Mammalian cell lines are important host cells for the industrial production of pharmaceutical proteins owing to their capacity for correct folding, assembly and post-translational modification. In particular, Chinese hamster ovary (CHO) cells are the most dependable host cells for the industrial production of therapeutic proteins. Growing demand for therapeutic proteins promotes the development of technologies for high quality and productivity in CHO expression systems. The following are fundamentally important for effective production. 1) Construction of cultivation process. The CHO-based cultivation process is well established and is a general platform of therapeutic antibody production. The cost of therapeutic protein production using CHO cells is equivalent to that using microbial culture. 2) Cell line development. Recent developments in omics technologies have been essential for the development of rational methods of constructing a cell line. 3) Cell engineering for post-translational steps. Improvement of secretion, folding and glycosylaiton is an important key issue for mammalian cell production systems. This review provides an overview of the industrial production of therapeutic proteins using a CHO cell expression system.

Previously, we established an easy and quick construction method for obtaining a stable and highly productive gene‐amplified recombinant Chinese hamster ovary (CHO) cell line. With a gradual increase in methotrexate (MTX) concentration, gene‐amplified cell pools had high and stable specific growth and production rates. Moreover, the phenotype of gene‐amplified cells seemed to be affected by the location of the amplified gene in chromosomal DNA. We suspected that various kinds of gene‐amplified cells might appear during the long‐term selection to construct gene‐amplified cell pools. To clarify the behavior of gene‐amplified cell pools during a stepwise increase of MTX concentration, we isolated gene‐amplified clones derived from gene‐amplified cell pools. We compared the characteristics of isolated clones, such as the productivity of recombinant protein, stability of amplified genes, and the location of amplified genes. As a result, telomere‐type clones, in which the amplified gene was located near the telomeric region, were found to be more stable and productive than other types of clones. Telomere‐type clones had over 100 copies of amplified genes in the chromosomal DNA. In contrast, a large number of other types of clones had less than 10 copies of amplified genes. During long‐term cultivation in the absence of MTX, in other types of clones, amplified genes rapidly decreased in the chromosomal DNA.

Background: The integration of biotechnology into chemical manufacturing has been recognized as a key technology to build a sustainable society. However, the practical applications of biocatalytic chemical conversions are often restricted due to their complexities involving the unpredictability of product yield and the troublesome controls in fermentation processes. One of the possible strategies to overcome these limitations is to eliminate the use of living microorganisms and to use only enzymes involved in the metabolic pathway. Use of recombinant mesophiles producing thermophilic enzymes at high temperature results in denaturation of indigenous proteins and elimination of undesired side reactions; consequently, highly selective and stable biocatalytic modules can be readily prepared. By rationally combining those modules together, artificial synthetic pathways specialized for chemical manufacturing could be designed and constructed.Results: A chimeric Embden-Meyerhof (EM) pathway with balanced consumption and regeneration of ATP and ADP was constructed by using nine recombinantE. colistrains overproducing either one of the seven glycolytic enzymes ofThermus thermophilus, the cofactor-independent phosphoglycerate mutase ofPyrococcus horikoshii, or the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase ofThermococcus kodakarensis. By coupling this pathway with theThermusmalate/lactate dehydrogenase, a stoichiometric amount of lactate was produced from glucose with an overall ATP turnover number of 31.Conclusions: In this study, a novel and simple technology for flexible design of a bespoke metabolic pathway was developed. The concept has been testified via a non-ATP-forming chimeric EM pathway. We designated this technology as “synthetic metabolic engineering”. Our technology is, in principle, applicable to all thermophilic enzymes as long as they can be functionally expressed in the host, and thus would be potentially applicable to the biocatalytic manufacture of any chemicals or materials on demand.