Naming CHO cells for bio‐manufacturing: Genome plasticity and variant phenotypes of cell populations in bioreactors question the relevance of old names

Wurm, Florian M.

doi:10.1002/biot.202100165

Cited by 23 publications

(17 citation statements)

References 46 publications

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“…Another aspect that hampered genomic analyses in CHO cells was the lack of a high-quality genomic reference. In contrast to the human genome the first CHO genome was published a decade later in 2011 (Xu et al, 2011). Since then, major improvements have been made, including the assembly of a greatly improved CHO reference genome (CHOK1GS_HDv1) and re-sequencing of the Chinese hamster (CH) genome using both short and long read methods (PICR genome assembly (Brinkrolf et al, 2013;Rupp et al, 2014Rupp et al, , 2018.…”

Section: Introductionmentioning

confidence: 99%

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

Stadermann

Gamer

Fieder

et al. 2022

Biotech & Bioengineering

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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.

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Section: Introductionmentioning

confidence: 99%

“…(Frye et al, 2016; F. M. Wurm & Wurm, 2017; M. J. Wurm & Wurm, 2021). This genetic plasticity can translate into production instability and the need for extensive clone screening during cell line development, when manufacturing CHO cell lines are generated by random integration of transgenes into the structurally instable CHO genome.…”

Section: Introductionmentioning

confidence: 99%

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

Stadermann

Gamer

Fieder

et al. 2022

Biotech & Bioengineering

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“…First, clonal CHO cell populations exhibit natural heterogeneity, which could enable the isolation of a re-clone with desired features (i.e., higher productivity) [20,23,29,30,36]. This heterogeneity is generally accepted in the field and, importantly, is not indicative of instability in upstream process performance or product quality [6,23,37,38]. More recently, it has been suggested that the process of single cell cloning itself can impact CHO cell epigenetics in a heritable manner, leading to altered (and possibly improved) performance [39].…”

Section: Discussionmentioning

confidence: 99%

Improved Titer in Late-Stage Mammalian Cell Culture Manufacturing by Re-Cloning

Rehmann

Tian

et al. 2022

Bioengineering

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Improving productivity to reduce the cost of biologics manufacturing and ensure that therapeutics can reach more patients remains a major challenge faced by the biopharmaceutical industry. Chinese hamster ovary (CHO) cell lines are commonly prepared for biomanufacturing by single cell cloning post-transfection and recovery, followed by lead clone screening, generation of a research cell bank (RCB), cell culture process development, and manufacturing of a master cell bank (MCB) to be used in early phase clinical manufacturing. In this study, it was found that an additional round of cloning and clone selection from an established monoclonal RCB or MCB (i.e., re-cloning) significantly improved titer for multiple late phase monoclonal antibody upstream processes. Quality attributes remained comparable between the processes using the parental clones and the re-clones. For two CHO cells expressing different antibodies, the re-clone performance was successfully scaled up at 500-L or at 2000-L bioreactor scales, demonstrating for the first time that the re-clone is suitable for late phase and commercial manufacturing processes for improvement of titer while maintaining comparable product quality to the early phase process.

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“…In recent years, the scale of cell-culture-based production of biologicals in the biopharmaceutical industry has continued to expand. For example, the value of >USD 100 billion in revenue per year generated by antibodies and similar molecules from CHO cells [ 37 , 38 ]. There is large demand for Vero cell substrates in the production of five billion doses of inactivated SARS-CoV-2 vaccine [ 17 , 39 , 40 ].…”

Section: Discussionmentioning

confidence: 99%

Integrated Multi-Omic Characterization of the Detachment Process of Adherent Vero Cells with Animal-Based and Animal-Origin-Free Enzymes

Huang

et al. 2022

Cells

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Cell detachment techniques using animal-derived enzymes are necessary for the production of biopharmaceuticals that are made with the help of adherent cell cultures, although the majority of protein therapeutics (>USD 100 billion of income per year) are made under suspension cultures that do not require animal-derived proteins for manufacture. In this study, we establish the optimal Vero cell detachment process, and analyze physiological changes during cell detachment at the cellular and molecular levels. Using flow cytometry, we find that animal-based enzymes are more likely to induce apoptosis than animal-origin-free enzymes. We analyze the levels of RNAs, proteins, and metabolites in cells treated with two detachment strategies, and identify 1237 differentially expressed genes, 2883 differential proteins, and 210 differential metabolites. Transcriptomic analysis shows that animal-origin-free enzymes have a less significant effect on gene expression levels. Combined with proteomic analysis, animal-based enzymes affect the oxidative phosphorylation process and reduce the mRNA and protein levels of Cytochrome C Oxidase Assembly Protein 17 (COX17), which is a Cytochrome C Oxidase Copper Chaperone involved in the mitochondrial respiratory chain. Metabolomics analysis indicates that the levels of spermine and spermidine, which are involved in the glutathione metabolism pathway and apoptosis inhibition, are significantly reduced. Therefore, COX17, spermine, and spermidine may be biomarkers for evaluating the cell subculture process. In conclusion, we have deeply characterized the cell subculture process through multi-omics, which may provide important guidance for research and process evaluation to optimize cell detachment processes.

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Naming CHO cells for bio‐manufacturing: Genome plasticity and variant phenotypes of cell populations in bioreactors question the relevance of old names

Cited by 23 publications

References 46 publications

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

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

Improved Titer in Late-Stage Mammalian Cell Culture Manufacturing by Re-Cloning

Integrated Multi-Omic Characterization of the Detachment Process of Adherent Vero Cells with Animal-Based and Animal-Origin-Free Enzymes

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