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• W4200360858 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." @default.
• W4200360858 created "2021-12-31" @default.
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• W4200360858 date "2022-01-19" @default.
• W4200360858 modified "2023-10-13" @default.
• W4200360858 title "Structural analysis of random transgene integration in CHO manufacturing cell lines by targeted sequencing" @default.
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• W4200360858 doi "https://doi.org/10.1002/bit.28012" @default.
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