明双金鸡宁酸测定法（BCA）测定抗体浓度。 </li><li>如果另一个净化运行是要执行，主要在结合缓冲液，并重复采样泵管步骤4.9-4.14。 </li><li>在纯化轮次的完成，沉浸A，B和样品泵的入口管在超纯水中，并如在步骤3中进行清洗系统和柱。 </li><li>淹没A，B和泵管在20％乙醇，重复洗涤程序对系统和列存储。 </li><li>断开从下游出口列和替换柱塞，然后在入口断开柱和更换塞子，重新连接管连接到该系统。储存在4℃下的列。 </li></ol>

<ol>
	<li>Nelson, A. L., Reichert, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+trends+for+therapeutic+antibody+fragments.">Development trends for therapeutic antibody fragments.</a> Nat Biotechnol. 27 (4), 331-337 (2009).</li><li>Drake, P. M., Rabuka, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+emerging+playbook+for+antibody-drug+conjugates:+lessons+from+the+laboratory+and+clinic+suggest+a+strategy+for+improving+efficacy+and+safety.">An emerging playbook for antibody-drug conjugates: lessons from the laboratory and clinic suggest a strategy for improving efficacy and safety.</a> Curr Opin Chem Biol. 28, 174-180 (2015).</li><li>Kontermann, R. E., Brinkmann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bispecific+antibodies.">Bispecific antibodies.</a> Drug Discov Today. 20 (7), 838-847 (2015).</li><li>Beck, A., Wurch, T., Bailly, C., Corvaia, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+and+challenges+for+the+next+generation+of+therapeutic+antibodies.">Strategies and challenges for the next generation of therapeutic antibodies.</a> Nat Rev Immunol. 10 (5), 345-352 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Food and Drug Administration. 22, (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> European Medicines Agency. 13, (2014).</li><li>Lopez-Morales, C. A., 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=Physicochemical+and+biological+characterization+of+a+biosimilar+trastuzumab.">Physicochemical and biological characterization of a biosimilar trastuzumab.</a> Biomed Res Int. 2015, 427235 (2015).</li><li>Jung, S. K., 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=Physicochemical+characterization+of+Remsima.">Physicochemical characterization of Remsima.</a> MAbs. 6 (5), 1163-1177 (2014).</li><li>Visser, J., 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=Physicochemical+and+functional+comparability+between+the+proposed+biosimilar+rituximab+GP2013+and+originator+rituximab.">Physicochemical and functional comparability between the proposed biosimilar rituximab GP2013 and originator rituximab.</a> BioDrugs. 27 (5), 495-507 (2013).</li><li>Li, J., 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=A+comparative+study+of+different+vector+designs+for+the+mammalian+expression+of+recombinant+IgG+antibodies.">A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies.</a> J Immunol Methods. 318 (1-2), 113-124 (2007).</li><li>Fitzgerald, K. D., Semler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bridging+IRES+elements+in+mRNAs+to+the+eukaryotic+translation+apparatus.">Bridging IRES elements in mRNAs to the eukaryotic translation apparatus.</a> Biochim Biophys Acta. 1789 (9-10), 518-528 (2009).</li><li>Ho, S. C., 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=IRES-mediated+Tricistronic+vectors+for+enhancing+generation+of+high+monoclonal+antibody+expressing+CHO+cell+lines.">IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines.</a> J Biotechnol. 157 (1), 130-139 (2012).</li><li>Dodev, T. S., 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=A+tool+kit+for+rapid+cloning+and+expression+of+recombinant+antibodies.">A tool kit for rapid cloning and expression of recombinant antibodies.</a> Sci Rep. 4, 5885 (2014).</li><li>Walsh, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modifications+of+protein+biopharmaceuticals.">Post-translational modifications of protein biopharmaceuticals.</a> Drug Discov Today. 15 (17-18), 773-780 (2010).</li><li>Backliwal, G., 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=Rational+vector+design+and+multi-pathway+modulation+of+HEK+293E+cells+yield+recombinant+antibody+titers+exceeding+1+g/l+by+transient+transfection+under+serum-free+conditions.">Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions.</a> Nucleic Acids Res. 36 (15), 96 (2008).</li><li>Bebbington, C. R., 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=High-level+expression+of+a+recombinant+antibody+from+myeloma+cells+using+a+glutamine+synthetase+gene+as+an+amplifiable+selectable+marker.">High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker.</a> Biotechnology (N Y). 10 (2), 169-175 (1992).</li><li>Kaufman, R. J., 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=Coamplification+and+coexpression+of+human+tissue-type+plasminogen+activator+and+murine+dihydrofolate+reductase+sequences+in+Chinese+hamster+ovary+cells.">Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells.</a> Mol Cell Biol. 5 (7), 1750-1759 (1985).</li><li>Dalton, A. C., Barton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over-expression+of+secreted+proteins+from+mammalian+cell+lines.">Over-expression of secreted proteins from mammalian cell lines.</a> Protein Sci. 23 (5), 517-525 (2014).</li><li>Pham, P. L., 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=Transient+gene+expression+in+HEK293+cells:+peptone+addition+posttransfection+improves+recombinant+protein+synthesis.">Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis.</a> Biotechnol Bioeng. 90 (3), 332-344 (2005).</li><li>Pham, P. L., 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=Large-scale+transient+transfection+of+serum-free+suspension-growing+HEK293+EBNA1+cells:+peptone+additives+improve+cell+growth+and+transfection+efficiency.">Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency.</a> Biotechnol Bioeng. 84 (3), 332-342 (2003).</li><li>Yang, W. C., 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=Addition+of+valproic+acid+to+CHO+cell+fed-batch+cultures+improves+monoclonal+antibody+titers.">Addition of valproic acid to CHO cell fed-batch cultures improves monoclonal antibody titers.</a> Molecular Biotechnology. 56 (5), 421-428 (2014).</li><li>Backliwal, G., 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=Valproic+acid:+a+viable+alternative+to+sodium+butyrate+for+enhancing+protein+expression+in+mammalian+cell+cultures.">Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures.</a> Biotechnol Bioeng. 101 (1), 182-189 (2008).</li><li>Jiang, Z., Sharfstein, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+butyrate+stimulates+monoclonal+antibody+over-expression+in+CHO+cells+by+improving+gene+accessibility.">Sodium butyrate stimulates monoclonal antibody over-expression in CHO cells by improving gene accessibility.</a> Biotechnol Bioeng. 100 (1), 189-194 (2008).</li><li>Jordan, M., Wurm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+adherent+and+suspended+cells+by+calcium+phosphate.">Transfection of adherent and suspended cells by calcium phosphate.</a> Methods. 33 (2), 136-143 (2004).</li></ol>

