Recombinant expression of antibody molecules in mammalian cells offers important advantages over traditionally utilized bacterial expression, including glycosylation required for antibody functionality and markedly reduced levels of endotoxin contamination. Advances in transient mammalian expression systems enable high yields (>100 mg/liter) that now allow for effective recombinant antibody production at a reasonable cost. Here, we provide step-by-step protocols for the design and recombinant expression of full-length IgG antibodies and antibody-derived constructs (including Fab, Fc-fusions and bispecifics) in mammalian cells. Antibody constructs are designed by combining antibody variable domains, generated by phage display or derived from human/humanized monoclonals, with constant regions. The constructs are then expressed from mammalian vectors, secreted into culture media, purified by affinity chromatography and characterized by biolayer interferometry. This article provides detailed protocols, sequences and strategies that allow the expression and purification of endotoxin-free antibody reagents suitable for testing in animal models within a 3-week time frame.

中文翻译：

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人抗体在哺乳动物细胞中的瞬时表达。

与常规利用的细菌表达相比，抗体分子在哺乳动物细胞中的重组表达具有重要的优势，包括抗体功能所需的糖基化作用和内毒素污染水平的显着降低。瞬时哺乳动物表达系统的进步实现了高产量（> 100 mg / L），现在可以以合理的成本有效地生产重组抗体。在这里，我们为哺乳动物细胞中全长IgG抗体和抗体衍生的构建体（包括Fab，Fc融合和双特异性抗体）的设计和重组表达提供了分步操作规程。通过将通过噬菌体展示产生或衍生自人/人源化单克隆抗体的抗体可变域与恒定区相结合来设计抗体构建体。然后从哺乳动物载体表达构建体，将其分泌到培养基中，通过亲和层析纯化，并通过生物层干涉术表征。本文提供了详细的方案，序列和策略，可在3周内表达和纯化无内毒素的抗体试剂，这些试剂适合在动物模型中进行测试。