Unraveling what makes a monoclonal antibody difficult‐to‐express: From intracellular accumulation to incomplete folding and degradation via ERAD

摘要

Abstract >Although most therapeutic monoclonal antibodies (mAbs) can routinely be produced in the multigram per litre range, some mAb candidates turn out to be difficult‐to‐express (DTE). In addition, the class of more complex biological formats is permanently increasing and mammalian expression systems like Chinese hamster ovary (CHO) cell lines can show low performance. Hence, there is an urgent need to identify any rate limiting processing step during cellular synthesis. Therefore, we assessed the intracellular location of the DTE antibody mAb2 by fluorescence and electron microscopy (EM) and revealed an accumulation of the antibody, which led to an aberrant morphology of the endoplasmic reticulum (ER). Analysis of underlying cellular mechanisms revealed that neither aggregation nor antibody assembly, but folding represented the reason for hampered secretion. We identified that the disulfide bridge formation within the antibody light chain (LC) was impaired due to less recognition by protein disulfide isomerase (PDI). As a consequence, the DTE molecule was degraded intracellularly by the ubiquitin proteasome system via ER‐associated degradation (ERAD). This study revealed that with the continuous emergence of DTE therapeutic protein candidates, special attention needs to be drawn to optimization processes to ensure manufacturability. </abstract> </span> <span class="z_kbtn z_kbtnclass hoverxs" style="display: none;">展开▼</span> </div> <div class="translation abstracttxt"> <span class="zhankaihshouqi fivelineshidden" id="abstract"> <span>机译：</span><Abstract XML：lang =“en”type =“main”> <标题类型=“main”>抽象</ title> 虽然大多数治疗性单克隆抗体（mAbs）可以在每升多餐厅中常规生产，但是一些MAB候选结果难以表达（DTE）。此外，更复杂的生物形式的类是永久性增加的，哺乳动物表达系统如中国仓鼠卵巢（CHO）细胞系可以显示出低性能。因此，迫切需要识别蜂窝合成期间的任何速率限制处理步骤。因此，我们通过荧光和电子显微镜（EM）评估了DTE抗体MAB2的细胞内位置，并揭示了抗体的积累，这导致了内质网（ER）的异常形态。潜在的细胞机制分析表明，既不聚集也不是抗体组装，但折叠代表了妨碍分泌的原因。我们认为抗体轻链（LC）内的二硫键形成由于蛋白二硫化物异构酶（PDI）的识别较小而受到损害。因此，通过ER相关的降解（ERAD），通过泛素蛋白酶体系（ERAD）细胞内分子细胞内分子降解。本研究表明，随着DTE治疗蛋白候选者的连续出现，需要特别注意优化过程以确保可制造性。</ p> </摘要> </span> <span class="z_kbtn z_kbtnclass hoverxs" style="display: none;">展开▼</span> </div> </div> <div class="record"> <h2 class="all_title" id="enpatent33" >著录项</h2> <ul> <li> <span class="lefttit">来源</span> <div style="width: 86%;vertical-align: text-top;display: inline-block;"> <a href='/journal-foreign-15017/'>《Biotechnology and Bioengineering》</a> <b style="margin: 0 2px;">|</b><span>2020年第1期</span><b style="margin: 0 2px;">|</b><span>共12页</span> </div> </li> <li> <div class="author"> <span class="lefttit">作者</span> <p id="fAuthorthree" class="threelineshidden zhankaihshouqi"> </p> <span class="z_kbtnclass z_kbtnclassall hoverxs" id="zkzz" style="display: none;">展开▼</span> </div> </li> <li> <div style="display: flex;"> <span class="lefttit">作者单位</span> <div style="position: relative;margin-left: 3px;max-width: 639px;"> <div class="threelineshidden zhankaihshouqi" id="fOrgthree"> </div> <span class="z_kbtnclass z_kbtnclassall hoverxs" id="zhdw" style="display: none;">展开▼</span> </div> </div> </li> <li > <span class="lefttit">收录信息</span> <span style="width: 86%;vertical-align: text-top;display: inline-block;"></span> </li> <li> <span class="lefttit">原文格式</span> <span>PDF</span> </li> <li> <span class="lefttit">正文语种</span> <span>eng</span> </li> <li> <span class="lefttit">中图分类</span> <span><a href="https://www.zhangqiaokeyan.com/clc/180.html" title="生物工程学（生物技术）">生物工程学（生物技术）;</a></span> </li> <li class="antistop"> <span class="lefttit">关键词</span> <p style="width: 86%;vertical-align: text-top;"> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=CHO cell line engineering&option=203" rel="nofollow">CHO cell line engineering;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=difficult‐to‐express mAbs&option=203" rel="nofollow">difficult‐to‐express mAbs;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=ER‐associated degradation&option=203" rel="nofollow">ER‐associated degradation;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=incomplete domain folding&option=203" rel="nofollow">incomplete domain