The authors declare that they have no competing financial interests.

该协议细节的转染，稳定表达和治疗性抗体的纯化在HEK-293细胞。抗体基因的稳定表达是在产生用于治疗性抗体的开发和制造的抗体产生细胞系的第一步。而中国仓鼠卵巢（CHO）细胞仍然是治疗性蛋白质的选择的表达平台，HEK-293细胞系获得突出的实现，在这些细胞中产生的蛋白质是天然存在的人类蛋白质更匹配，在后方面-translational修改和功能14,15。 ...

治疗性抗体的成功将继续推动大量投资进入的抗体开发为下一代疗法的浪潮开始。抗体市场预期由抗体片段1，抗体-药物偶联物2，双特异性抗体3和工程化抗体具有有利性质4重塑。另一类获得医药感兴趣的是生物仿制药。生物仿制药抗体是"高度相似"复制这种已经获得监管部门的批准治疗性抗体产品。拟议的生物仿制药必须是就其结构，功能，动物毒性，临床安全性和有效性，人体药代动力学（PK），药效学（PD）和免疫原性5,6鼻祖抗体相媲美。 批准ř生物仿制药的抗体的茨一直缓慢由于在产品的最终质量的严格限制。确切的制造过程，如通过对最终的处理步骤的特定细胞系和培养条件可以保持专有的。更重要的是，抗体的制造固有涉及程度可变性的可加至生产高度类似产品的挑战。全面理化和生物物理表征和比较是相当困难的，但许多研究表明生物仿制药的抗体的特性出现在文献7，8，9。 产生治疗性抗体便从哺乳动物宿主细胞用携带的基因的各抗体的载体的转染。载体设计，细胞系和培养条件是建立前的关键考虑因素PRESSION系统。 抗体的DNA序列可以从药物银行（www.drugbank.ca），IMGT（www.igmt.org）或研究的出版物，包括专利中采购。例如，曲妥单抗的序列，可通过药物银行（DB编号:DB00072）。可变区的氨基酸序列可经历基因设计和优化用于合成在所需宿主物种。它是没有修饰的氨基酸序列制成的生物仿制药抗体重要。一旦合成，抗体基因可亚克隆到所选择的合适的载体。 人IgG抗体由两条相同的重链和两个相同的轻链组成。两条链的紧密调节表达是用于在哺乳动物细胞10的异源抗体蛋白的最佳生产的关键。帧内以及链间二硫键必须形成和一些翻译后修饰的需要成为在蛋白质的生物合成过程中troduced。许多载体是已专为表达抗体基因（参考材料表）可用。这些特定抗体 - 载体通常表示为重链和轻链，以便仅在每个链的可变区所需要克隆的恒定区。 有两个独立的构建体（共转染）细胞的转染是用于输送重和轻链编码基因的最常见的方法。也就是说，每个基因通过其自身的启动子被组装的内质网前驱动和转录为单独的抗体链。另一方面，多顺反子载体具有掺入内部核糖体进入位点（IRES）元件，使多个基因作为与由mRNA 11的内部区域允许翻译单一的mRNA转录物的表达。在这种情况下，重链和轻链编码基因被连接在一个arrangEMENT同时实现抗体链10,12的共表达。 而瞬时转染细胞产生足够的蛋白来执行的实验的数量有限，已经经过选择基因组整合稳定转染的细胞系可以提供更高的产量。较高的蛋白质的量允许为涉及体外表征检测开发，并能提供在考虑抗体质量为下游应用的指示，如克隆细胞系和铅候选选择。 本文的目的是描述在哺乳动物表达系统中产生的治疗性抗体的稳定表达和纯化。事实上，这种方法可以适用于一生物仿制药抗体的表达。该方法可用于抗体的初始特性继续到关键的，尽管是费时STE之前确定一个理想的克隆用于大规模生产的PS。此外，这种方法可用于表达其它蛋白质，而不只是抗体。 下面的详细方案描述的治疗性抗体曲妥单抗的表达。这包括制备载体DNA随后在HEK-293细胞系和抗体蛋白质的纯化稳定转染通过自动化色谱法的。

