The Evolution of Protein Expression and Cell Culture

Sep 30, 2007


The biopharmaceutical industry has seen an extraordinary evolution over the last 30 years since the first successes of recombinant DNA technology. Today, it represents the highest growth rate sector for the pharmaceutical industry. Developments in protein expression systems and cell culture technologies, along with major advances in analytical characterization capabilities, are at the core of the biopharmaceutical industry's rapid growth. This article presents an overview of how protein expression and cell culture have evolved over the last 20 years and highlights the emerging areas for development in each of these fields.

It is commonly believed that technologies in the next 10–15 years will enable sequencing an individualized human genome for less than $1,000. With innovations like these, the twenty-first century will certainly belong to biotechnology. From an industrial standpoint, the discovery of therapeutic molecules and the development of cell lines and processes to produce these molecules will be of paramount importance. This article describes various approaches that have been prevalent in the industry or are likely to be used in the future for generating cell lines with desirable traits and developing high titer cell culture processes.


The factors that dictate the choice of a cell line include the expression level, economic considerations, and desired product quality for a given biological function. Because of the ease and the speed with which E. coli processes could be scaled up, this bacterium was a dominant host until the mid 1990s. Fermentation systems for E. coli with capacities in the range of 40,000 L were operational in the 1980s. As the need for producing properly folded protein molecules and antibodies with appropriate glycosylation patterns grew, the industry shifted toward the use of eukaryotes, particularly mammalian cells. The processes developed in the 1980s using mammalian cells often resulted in lower product yields. However, product yields of 1–2 g/L are currently commonplace, and industrial claims of product titers as high as 10 g/L have been recently made. The two cell lines that are primarily used for recombinant protein production in the biotechnology industry are Chinese hamster ovary (CHO) and mouse myeloma (NS0) cells. At the same time, extensive use of and research in other expression systems, including bacteria,1 yeasts,2 insect cells,3 plants,4 human cells,5 and transgenic animals,6 are underway. Some notable applications that have emerged in the recent past include the glycoengineered yeast strains for controlled glycosylation,7 the use of duckweeds for producing protein therapeutics,8 and the cell-free production of recombinant proteins.9 Because the majority of investigational and clinical biotechnology products are currently being produced in mammalian cells, this article focuses on the use of mammalian cells to produce protein and antibody therapeutics.


Expression vectors commonly use a strong viral or cellular promoter and the gene of interest is generally isolated as a cDNA without introns. Because splicing is known to affect the cytoplasmic transport and translation of mRNA,10 most expression vectors will also include at least one intron sequence, usually located between the promoter and the cDNA coding sequence. The gene expression may also be improved by using the more abundant tRNA codons.11

It is known that the transcription of the inserted recombinant gene in the mammalian cell genome often can be influenced by the site of integration of the gene of interest in the mammalian cell genome. This may in turn affect the specific productivity and clonal stability of the resulting cell line. The incorporation of the recombinant gene in the host genome is a random process and several approaches have been demonstrated to either locally modify the chromatin structure or target a specific site of integration. Several protective cis-regulatory elements can be used to locally modify the chromatin structure used, e.g., insulators,12 locus control regions,13 scaffold or matrix attachment regions,14 ubiquitous chromatin opening elements,15 conserved antirepressor elements,16 and butyrate.17

The integration of the recombinant gene in the host genome is random, which makes it an inefficient process (approximately 1 in 10,000 events results in a successful recombination) and the chromosomal site of insertion and the copy number cannot be predicted.18 To overcome the problems associated with randomness of recombinant gene insertion in the host genome, the use of episomal vectors has been demonstrated as a viable strategy for recombinant protein production.19 However, the most notable development has been the demonstration of site-specific homologous recombination, which can be achieved by using recombinase enzymes (e.g., bacteriophage P1 Cre or yeast Flp enzymes). These enzymes exchange DNA between the transfected plasmid and the host genome at the sites thought to be relevant for high expression.20