Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives

Abstract

The expansion of the biologics pipeline depends on the identification of candidate proteins for clinical trials. Speed is one of the critical issues, and the rapid production of high quality, research-grade material for preclinical studies by transient gene expression (TGE) is addressing this factor in an impressive way: following DNA transfection, the production phase for TGE is usually 2-10 days. Recombinant proteins (r-proteins) produced by TGE can therefore enter the drug development and screening process in a very short time--weeks. With "classical" approaches to protein expression from mammalian cells, it takes months to establish a productive host cell line. This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studies.

A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications

“…The robustness, versatility and relative simplicity of PEI-mediated transient expression systems concomitant with the use of performing cell lines and episomal expression vectors [10][11][12] have made protein expression in mammalian cells a valuable alternative to bacterial systems. For a given cell line, the efficiency of PEI-mediated transfection depends on many parameters such as the culture medium in which the transfection occurs, the mode of cell culture (i.e.…”

“…Transient gene expression in mammalian cells is widely used for the fast production of recombinant proteins, mainly for pre-clinical in vitro and in vivo applications. Over the last 10 years, major improvements of the technique leading to better yields have made transient gene expression a competitive alternative to the establishment of stable cell lines as it shortens the time required to obtain the target protein in sufficient amount from several months to a few weeks only [7][8][9][10][11][12]. The capacity to quickly express recombinant proteins in mammalian cells by transfection is thus of great benefit not only for large-scale applications but also for highthroughput expression for functional screening and crystallization purposes [3,[11][12][13][14][15].…”

“…The human embryonic kidney 293 cell line (HEK293) and its derivatives, such as the HEK293-EBNA1 that stably expresses the Epstein-Barr virus Nuclear Antigen 1 (293E) or the simian virus 40 Large-T antigen (293T), are the most commonly used cell lines for transient gene expression [10][11][12]. Indeed, HEK293 cells allow proper folding and relevant post-translational modifications of the proteins.…”

“…They are also easy to grow in serum-free suspension culture and highly transfectable with most gene transfer agents [11]. Polyethylenimine (PEI) is a widely used transfection agent for small to large-scale protein expression as it is highly efficient and cost-effective [10][11][12]. PEI is a cationic polymer which binds to and condensates DNA into small cationic nanoparticles, thus facilitating DNA cell uptake, most likely via their interaction with cell-surface heparan sulfate proteoglycans [16,17].…”

Shear stress increases cytotoxicity and reduces transfection efficiency of liposomal gene delivery to CHO-S cells

“…Such scenarios are relevant to largescale transfections carried out in bioreactors for transient expression of recombinant protein, and to liposomal gene and drug delivery to blood cells where the liposomal complex is injected into blood vessels. The primary focus of this study is on suspension adapted CHO-S cells which have been used for large scale transfection in bioreactors to transiently express recombinant protein (Baldi et al 2007). Mardikar and Niranjan subjected various animal cells in suspension to shear stress and depending on the shear stress level report formation of pores, population and cell shrinkage (Mardikar and Niranjan 2000).…”