Heavy chain dimers stabilized by disulfide bonds are required to promote in vitro assembly of trastuzumab

BMC Molecular and Cell Biology

article

Farràs, Mercè¹  Román, Ramón²  Camps, Marc¹  Miret, Joan²  Martínez, Óscar¹  Pujol, Xavier¹  Casablancas, Antoni²  Cairó, Jordi Joan² 

[1] Department of Biotechnology, Farmhispania SA;Department of Chemical, Biological and Environmental Engineering, Autonomous University of Barcelona

关键词: mAb;    Protein structure;    Folding;    Disulfide bonds;    Renaturalization;    LC;    HC;    mAb assembly;    Urea;    Anti-HER2;    Non-covalent;    Slow dialysis;    Glutathione;    2-mercaptoethanol;    Affinity chromatography;    Trastuzumab;    Immunoglobulin;   

DOI  :  10.1186/s12860-019-0244-x

学科分类：内科医学

来源: Colegio Oficial de Psicologos

【 摘 要 】

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. 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. 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.

【 授权许可】

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