<table><tbody><tr><td>pFUSE vector series</td><td>InvivoGen</td><td>N/A</td><td>Heavy and light antibody genes expressed in separate vectors that require co-transfection.</td></tr><tr><td>mAbXpress vector series</td><td>ACYTE Biotech Pty Ltd.</td><td></td><td>Heavy and light antibody genes expressed in separate vectors that require co-transfection. Refer to: Jones, M. L. et al. A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies. J Immunol Methods. 354 (1-2), 85-90, doi:10.1016/j.jim.2010.02.001, (2010).</td></tr><tr><td>pVITRO1 vector</td><td>N/A</td><td>N/A</td><td>Heavy and light antibody genes are each driven by a separate promoter in a single vector.&amp;nbsp; Refer to: Dodev, T. S. &lt;em&gt;et al.&lt;/em&gt; A tool kit for rapid cloning and expression of recombinant antibodies. Sci Rep. 4 5885, doi:10.1038/srep05885, (2014).</td></tr><tr><td>GS vector series</td><td>Lonza</td><td></td><td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript.</td></tr><tr><td>Multi-cistronic vector series 1</td><td>N/A</td><td>N/A</td><td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Li, J. &lt;em&gt;et al.&lt;/em&gt; A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies. J Immunol Methods. 318 (1-2), 113-124, doi:10.1016/j.jim.2006.10.010, (2007).</td></tr><tr><td>Multi-cistronic vector series 2</td><td>N/A</td><td>N/A</td><td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Ho, S. C. &lt;em&gt;et al.&lt;/em&gt; IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol. 157 (1), 130-139, doi:10.1016/j.jbiotec.2011.09.023, (2012).</td></tr><tr><td>pVITRO1-Trastuzumab-IgG1/&amp;kappa;</td><td>Addgene</td><td>61883</td><td>Mammalian expression vector containing trastuzumab antibody genes with hygromycin resistance gene; pVITRO1-Trastuzumab-IgG1/&amp;kappa; was a gift from Andrew Beavil.</td></tr><tr><td>Fast-Media Hygro Agar</td><td>Jomar Life Research</td><td>fas-hg-s</td><td>Used to prepare low salt LB agar containing 75 &amp;micro;g/ml hygromycin.</td></tr><tr><td>Fast-Media Hygro TB</td><td>Jomar Life Research</td><td>fas-hg-l</td><td>Used to prepare low salt TB broth containing 75 &amp;micro;g/ml hygromycin.&amp;nbsp;</td></tr><tr><td>Glycerol, BioXtra, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>G6279</td><td>Prepare to 80% with water and autoclave. Store at room temperature.</td></tr><tr><td>Jestar 2.0/LFU Plasmid Maxi Kit</td><td>Astral Scientific</td><td>G221020</td><td>Plasmid Maxi Prep Kit; elute or resuspend DNA in water (pH 7.0-8.5).</td></tr><tr><td>FreeStyle 293-F Cells</td><td>Life Technologies</td><td>R790-07</td><td>HEK-293 cell line adapted to suspension culture in serum-free media.</td></tr><tr><td>FreeStyle 293 Expression Medium</td><td>Life Technologies</td><td>12338-018</td><td>Serum-free media specially formulated for maintaing 293-F cell line and high protein expression.