Introduction

Pool Cell Lines for Development

Biologic pharmaceutical drug developers have been contemplating using uncloned stable mammalian cell pools for GLP toxicology production, GMP production, and Phase 1 clinical products for a number of years (Rajendra, 2017; Stuible, 2018). The advantages of stable mammalian cell pools include faster timelines and reduced costs to initiate clinical trials; however, until COVID-19 very few programs used this approach. In addition to traditional biologic therapeutics, other biologic products requiring cGMP production (e.g. molecules that are used in diagnostic assays, medical devices, or cell or gene therapy production) may also benefit from this type of approach. The main concerns around cell pool production have been both product and process consistency. As compared to clonal cell lines, there are limited published data showing that cell pools can reliably provide the product and process consistency necessary for clinical production. To our knowledge, there are few successful examples of this approach, with the documented ones published only recently (Agostinetto, 2022; Zhang, 2021; Xu et al, 2022). If cell pools are going to be used as outlined above, the characteristics of the product from a cell pool should mimic a clonal production cell line. The characteristics requiring consistency are high titer, production behavior, production stability, scalability, and protein quality. In addition, the process to produce pools should be fast and work well for a variety of molecule types. The technology described here meets these criteria to accelerate biologic product development.

Targeted Integration Cell Line Development Overview

The generation of high expressing, stable cell lines producing proteins with the desired product quality is the goal of any cell line development program. Typically, these requirements need to be achieved on a shortened timeline with the goal of getting to First in Human as quickly as possible. Toward this goal, many novel cell line engineering technologies have been developed for integration of transgenes into the CHO genome. These technologies can be broadly grouped into three main categories: random integration (Grandjean, 2011), semi-targeted integration (Bleck, 2005; Rajendran, 2021), and targeted recombination (Feary, 2020; Ng, 2021). Here, we explore the advantages of combining the latter two technologies by placing a relatively high number (>200) of Dock sites (aka landing pads) throughout the genome, followed by targeted recombination of an expression construct (Boat) into these dock sites.

GPEx® and GPEx® Lightning Technology

GPEx® technology (Figure 1A) is a versatile system designed to insert genes of interest into a wide variety of mammalian host cells (Bleck, 2005). It is based on the use of replication incompetent retroviral vectors (retrovectors) to actively insert the desired genes into or around the transcription start point of genes (Wu, 2003; Mitchell,2004). This preference for transcriptionally “active” regions of the genome allows for higher, more consistent levels of expression per copy of the gene inserted as compared to other methods of gene insertion. These integrated genes are maintained in an extremely stable manner through subsequent cell divisions as if they were small endogenous cellular genes (Bleck, 2012).
GPEx® Lightning technology leverages the GPEx® technology to insert Docks throughout the CHO genome. This parental Dock cell line is then co-transfected with a Boat plasmid containing the gene(s) of interest (GOI), and the recombinase plasmid. This leads to the targeted integration of multiple copies of the GOI into Dock sites throughout the genome. To select for cells containing a high number of targeted recombination events, we utilized a unique glutamine synthetase (GS) selection method in a GS knock-out cell line. This allows for enrichment of high producing cells without the use of selective chemicals (i.e. methionine sulphoximine, methotrexate). Here we demonstrate that GPEx® Lightning technology produces stable pools with high titer and consistent product quality which is maintained between pools and clones. This enables the use of cell pool-produced drug substance for toxicology, and potentially for clinical studies, to reduce product development timelines and costs.