Genmab, Utrecht, the Netherlands.
Nat Protoc. 2014 Oct;9(10):2450-63. doi: 10.1038/nprot.2014.169. Epub 2014 Sep 25.
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).
以实用且高效的方式生成具有天然 IgG 结构的双特异性抗体（bsAb）一直是一个长期存在的挑战。在这里，我们描述了一种易于使用的方法来生成双特异性 IgG1（bsIgG1），即 Fab 臂交换的控制（cFAE）。该方案包括以下步骤：（i）分别表达两种亲本 IgG1，它们在 CH3 结构域中含有单个匹配点突变；（ii）在体外允许半分子重组的允许氧化还原条件下混合亲本 IgG1；（iii）去除还原剂以允许链间二硫键重新氧化；（iv）使用基于色谱或基于质谱（MS）的方法分析交换效率和最终产物。该方案可生成具有常规 IgG 结构、特征和质量属性的 bsAb，无论是在台架规模（微克至毫克）还是在小型生物反应器规模（毫克至克）下，该规模旨在模拟大规模生产（千克）。从高质量的纯化蛋白开始，在 2-3 天（包括质量控制）内通常可以获得≥95%的交换效率。
