Development of a new CHO Transient Expression Platform for mAb Production, Evaluation of Novel Viral Episome Maintenance Proteins for Transient Gene Expression

Chinese hamster ovary (CHO) cells are the production system of choice for the expression of monoclonal antibodies (mAbs) and other biotherapeutics. Transient gene expression (TGE) platforms allow the rapid supply of high-quality pre-clinical material and are therefore the expression method of choice for early phase drug development over stable expression methods, which are time and resource intensive. To ensure consistent protein characteristics from discovery to development, it is desirable to express early material in cells closely related to those used during stable cell line development (CLD) and manufacturing. Therefore, adaptation of CLD CHO cell lines that are used for stable expression for early stage TGE is important in the drug discovery and manufacturing pipeline. TGE is characterised by the presence of an extra-chromosomal plasmid which is not integrated into the genome and therefore is lost over time. To establish latency, Epstein Barr virus (EBV) uses the Epstein Barr nuclear antigen 1 (EBNA-1) protein to retain its genome as an episome within a host cell, and has been widely adapted for use in TGE systems leading to improved plasmid retention and increased transient protein yields. EBV however does not naturally infect rodents, demonstrating human tropism, and therefore CHO cell TGE systems that rely on EBNA-1 are unable to achieve the episomal replication that EBV can accomplish during natural infection. This project describes the development of a new TGE system suitable for pre-clinical material supply using a CHO cell line. To improve transient protein expression from this host, the use of two alternative episome maintenance proteins (EMPs) was evaluated: latency associated nuclear antigen (LANA) derived from both KSHV and MHV68. The work describes the successful development of a new CHO cell TGE system through a holistic approach focusing on vector optimisation, bioprocess engineering and cell engineering, achieving comparable titres to other well established TGE systems. Moreover, the results from this work open up promising new areas of research to further increase transient expression yields from CHO and other mammalian cell expression systems.