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

Mercè Farràs,Ramón Román,Xavier Pujol,Joan Miret,Jordi Joan Cairó,Marc Camps,Óscar Martínez,Antoni Casablancas

doi:10.1186/s12860-019-0244-x

Mercè Farràs, Ramón Román + Show 6 more

Open Access

https://doi.org/10.1186/s12860-019-0244-x

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Abstract

BackgroundMonoclonal 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.ResultsIn 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.ConclusionsOur 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.

Highlights

Monoclonal antibodies and their derivatives have become one of the most important classes of therapeutic drugs
Unfolding and folding a Monoclonal antibody (mAb) without physical separation Firstly, we adapted the Maeda et al method [20] based on slow dialysis and appropriate redox buffer in order to test the ability of the method to refold and reoxidize denatured and reduced trastuzumab, without physical chain separation
If the same protocol was applied with physical separation of Heavy Chain (HC) and Light Chain (LC) followed by the refolding of each chain independently, HC showed precipitation and, the original mAb structure could not be recovered

Summary

Introduction

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. Therapeutic antibodies have risen to prominence over the past three decades and are the fastest growing drug class, with currently more than 70 monoclonal antibodies (mAbs) approved by FDA and EMA [1,2,3]. Mabs in vivo structure The complex structure of mAbs lead to further efforts in order to understand their folding, denaturation and refolding [9, 10]. Heavy chains (HCs) and light chains (LCs) are co-translationally translocated into the endoplasmic reticulum (ER) and folding begins even before the polypeptide chains are completely translated. The Ig fold is characterized by a greek-key β-barrel topology in which the barrel is not continuously hydrogen bonded, but instead composed of two sheets, forming a sandwich-like structure [11]

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