Structural analysis of random transgene integration in CHO manufacturing cell lines by targeted sequencing

Abstract

Genetically modified CHO cell lines are traditionally used for the production of biopharmaceuticals. However, an in-depth molecular understanding of the mechanism and exact position of transgene integration into the genome of pharmaceutical manufacturing cell lines is still scarce. Next-generation sequencing (NGS) holds great promise for strongly facilitating the understanding of CHO cell factories, as it has matured to a powerful and affordable technology for cellular genotype analysis. Targeted Locus Amplification (TLA) combined with NGS allows for robust detection of genomic positions of transgene integration and structural genomic changes occurring upon stable integration of expression vectors. TLA was applied to generate comparative genomic fingerprints of several CHO production cell lines expressing different monoclonal antibodies. Moreover, high producers resulting from an additional round of transfection of an existing cell line (supertransfection) were analyzed to investigate the integrity and the number of integration sites. Our analyses enabled detailed genetic characterization of the integration regions with respect to the number of integrates and structural changes of the host cell's genome.Single integration sites per clone with concatenated transgene copies could be detected and were in some cases found to be associated with genomic rearrangements, deletions or translocations. Supertransfection resulted in an increase in titer associated with an additional integration site per clone. Based on the TLA fingerprints, CHO cell lines originating from the same mother clone could clearly be distinguished.Interestingly, two CHO cell lines originating from the same mother clone were shown to differ genetically and phenotypically despite their identical TLA fingerprints. Taken together, TLA provides an accurate genetic characterization with respect to transgene integration sites compared with conventional methods and represents a valuable tool for a comprehensive evaluation of CHO production clones early in cell line development.

A CHO stable pool production platform for rapid clinical development of trimeric SARS‐CoV‐2 spike subunit vaccine antigens

“…Interestingly, although the number of data points is limited, there appears to be a negative correlation between copy number and protein productivity for these Delta clones: cells within a stable pool that have integrated a greater number of plasmid copies appear less likely to be among those producing the most recombinant protein (Figure 10b). A wide range of integrated transgene copy numbers has been reported in the literature for CHO pools and clones produced using a variety of methods, including many reports of high-producing cells with copy numbers similar to those reported here (Balasubramanian et al, 2018;Derouazi et al, 2006;Dhiman et al, 2020;Schmieder et al, 2022;Stadermann et al, 2022). Therefore, the copy number results we have obtained are not unexpected.…”

“…The random integration nature of the stable plasmid transfection approach used to generate our CHO pools dictates that integration of the plasmid into the host cell genome is a less well‐controlled process than for targeted integration approaches (transposases/RMCE): genome‐integrated plasmid sequences are expected to be found in different orientations, as concatemers and in truncated forms (Wurm & Petropoulos, 1994). Breakpoints between concatemerized plasmids as well as between plasmids and flanking genomic CHO DNA are also not predictable (Dhiman et al, 2020; Stadermann et al, 2022). Despite this, we have found production of non‐full‐length protein products from randomly integrated stable CHO pools to be quite infrequent: in the over 30 pools expressing spike proteins generated over the last 2 years, there was only a single pool expressing a truncated, discrete secreted product detected at low levels (shown in Supporting Information: Figure S4).…”

“…The random integration nature of the stable plasmid transfection approach used to generate our CHO pools dictates that integration of the plasmid into the host cell genome is a less well-controlled process than for targeted integration approaches (transposases/RMCE): genome-integrated plasmid sequences are expected to be found in different orientations, as concatemers and in truncated forms (Wurm & Petropoulos, 1994). Breakpoints between concatemerized plasmids as well as between plasmids and flanking genomic CHO DNA are also not predictable (Dhiman et al, 2020;Stadermann et al, 2022).…”

Nanopore Cas9‐targeted sequencing enables accurate and simultaneous identification of transgene integration sites, their structure and epigenetic status in recombinant Chinese hamster ovary cells

“…The identification of transgene ISs, however, is challenging as usually only the plasmid sequence before transfection is known. Targeted‐locus‐amplification (TLA) coupled with next‐generation sequencing (NGS) is currently the method of choice to identify ISs of transgenes in mammalian cells and is readily used in industry (Schmieder et al, 2022; Stadermann et al, 2022). The technology is based on crosslinking live cells and proximity ligation followed by Illumina short‐read NGS (de Vree et al, 2014).…”

Nanopore Cas9-targeted sequencing enables accurate and simultaneous identification of transgene integration sites, their structure and epigenetic status in recombinant Chinese hamster ovary cells

“…The identification of transgene integration sites, however, is challenging as usually only the plasmid sequence prior to transfection is known. Targeted-Locus-Amplification (TLA) coupled with Next Generation Sequencing (NGS) is currently the method of choice to identify ISs of transgenes in mammalian cells and is readily used in industry (Schmieder et al, 2022;Stadermann et al, 2022). The technology is based on crosslinking live cells and proximity ligation followed by Illumina short-read NGS (de Vree et al, 2014).…”