Large-Scale Transient Gene Expression

The market for therapeutic proteins, including antibodies, currently exceeds $50 billion and is growing faster than any other sector of the pharmaceutical industry (1). More than 50 per cent of the processes approved for manufacturing such proteins rely on mammalian expression hosts, and this percentage is growing quickly (2). Such manufacturing processes typically use stable cell lines, where the gene for the protein of interest is integrated into the host chromosome as part of an appropriate expression vector. These stable cell lines are generated by the labour and time-intensive steps of gene transfer, selection, screening and stability testing, work that can take as long as a year after transfection. Transient gene expression (TGE) offers an alternative means of protein expression, where the protein production occurs right after transfection, allowing the recovery of purified protein within days or weeks.

While the concept of TGE is not new, due to a lack of scalable methods and low expression levels, the technology was used until recently only in research labs. However, with the race to launch antibody products on the market, there arose the need to develop high-throughput methods to produce mg-gram quantities of multiple antibodies or antibody fragments. This requirement has acted as a further impetus to TGE technology and led to its adoption by a number of biopharmaceutical companies, which now implement this technology at scales of up to 100 litres. TGE has emerged as a powerful technology, which can be used to:

• Support product discovery and development – not only could TGE allow high-throughput access to different protein candidates or antigens, but it could also predict possible issues with expression of a candidate protein like low expression levels, excessive aggregation, rapid degradation or lack of processing leading to non-activated protein. It could also help in early identification of process conditions, which might alleviate such issues.
• Speed-up project execution by providing early access to proteins for physical, chemical and biological (in vitro and in vivo) characterisation. Such data could already be used for filing IND applications, which would otherwise be delayed until the development of an early-stage process (some months after stable cell line generation) before such studies could begin.
• Provide early access to candidate protein in order to develop and optimise purification processes and to start performing formulation and stability studies.
• Manufacture cytotoxic proteins of pharmaceutical interest for which making cell lines is either difficult or impossible. While expression driven from inducible promoters could be an alternative, due to high uninduced level of expression from such promoters, they are often not a good solution.
• Manufacture proteins as vaccines or therapeutics for emergencies.
• Manufacture therapeutics for treating rare or orphan diseases or for use in individualised medicine where economics do not justify large investments of time, infrastructure and money for such small market applications.

By virtue of its speed and flexibility, TGE can add value and provide solutions to a host of situations and requirements. Not only does TGE allow rapid protein expression, it also does not require the large upfront investment necessary for the establishment of stable cell lines for those protein candidates which would not make it to clinical testing. With greater availability and acceptance of complementary technology like disposable cell culture systems, TGE can enable rapid production of large amounts of proteins with GMP-like quality without significant investment, expertise or manufacturing infrastructure. In order to improve its potential speed and flexibility, over the years, research in this field has focused on developing scalable transfection methods using improved transfection reagents, protocols and expression processes in order to obtain even higher transfection efficiencies and higher protein productivities. This article describes such developments and various other aspects of the TGE technology, and points out hurdles to its implementation and widespread adoption.