</td></tr><tr><td>Kolliphor P188</td><td>Sigma-Aldrich</td><td>K4894</td><td>Non-ionic surfactant; pluronic F-68; prepare to 10% in water and filter-sterilize using 0.22 &amp;mu;m filter. Store at 4 &amp;deg;C.</td></tr><tr><td>DMEM, high glucose&amp;nbsp;</td><td>Life Technologies</td><td>11995-065</td><td></td></tr><tr><td>Heat-Inactivated Foetal Bovine Serum</td><td>Life Technologies</td><td>10082-147</td><td></td></tr><tr><td>Polyethylenimine, Linear, MW 25,000&amp;nbsp;</td><td>Polysciences, Inc.</td><td>23966</td><td>Prepare to 1 mg/ml in water. Adjust to pH 7.0 with 1 M HCl (solution becomes clear) and filter-sterilize using 0.22 &amp;mu;m filter. Store at -80 &amp;deg;C until use.</td></tr><tr><td>OptiPro SFM</td><td>Life Technologies</td><td>12309-050</td><td>Transfection formulated serum-free media</td></tr><tr><td>Hygromycin B Solution</td><td>Jomar Life Research</td><td>ant-hg-1</td><td></td></tr><tr><td>Dimethylsulphoxide (DMSO)</td><td>Thermo Fisher Scientific</td><td>AJA2225</td><td></td></tr><tr><td>Tryptone (casein peptone)</td><td>Thermo Fisher Scientific</td><td>LP0042B</td><td>Prepare to 20% in PBS and filter-sterilize using 0.22 &amp;mu;m filter. Store at 4 &amp;deg;C.&amp;nbsp;</td></tr><tr><td>Phosphate Buffered Saline (PBS) Tablets, pH 7.4, 100 ml</td><td>Astral Scientific</td><td>09-2051-100</td><td></td></tr><tr><td>HiTrap Protein A High Performance, 1 x 5 ml column</td><td>Sigma-Aldrich</td><td>GE17-0403-01</td><td></td></tr><tr><td>AKTApurifier 100</td><td>GE Healthcare</td><td>28406266</td><td>Automated FPLC system, which can include a P-960 sample pump and Frac-920 fraction collector.</td></tr><tr><td>Glycine-HCl</td><td>Sigma-Aldrich</td><td>G2879</td><td></td></tr><tr><td>Citric Acid, monohydrate</td><td>Astral Scientific</td><td>BIOC2123</td><td></td></tr><tr><td>Sodium Citrate, trisodium salt dihydrate&amp;nbsp;</td><td>Astral Scientific</td><td>BIOCB0035</td><td></td></tr><tr><td>1 M Tris-HCl solution pH 9.0</td><td>Astral Scientific</td><td>BIOSD8146</td><td></td></tr><tr><td>Amicon Ultra Centrifugal Filters (30 MWCO)</td><td>Merck Millipore</td><td>UFC803008/UFC903008</td><td>Used to buffer exchange and concentrate purified protein.</td></tr><tr><td>Pierce Bicinchoninic Acid (BCA) Assay Kit</td><td>Thermo Fisher Scientific</td><td>23227</td><td></td></tr><tr><td>BLItz System</td><td>fort&amp;eacute;BIO</td><td>45-5000</td><td>Instrument used for bio-layer interferometry (BLI) measurements.</td></tr><tr><td>Protein A biosensors</td><td>fort&amp;eacute;BIO</td><td>18-5010</td><td></td></tr><tr><td>Acrylamide/Bisacrylamide (37.5:1), 40% solution</td><td>Astral Scientific</td><td>786-502</td><td></td></tr><tr><td>Ammonium Persulfate (APS)</td><td>Astral Scientific</td><td>AM0486</td><td></td></tr><tr><td>TEMED</td><td>Astral Scientific</td><td>AM0761</td><td></td></tr><tr><td>Coomassie Brilliant Blue R-250</td><td>Astral Scientific</td><td>786-498</td><td></td></tr><tr><td>Precision Plus Dual-Color Protein Standard</td><td>Bio-Rad</td><td>1610374</td><td></td></tr></tbody></table>