folding;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=production bottleneck&option=203" rel="nofollow">production bottleneck;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=protein disulfide isomerase&option=203" rel="nofollow">protein disulfide isomerase;</a> </p> <div class="translation"> 机译：CHO细胞线工程;难以表达的mAb;ER相关的降解;不完全域折叠;生产瓶颈;蛋白质二硫化物异构酶; </div> </li> </ul> </div> </div> <div class="literature cardcommon"> <div class="similarity "> <h3 class="all_title" id="enpatent66">相似文献</h3> <div class="similaritytab clearfix"> <ul> <li class="active" >外文文献</li> <li >中文文献</li> <li >专利</li> </ul> </div> <div class="similarity_details"> <ul > <li> <div> <b>1. </b><a class="enjiyixqcontent" href="/journal-foreign-detail/0704021881682.html">Unraveling what makes a monoclonal antibody difficult‐to‐express: From intracellular accumulation to incomplete folding and degradation via ERAD</a> <b>[J]</b> . <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> </a> <a href="/journal-foreign-15017/" target="_blank" rel="nofollow" class="tuijian_authcolor">Biotechnology and Bioengineering .</a> <span>2020</span><span>,第1期</span> </span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：解开是什么使单克隆抗体难以表达：从细胞内积聚到通过Erad的不完全折叠和降解</span> </p> </li> <li> <div> <b>2. </b><a class="enjiyixqcontent" href="/journal-foreign-detail/07040253572.html">Model-Directed Engineering of ‘‘Difficult-to- Express’’ Monoclonal Antibody Production by Chinese Hamster Ovary Cells</a> <b>[J]</b> . <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Leon P. 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serum or monoclonal antibody, method for in vitro targeting a molecule of interest with cd11b expressing cells, cd11b expressing cells, cell therapy product for immunizing a human or animal against an antigen use of cd11b expressing cells, and method for immunizing a patient against antigen</a> <b>[P]</b> . <span> 外国专利：  BRPI0411510A </span> <span> . 2006-07-25</span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：蛋白质，药物组合物，蛋白质用途，多肽，其用途，载体，核酸，宿主细胞，多克隆血清，单克隆抗体，多克隆血清或单克隆抗体的用途，用cd11b表达细胞体外靶向目标分子的方法， cd11b表达细胞，用于针对人或动物针对抗原的免疫的细胞治疗产品以及针对cd11b表达细胞的抗原免疫的方法 </span> </p> </li> <li> <div> <b>2. </b><a class="enjiyixqcontent" href="/patent-detail/06130401301739.html">isolated anti-edar agonist monoclonal antibody or isolated monoclonal antibody fragment or antigen-binding portion or fragment thereof, isolated nucleic acid molecule, expression vector, host cell, transgenic non-human animal, hybridoma, pharmaceutical composition, pharmaceutical kit and, method for producing agonist anti-edar monoclonal antibodies or fragments of isolated monoclonal antibody or antigen binding portions or fragments thereof</a> <b>[P]</b> . <span> 外国专利：  BRPI1013373A2 </span> <span> . 2020-07-28</span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：分离的抗爱德华激动剂单克隆抗体或其分离的单克隆抗体片段或其抗原结合部分或片段，分离的核酸分子，表达载体，宿主细胞，转基因非人类动物，杂交瘤，药物组合物，药物试剂盒及其生产方法激动剂抗爱德华单克隆抗体或分离的单克隆抗体的片段或其抗原结合部分或片段 </span> </p> </li> <li> <div> <b>3. </b><a class="enjiyixqcontent" href="/patent-detail/06130440124381.html">Isolated human antibody, hybridoma, transfectoma, eukaryotic or prokaryotic host cell, non-human animal or transgenic plant, method for producing a human monoclonal antibody, composition, immunoconjugate, bispecific or multispecific molecule, method for inhibiting the development and / or proliferation of a cd25 expressing cell, method for eliminating a cd25 expressing cell, method for treating or preventing a disorder involving cd25 expressing cells, method and diagnostic kit for detecting the presence of cd25 antigen or a cd25 expressing cell, vector of expression, anti-idiotic antibody, and use of it</a> <b>[P]</b> . <span> 外国专利：  BR0316282A </span> <span> . 2005-10-11</span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：分离的人抗体，杂交瘤，转染瘤，真核或原核宿主细胞，非人动物或转基因植物，生产人单克隆抗体的方法，组合物，免疫缀合物，双特异性或多特异性分子，抑制肿瘤的发展和/或增殖的方法cd25表达细胞，消除cd25表达细胞的方法，治疗或预防涉及cd25表达细胞的疾病的方法，用于检测cd25抗原或cd25表达细胞的存在的方法和诊断试剂盒，表达载体，抗惯性抗体，和使用它 </span> </p> </li> </ul> </div> </div> </div> <div class="theme cardcommon" style="overflow: auto;display:none"> <h3 class="all_title" id="enpatent55">相关主题</h3> <ul id="subject"> </ul> </div> </div> </div> </div> <div class="right 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