Mammalian Expression System and Improvisation for High Production

Mammalian protein expression system has the function of protein folding and post-translational modification which let the protein closer to the natural protein, so that to obtain the same biological activity with natural protein. Therefore, the Mammalian Cell Expression System is the most widely used in the development and production of recombinant protein drugs, particularly in the therapeutic monoclonal antibodies. Email :marketing@medicilon.com.cn web:www.medicilon.com

Abstract: Recombinant proteins are of immense value both for research as well as for therapeutic use. Ever since insulin was introduced for therapeutic use there is lot of interest in the development of recombinant proteins as therapies. Recombinant proteins are often produced in heterologous host such as Bacteria, Yeast, Mammalian Cells, Transgenic Plants and Animals. Each one of these host cell lines have their own advantages and disadvantages. With the increase in the number of monoclonal antibody (mAb) based therapeutics at different stages of clinical trials is demanding for an efficient mammalian expression system. Mammalian expression system is often known to produce proteins with proper post-translational modification that were equivalent to that of humans. This review article is focused on strategies for increasing proteins production, host cell lines and the applications and products that were produced so far using mammalian expression systems. The purpose of writing this article is to summarise the advances in mammalian expression systems and their use. 1. Introduction The major part of biopharmaceuticals is protein based and extracted in recombinant hosts. For extracting some biopharmaceutical proteins, bacteriological systems are useful because they are cheap with higher output, and fast execution. In 1980 the industry has shifted towards the utilization of mammalian cells for the correctly folded protein production with suitable glycosylated patterns. Mammalian cells are favoured hosts for manufacturing of complex protein therapeutics, as the post translational modifications are functionally and pharmacokinetically compatible with humans. Stable cell lines which are an important requirement for production of biopharmaceutical proteins are developed by transfecting host cells with the suitable vector system with the gene of interest .The normal productivity scale achieved by using mammalian cells is 1 to 2 g/L and recently it has been claimed to 10g/L in the production scale. Thus, during the last two centuries, cultured mammalian cells have became the platform for the production of recombinant proteins or therapeutics in spite of low productivity, high production cost, and other difficulties when compared with bacteriological systems. With the huge demand for production of monoclonal antibodies the capacity of cell culture volume has been increased from < 300,000 litres to approximately 3 million litres. In case of mammalian and animal cell lines, most of the proteins are secreted with subsequent refolding rather than demanding cell lysis to extract as in case with prokaryotic system. The common nonhuman mammalian cell lines used for manufacturing of therapeutic proteins are Chinese hamster ovary (CHO) cells, mouse myeloma cells (NS0 and Sp2/0) and baby hamster kidney (BHK21) cells . The post translation modifications like galactose-a1, 3-galactose(agal) and N-glycolylneuraminic acid (NGNA) which are not expressed in human are also been produced by these nonhuman mammalian cell lines. As humans have circulating antibodies against to these N-glycans, the clones with suitable glycan profiles are screened with utmost care when non-human mammalian cell lines are used for production of recombinant proteins. 2. Improvements in Mammalian Expression System Mammalian expression improvements are accomplished by suitable vector design. Vectors possessing strong promoter for example cytomegalovirus (CMV) promoter to obtain higher level transcription of messenger RNA, correct signal peptide, specific introns and optimization of product gene codon for target cell type, balance of GC/AT ratio have shown to achieve the high level of gene expression by improving the secretion process and processing of mRNA. Utilization of transcription control units are also essential . Gene amplification system is the most reliable method brought about by selective marker utilization for example DHFR or glutamine synthetase (GS) for the generation of cell lines. There are around 200 recombinant biopharmaceutical products available in the market and many more are under clinical improvement. Most of the proteins products are glycosylated. An expression system permitting effective production of quality glycoprotein is very important. For the maintenance of long time residence in circulation the sialylation of glycoproteins is very essential. The degree of sialylation varies depending on the type of product, the hose cell line used and the culture conditions maintained for the growth. The biosynthesis of sialic acid, obtainability of nucleotide-sugars, and CMP sialic acid transporter and the sialyl-transferase are the limiting steps for sialylation. 