BACKGROUND Monoclonal antibodies (mAbs) and their derivatives have become one of the most important classes of therapeutic drugs. Their multiple applications increased the interest for understanding their complex structure. In vivo, animal cells are able to fold mAbs correctly (Song et al, J Biosci Bioeng 110:135-40, 2010), whereas previous in vitro approaches were scarce and mostly unsuccessful. RESULTS In this work, we compared in vitro assembly characteristics of trastuzumab, produced either by A) physical separation and refolding of its sub-units or B) direct joining of individually produced heavy and light chains. Native and denatured structures of trastuzumab were determined by SEC-HPLC, HIC-HPLC and SDS-PAGE. CONCLUSIONS Our results demonstrate the requirement of correctly folded HC, forming disulfide-bonded dimers, in order to form a fully functional mAb. Otherwise, the unfolded HC tend to precipitate. We were able to assemble trastuzumab in this fashion by only mixing them to LC in pH-buffered conditions, while monomeric HC structure was too unstable to render a functional mAb. This approach has been used in the generation of homogeneous ADC, with results pending to be published.

中文翻译：

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需要通过二硫键稳定的重链二聚体来促进曲妥珠单抗的体外组装。

背景技术单克隆抗体（mAb）及其衍生物已成为治疗药物的最重要类别之一。它们的多种应用增加了对了解其复杂结构的兴趣。在体内，动物细胞能够正确折叠mAb（Song等人，J Biosci Bioeng 110：135-40，2010），而以前的体外方法很少，而且大多没有成功。结果在这项工作中，我们比较了曲妥珠单抗的体外组装特性，该特性是通过A）物理分离和亚单位的折叠或B）直接连接单独产生的重链和轻链而产生的。曲妥珠单抗的天然和变性结构通过SEC-HPLC，HIC-HPLC和SDS-PAGE确定。结论我们的结果证明了正确折叠HC，形成二硫键二聚体的要求，为了形成功能齐全的mAb。否则，展开的HC易于沉淀。我们仅通过在pH缓冲条件下将曲妥珠单抗与LC混合即可组装成曲妥珠单抗，而单体HC结构太不稳定而无法提供功能性mAb。该方法已用于生成同类ADC，其结果有待公布。