Heavy chain dimers stabilized by disulfide bonds are required to promote in vitro assembly of trastuzumab
Farràs Mañé, Marta (Department of Biotechnology. Farmhispania SA)
Román, Ramón (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Camps, Marc (Department of Biotechnology. Farmhispania SA)
Miret, Joan (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Martínez, Óscar (Department of Biotechnology. Farmhispania SA)
Pujol, Xavier (Department of Biotechnology. Farmhispania SA)
Casablancas, Antoni (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Cairó i Badillo, Jordi Joan (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)

Data:	2020
Resum:	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.
Nota:	Altres ajuts: CDTI/IDI-20170546
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	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
Publicat a:	BMC Molecular and Cell Biology, Vol. 21 (January 2020) , art. 2, ISSN 2661-8850

DOI: 10.1186/s12860-019-0244-x
PMID: 31964343

9 p, 1.8 MB

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