Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

Bin Li,Ryan Haryadi,Yee Jiun Kok,Xuezhi Bi,Helen X Pu,Zhiwei Song,Lin-Tang Goh,Ellen R Goldman,Lu Zheng,Steven Ho,Yuansheng Yang,Natasha A Pereira

doi:10.1371/journal.pone.0116878

Abstract

Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

Highlights

Recombinant monoclonal antibodies produced by CHO cells represent the most rapidly growing class of biotherapeutics
The phylogenetic trees of the heavy chain (HC) and light chain (LC) signal peptides are shown in S1 Fig. Detailed sequence information of all the signal peptides shown in S1 Fig. is shown in S1 Table
Sequences of human Ig HC and kappa LC cDNAs with complete coding regions were collected from the PubMed database

Summary

Introduction

Recombinant monoclonal antibodies produced by CHO cells represent the most rapidly growing class of biotherapeutics. Optimized Signal Peptides for Producing Antibody Drugs in CHO Cells increased dramatically and extensive efforts have been directed at improving their yields in mammalian cells [3]. A signal peptide that contains 5–30 amino acids present at the N-terminus of nascent proteins is recognized by the signal recognition particle (SRP) in the cytosol while the protein is still being synthesized on the ribosome. The SRP delivers the SRP-ribosome-nascent chain (SRP-RNC) complex to the SRP-receptor (SR) in the endoplasmic reticulum (ER) membrane. GTP-dependent mechanisms deliver the RNC complex to a membrane-bound translocon which allows translocation of the growing polypeptide chain into the lumen of the ER. After crossing the ER membrane, the signal peptide is cleaved off by a signal peptide peptidase (SPP) for reviews see [4,5,6,7,8,9,10]

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