Controlled Fab-arm exchange for the generation of stable bispecific IgG1
Controlled Fab-arm exchange for the generation of stable bispecific IgG1
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DOI：
10.1038/nprot.2014.169
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发表时间：
2014-10-01
期刊：
影响因子：
14.8
通讯作者：
Parren, Paul W. H. I.
中科院分区：
文献类型：
--
作者：
Labrijn, Aran F.;Meesters, Joyce I.;Parren, Paul W. H. I.
以实用和有效的方式产生具有天然IgG结构的双特异性抗体（bsAbs）一直是一个长期的挑战。在这里，我们描述了受控fab臂交换（cFAE），这是一种易于使用的生成双特异性IgG1 （bsIgG1）的方法。该方案包括以下内容：(i)分别表达两个亲本IgG1s，其中包含CH3结构域的单个匹配点突变；（ii）在体外允许氧化还原条件下混合亲本IgG1s，使半分子重组；（iii）去除还原剂，使链间二硫键重新氧化；（iv）使用基于色谱或质谱（MS）的方法分析交换效率和最终产品。该方案生成具有常规IgG结构、特征和质量属性的bsab，在实验规模（微克到毫克）和小型生物反应器规模（毫克到克），设计用于模拟大规模生产（公斤）。从优质纯化蛋白开始，通常在2-3天内（包括质量控制）可获得>= 95%的交换效率。
The generation of bispecific antibodies (bsAbs) with natural IgG architecture in a practical and efficient manner has been a longstanding challenge. Here we describe controlled Fab-arm exchange (cFAE), which is an easy-to-use method to generate bispecific IgG1 (bsIgG1). The protocol involves the following: (i) separate expression of two parental IgG1s containing single matching point mutations in the CH3 domain; (ii) mixing of parental IgG1s under permissive redox conditions in vitro to enable recombination of half-molecules; (iii) removal of the reductant to allow reoxidation of interchain disulfide bonds; and (iv) analysis of exchange efficiency and final product using chromatography-based or mass spectrometry (MS)-based methods. The protocol generates bsAbs with regular IgG architecture, characteristics and quality attributes both at bench scale (micrograms to milligrams) and at a mini-bioreactor scale (milligrams to grams) that is designed to model large-scale manufacturing (kilograms). Starting from good-quality purified proteins, exchange efficiencies of >= 95% can routinely be obtained within 2-3 d (including quality control).