Development of a CHOK1SV transient expression system for rapid generation of recombinant proteins

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The biopharmaceutical industry relies upon screening and adaptation procedures to identify stable, productive transfectants able to grow in defined media to produce a recombinant protein (rP). This procedure is not ideal early in the drug development pipeline when it is necessary to screen and evaluate potential rP drug targets and/or when the more rapid generation of recombinant material would allow more molecules into first-in-human studies faster. Transient gene expression therefore provides a means for the generation of rapid amounts of rP for such studies. Transient expression technology for the production of biotherapeutic relevant rPs has mostly utilised suspension adapted human embryonic kidney cells (HEK293 cells) and calcium phosphate precipitation although recently Chinese hamster ovary (CHO) cells have been used. Indeed, a number of companies are investigating transient expression technology to produce hundreds of mgs of rPs in a matter of days using non-viral DNA delivery systems and cells cultured in bioreactors. Recent reports also demonstrate the potential to improve titres in transient processes using a systematic approach including optimisation of the vector and transfection conditions, use of cell cycle regulators (p18, p21) and fibroblast growth factor, and addition of valproic acid to the media. The optimisation of the ratio of IgG heavy and light chain genes and the use of fed-batch cultures when generating IgG material using CHO transient monoclonal antibody (mAb) expression have been reported to improve yields whilst gene optimisation of heavy and light chain transcripts also increases mAb production levels during transient expression in CHO cells. The project Lonza Biologics has a CHO suspension adapted host cell line (CHOK1SV) which is able to achieve high cell concentrations and maintain viability for long periods of time compared to other suspension adapted CHO lines. Lonza also has its own proprietary vector expression system, the GS System. In this project the student will develop a novel CHOK1SV and GS vector based (DNA) transient expression system for the rapid production of 100's of mg's of rPs. This will build upon preliminary work from both the academic and industrial labs that show manipulation of transient systems can lead to increased yields. The test molecules will include a model mAb (gene optimised and non-optimised), tPA and rhEPO. Initially the student will investigate delivery systems and internalisation of the DNA to the nucleus after modification of the vector (e.g. via use of nuclear localisation sequences and partitioning elements). The student will also determine the influence of environmental conditions (e.g. temperature, pH) and media composition and feeding on product yield. The student will also investigate host cell engineering strategies to improve yield and speed of the transient expression process. In particular the student will focus upon cell cycle engineering (e.g. p21) and chaperones and foldases that we have shown in the laboratory in transient studies increases the high-level transient expression of model proteins (e.g. Torsin A, translation initiation factors). Finally, expression of the Vaccinia E3L protein in CHO strains has a positive effect upon transient expression of mAb. E3L acts to block the activation of cellular genes that respond to viral infection, in particular PKR. Work in the lab at Kent has shown that PKR activity can also be modulated by manipulation of p58 involved in preventing or modulating the activity of this kinase. The student will therefore investigate the effect of modulating PKR activity on transient yields. The outcomes: 1. Development of novel vector, CHOK1SV derived host and processing (media/feeds) technology for the expression of 100's mg of rPs 2. An understanding of the limitations upon transient expression of rPs from CHOK1SV cells