Cirmirakis, AK; (2014) An Investigation of the Artificial Chromosome Expression System for Production of Recombinant Proteins. Doctoral thesis , UCL (University College London).

Abstract

The requirement to produce complex, therapeutic proteins that possess human-like post-translational modifications for treatment of disease has accelerated the use of mammalian cell lines, in particular Chinese hamster ovary (CHO) cells, in the biopharmaceutical industry. However, producing stable, high expressing cell lines quickly, reproducibly and with relative simplicity whilst achieving a desired product quality profile proves challenging. Conventional methods involve random integration of the expression vector carrying the gene of interest (GOI) into the host genome, often resulting in clonal lines with highly variable protein expression. In order to overcome some of these challenges the artificial chromosome expression (ACE) system, comprising a functional artificial chromosome (AC) present in a CHO-K1 cell line, ACE targeting vector (ATV) with GOI, and a mutated λ integrase which catalyses site-specific integration of the ATV into the AC, has been developed. This targeted integration negates ‘position effects’ and reduces phenotypic variation. In this study this novel, minimally characterised system was investigated; the number of AC recombination sites was determined and optimal selection pressure and vectorology was established for the generation of highly productive cell lines. In an attempt to further improve protein production, two consecutive rounds of gene delivery were performed and its potential evaluated. Further, stability of the AC over long-term culture and its uncompromised structure over 50 cell passages was proven. In addition, a series of cell lines expressing recombinant proteins have consequently been investigated for stability over long-term culture, with the results underlining the need for optimal metabolite support for the cells to ensure consistent protein production. Another key feature of the system is the ability to isolate and transfer the AC into alternative host cell lines. To test this portability, the AC was isolated by FACS and an attempt to transfect into alternate CHO and HEK293 cell lines was made. Work presented in this study demonstrates the potential of the ACE system to become a predominant commercial platform for the production of recombinant proteins, owing to its high efficiency, competitive timelines for mammalian cell line generation, and low clonal variability.

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