2.1 Gene Integration The positional integration of recombinant gene affects the rate of transcription. The negative effect of random integrations like exogenous gene silencing has been overcome by uses the different strategies. A cis-regulated positive insulator element, Ubiquitous chromatin opening elements (UCOE) is used to increase transgene expression stability. UCOE is an un-methylated CpG island fragments which contains the chromatin in an open, non tissue specific manner that allows the gene integration in an independent manner for rapid expression in a vector. There is a significant increase in production even with the single copy integrations when vectors with UCOE are used for e.g. mAb production by six fold increase in CHO cells. In less than one month from transfection, rapid Production of 300mg has been reported when UCOE are used in combination with serum free media and suspension cell lines. Matrix attachment regions (MARs) are the attachment points of DNA which promotes the gene expression. The chromatin structure is maintained trancriptionally active by the MARS with the creation of chromatin loops. The demethylation of DNA may occur indirectly with increase in histone hyperacetylation by which it will be easily accessible for the transcription machinery by flanking the transgene. The site specific homologous recombination can be achieved by the use of particular recombinases which catalyzes the integration of gene of interest into the hot spot of host cell line. The specific attachment regions are crucial for this type of recombination. The specific enzymes like P1 Cre recombinase from bacteriophage, integrase from lambda phage and FLP from yeast are mostly used to enhance the integration of target genes. 2.2 Gene targeting The engineered chromosomes have to be considered for the improvisation of transfection process. For the targeted cell transfection, a mammalian-based artificial chromosome expression (ACE) System with an ACE targeting vector (ATV) and ACE λ integrase (attP attachment site) for recombination acceptor sites are used. This type of recombinant gene targeting without the use of gene amplification system helps to achieve high copy number producers under selection pressure by promoterless marker gene activation. More than 500 mg/L in batch terminal shake flask was produced by using ACE platform cell line for monoclonal antibodies.The cultivated mammalian cells can be integrated with the gene of interest by using the transposable elements like PiggyBac (PB) and sleeping beauty. In absence of selection pressure, the CHO cells transfected by PiggyBac showed stability for 3 month and an increase in the expression of fusion protein of tumor necrosis factor -Fc. 2.3 Cell Line Engineering The time integral of viable cell density (IVCD) and the productivity of specific protein(q) are the important factors which influence the quantity of the recombinant protein expression. The strategies for improvement of high production by cell line engineering include extension of cell culture longitivity, specific growth acceleration and modification of cellular functions.cell lines has been engineered by inserting the Proto-Oncogenes, apoptosis inhibitor genes, cell cycle controlling genes (cyclins) and various growth factor genes . The specific production and secretions has been enhanced by the expression of X-box binding protein 1 (XBP-1) in CHO cells by the utilization of combined technology of the anti apoptosis and secretion engineering. Introduction of X-linked inhibitor of apoptosis (XIAP) by transfecting the cell line to reduce the programmed cell death has increased the titers to 60%. The latest technologies with the help of protein domains like zinc finger proteins and the transcription activator like effectors (TALEs) are designed for the specific DNA sequence recognition. By fusing these protein domains with the specific endonucleases domains the target sites are created and can cut at particular DNA sequences. Fok I is the mostly used endonuclease domain for this purpose as it doesn't possess any specific site for cleavage and dimerization of DNA is required for cleavage.Triple knockout genes were developed in CHO cells using the Zinc Finger nucleases which includes the knockout of dihydrofolate reductase (DHFR) and glutamine synthase (GS) genes for the selection of high gene copy clones and α1, 6-fucosyltransferase8 (FUT8) for the production of non-fucosylated IgG1 with strong therapeutic potential increased antibody dependent cell mediated cytotoxicity (ADCC) in humans in vivo at very low dosages. Development of Faster cells lines were generated and launched by Lonza using knockout of GS gene in CHO cell lines by the ZFN technology. Use of Meganucleases and in silico modeling coupled with OMICS tools are the latest tools used in the cell line development for the enhanced stability and high expression of the recombinant proteins. . The latest technology with small interfering RNA is used to change the gene regulations in cells and the in silencing of genes for multiple targets for the better production in CHO cell lines . 2.4 Process development The important factors like optimization of medium and the feeding strategies have been employed to increase production rate. By using the high end pH - controlled feeding strategy for the lactate effect, the mAb and other recombinant protein production titers has been doubled in almost all CHO cell lines from bench to large scale production.The fed batch process with the highly defined chemical media reportedly increased the production rate from 44 p/c/d to 54 p/c/d using plant hydrolysate and the maximum titer of 10 -13 g/L of mAb and Fc-fusion protein . Raman spectroscopy is used to analyze and maintain the consistency of the media components for large scale production. Cryobag banking procedure was generated to shorten the time period from seed development to inoculums for production.