laboratory scale production and purification of a therapeutic antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

使用生物层干涉（BLI）转染HEK-293细胞的稳定生产单抗的结果确认， 如图1中。通过测量IgG抗体标准和蛋白质的生物传感器（ 图1A）之间的结合率产生的IgG的标准曲线。粗制上清液样品类似地测量，那么其浓度从标准曲线（ 图1B）内插。上清液的浓度测得为大约25微克/毫升双周（从传代培养收集50毫升取样）的无血清适应之后和之前为?...

Watch this Scientific Journal Video about Laboratory Scale Production and Purification of a Therapeutic Antibody at JoVE.com

Laboratory Scale Production and Purification of a Therapeutic Antibody

这个协议描述在哺乳动物表达系统中生产的治疗性抗体的。所描述的方法包括制备载体DNA，稳定转染和人胚肾293细胞系的无血清适应的，设置大规模培养和纯化使用亲和层析组成。

实验室规模的生产和治疗性抗体的纯化

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

laboratory-scale-production-and-purification-of-a-therapeutic-antibody

Research

JoVE Journal

Biochemistry

17.3K 次观看.  University of Sydney. 这个协议描述在哺乳动物表达系统中生产的治疗性抗体的。所描述的方法包括制备载体DNA，稳定转染和人胚肾293细胞系的无血清适应的，设置大规模培养和纯化使用亲和层析组成。 

视频: Laboratory Scale Production and Purification of a Therapeutic Antibody

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

一种高通量自动化的平台，制造蛋白质治疗的细胞系发展

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

科研

医学

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

用于检测实验方案<em&gt;蓝藻</em&gt;与抗体基因芯片液体和固体样品

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

环境科学

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

靶性抗原特异性治疗抗体的肿瘤及组织分布分析

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

癌症研究

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

高通量自动 Microbioreactor 系统在 CHO 细胞模型 IgG1 生产中的应用

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

生物工程

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 ...

