Mobile Affinity Selection Chromatography Analysis of Therapeutic Monoclonal Antibodies.
Mobile Affinity Selection Chromatography Analysis of Therapeutic Monoclonal Antibodies.
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DOI：
10.1021/acs.analchem.3c02180
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发表时间：
2023-11-07
影响因子：
7.4
通讯作者：
Regnier, Fred E.
中科院分区：
文献类型：
--
作者：
Narsimhan, Meena L.;Kim, Jinhee;Morris, Nathan A.;Bower, Mary A.;Gunawardena, Harsha P.;Bowen, Eric;Regnier, Fred E.
联邦监管机构要求持续验证重组治疗性单克隆抗体（mAb）的质量，通常通过两步过程实现。首先，通过蛋白A亲和层析将宿主细胞蛋白质组和代谢组从生产培养基中去除。其次，在用酸性洗涤从亲和柱中回收后，通过液相色谱-质谱（LC-MS）以多种方式评估单抗的质量。然而，冗长的样品制备和缺乏高阶结构分析是这种方法的局限性。为了解决这些问题，本报告提出了一种综合的方法来分析单克隆抗体的两个关键质量属性，即滴度和相对总含量。利用等压洗脱的移动亲和选择色谱（MASC）柱，在一个步骤中实现了样品制备和基于分子识别的分析的集成。MASC规避了蛋白A步骤，简化了样品制备。在10分钟内，(i)单抗荧光编码以进行特异性检测，（ii）单体和聚集体被分解，（iii）单抗滴度被定量，（iv）相对聚集体含量被确定，(v)分析物被检测，（vi）色谱柱准备用于下一个样品。本文建议，这种快速质量评估模式将在发现（筛选、克隆选择、表征）、工艺研发和制造的所有阶段都有价值。快速监测变异形成是质量评估的关键要素。
Federal regulatory agencies require continuous verification of recombinant therapeutic monoclonal antibody (mAb) quality that is commonly achieved in a two-step process. First, the host-cell proteome and metabolome are removed from the production medium by protein A affinity chromatography. Second, following recovery from the affinity column with an acidic wash, mAb quality is assessed in multiple ways by liquid chromatography–mass spectrometry (LC–MS). However, lengthy sample preparation and the lack of higher-order structure analyses are limitations of this approach. To address these issues, this report presents an integrated approach for the analysis of two critical quality attributes of mAbs, namely titer and relative aggregate content. Integration of sample preparation and molecular-recognition-based analyses were achieved in a single step utilizing an isocratically eluted mobile affinity selection chromatography (MASC) column. MASC circumvents the protein A step, simplifying sample preparation. Within 10 min, (i) mAbs are fluorescently coded for specific detection, (ii) monomers and aggregates are resolved, (iii) the mAb titer is quantified, (iv) relative aggregate content is determined, (v) analytes are detected, and (vi) the column is ready for the next sample. It is suggested herein that this mode of rapid quality assessment will be of value at all stages of discovery (screening, clone selection, characterization), process R&D, and manufacturing. Rapid monitoring of variant formation is a critical element of quality evaluation.
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影响因子：
5.3
作者：
Füssl F;Trappe A;Cook K;Scheffler K;Fitzgerald O;Bones J
通讯作者：
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影响因子：
7.4
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Li, ZhiYu;Kim, JinHee;Regnier, Fred E.
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Regnier, Fred E.
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影响因子：
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