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.