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins.
Several approaches have been taken to overcome the specificity issue of polyclonal antisera. One is by absorbing the unwanted antibodies by passing the antiserum through a chromatography column of immobilized antigens (2). This method is tedious and frequently unable to completely remove the unwanted antibodies. Another approach is to isolate individual antibody-producing B cells and expand them in culture. However, like most normal untransformed cells, B cells do not survive in long-term culture.
To overcome the inability of B cells to survive in culture, one approach is to prepare a myeloma-B cell hybridoma. In 1847, Henry Bence-Jones discovered that patients with multiple myeloma, a lymphoid tumor, produced a large quantity of antibodies (3). B cells in these patients have become malignant and grow uncontrollably. Since the malignant B cells are derived from a single clone, they are identical and produce only a single type of antibody (i.e., a monoclonal antibody, or mAb). However, most of these myeloma cells produce antibodies of unknown specificities. In 1975, by fusing a myeloma cell to a B cell, Cesar Milstein and Georges Kohler succeeded in producing a hybridoma that can be cultured indefinitely in vitro and produces an unlimited number of monoclonal antibodies of known antigenic specificity (4). The rationale behind their approach is to combine the immortal properties of the myeloma cell and the antibody producing properties of the B cell. Their technique revolutionized antibody production and provides a powerful means for identification and purification of biological molecules using monoclonal antibodies.
Generally, preparing a monoclonal antibody requires several months. The general procedure includes the following steps:
<ol>
	<li>Immunization and screening of antibody titer</li>
	<li>Fusion of antibody-producing B cells and myeloma cells</li>
	<li>Selective growth of the hybridoma</li>
	<li>Screening the hybridomas for producing the desired monoclonal antibody</li>
	<li>Cloning by limiting dilution - a process whereby cells are diluted to a concentration to statistically allow for less than 1 cell to be added to the wells of a 96-well plate. Some wells will end up with 0 cells and some will have 1 cell. The wells with seeded with 1 cell will eventually grow into a monoclonal population of cells.</li>
	<li>Growth of the hybridoma and preparation of monoclonal antibody</li>
</ol>
This protocol focuses on the last step - growth of the hybridoma and preparation of the monoclonal antibody. The antibody is purified from the culture supernatant by ammonium sulfate precipitation (often referred to as salting out) - a commonly used method of removing proteins from a solution. Proteins in solution form hydrogen bonds, along with other hydrophilic interactions, with water through their exposed polar and ionic groups. When concentrations of small, highly charged ions (such as ammonium or sulfate) are added, these groups compete with the proteins for binding to water. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation of the protein.

Producing Monoclonal Antibodies Using Hybridomas

抗体生成:使用杂交瘤生产单克隆抗体

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

教育

JoVE Science Education

高级生物学

免疫学

Production and Purification of Recombinant Antibodies from Mammalian Cells

Source: Shivange, Gururaj, et al. <a href="https://app.jove.com/v/60727">Analyzing Tumor and Tissue Distribution of Target Antigen-Specific Therapeutic Antibody.</a> J. Vis. Exp.(2020).
The video explains the process of producing antibodies in mammalian host cells, from gene expression to secretion. It involves transcription, translation, signal recognition, correct folding, and eventual release into the extracellular medium via secretory vesicles.

从哺乳动物细胞中生产和纯化重组抗体

1. Expression and purification of antibodies

	Maintenance of CHO cells
	
		Grow CHO cells in FreeStyle CHO Media supplemented with commercially available ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Production and Purification

A Technique to Purify Monoclonal Antibodies Produced by Chinese Hamster Ovary Cells

Source: Velugula-Yellela, S. R., et al. <a href="https://app.jove.com/v/58947">Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System.</a> J. Vis. Exp. (2019).
This video demonstrates an assay for purifying monoclonal antibodies from cell culture fluid harvested from Chinese Hamster Ovarian cells. It employs a porous glass resin with immobilized protein A, which binds to the Fc region of the antibodies in the sample, effectively separating them from contaminants. The use of a low-pH elution buffer weakens the interaction, facilitating the retrieval of purified antibodies.

一种纯化中国仓鼠卵巢细胞产生的单克隆抗体的技术

1. Purification of antibody
NOTE: The equilibration buffer for the in-house antibody is 25 mM Tris, 100 mM NaCl, pH 7.5. The elution buffer used is 0.1 M ...

Antibody Production from a Batch Overgrowth Culture of Transfected Mammalian Cells

Source: Elgundi, Z. et al., <a href="https://app.jove.com/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a>&#160;J. Vis. Exp.&#160;(2017)
The video demonstrates a method for producing therapeutic antibodies using stably transfected mammalian cells. Tryptone supplementation stimulates cell division, resulting in overgrowth and supporting a higher antibody yield. The antibody-containing medium is harvested, filtered, and stored for further use.

从转染哺乳动物细胞的批量生长培养物中生产抗体

NOTE: A suitable mammalian expression vector must be used for this protocol. Here, a single construct containing two expression cassettes is used ...

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 ...