Heavy chain dimers stabilized by disulfide bonds are required to promote in vitro assembly of trastuzumab
| Autor: | Antoni Casablancas, Jordi J. Cairó, Óscar Martínez, Ramón Román, Marc Camps, Xavier Pujol, Mercè Farràs, Joan Miret |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Non-covalent Protein Denaturation Protein Folding HC Protein Conformation Receptor ErbB-2 LC chemistry.chemical_compound 0302 clinical medicine Protein structure Trastuzumab Urea Disulfides 2-mercaptoethanol 2-Mercaptoethanol Chromatography High Pressure Liquid Mab lcsh:Cytology Glutathione Slow dialysis Monomer 030220 oncology & carcinogenesis Electrophoresis Polyacrylamide Gel Immunoglobulin Heavy Chains medicine.drug Research Article medicine.drug_class Monoclonal antibody Immunoglobulin light chain Affinity chromatography 03 medical and health sciences mAb assembly In vivo Renaturalization medicine Immunoglobulin Humans lcsh:QH573-671 Disulfide bonds Molecular Biology mAb Folding Cell Biology In vitro Anti-HER2 030104 developmental biology HEK293 Cells chemistry Mab assembly Immunoglobulin G Biophysics Immunoglobulin Light Chains |
| Zdroj: | BMC Molecular and Cell Biology Dipòsit Digital de Documents de la UAB Universitat Autònoma de Barcelona BMC Molecular and Cell Biology, Vol 21, Iss 1, Pp 1-9 (2020) Recercat. Dipósit de la Recerca de Catalunya instname Recercat: Dipósit de la Recerca de Catalunya Varias* (Consorci de Biblioteques Universitáries de Catalunya, Centre de Serveis Científics i Acadèmics de Catalunya) |
| ISSN: | 2661-8850 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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