Heterologous protein production using euchromatin-containing expression vectors in mammalian cells.

Wolfgang Sommeregger,Andreas Gili,Beatrice Grabner,Renate Kunert,Katharina Fauland,Patricia Stiedl,Katalin Zboray,Herwig P Moll,Anton Bauer,Emilio Casanova,Thomas Sterovsky,Edith Bogner

doi:10.1093/nar/gkv475

Abstract

Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.

Highlights

Recombinant protein production in mammalian cells is the predominant way of nowadays biologic drug production [1,2]
Cells were resuspended in 10% ice cold glycerol solution and aliquoted to Eppendorf tubes (100 ␮l) prior snap freezing in liquid N2 or directly used for electroporation. 50–100 ng of linearized plasmid DNA carrying the insert for Bacterial Artificial Chromosome (BAC) modification (Figures 1b and 4a plasmid-controls) flanked by 120–200 base pair homology regions (HR) to the respective BAC was electroporated to these cells with a Gene Pulser Xcell (BioRad) electroporator
We searched for BACs containing a permissive chromatin environment and combined them with strong ectopic promoters

Summary

Introduction

Recombinant protein production in mammalian cells is the predominant way of nowadays biologic drug production [1,2]. The recent sequencing of the CHO-K1 genome [7] combined with ‘Omics’ based systematic approaches (i.e. transcriptomics, proteomics and metabolomics) will result in a better understanding of these processes (Reviewed in [8,9,10]) Results of these studies will help to engineer CHO cells with improved culture characteristics, increased lifespan and production [11]. Another aspect strongly impacting protein yield and stability is the nature of the expression vector used to generate producer cell lines [11]. To circumvent chromatin positional effects, expression vectors can be flanked by cis-regulatory elements which reduce the positional ef-

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Conclusion