BioProcess International CELL LINE DEVELOPMENT September 2019 Introduction . .1 Reporting from the Frontiers of Cell Line Engineering at BPI Europe and BPI West FROM GENOME Cheryl Scott Creating New Cell Lines By Genome Editing . 10 Simplifying Cell-Based Assays and Improving EDITING TO MEDIA Production of Biomolecules Masahito Yamagata Streamlined Serum-Free Adaptation of ADAPTATION CHO-DG44 Cells . .14 Using a Novel Chemically Defined Medium N. Moroni, A. Di Nunzio, A. Perli, R. Puca, K. Hammett, G. Piras, J. Brooks, and L. Sibilio FEATURED REPORT Digital Edition Copyright Notice The content contained in this digital edition (“Digital Material”), as well as its selection and arrangement, is owned by Informa. and its affiliated companies, licensors, and suppliers, and is protected by their respective copyright, trademark and other proprietary rights. 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NEITHER Informa NOR ANY THIRD PARTY CONTENT PROVIDER OR THEIR AGENTS SHALL BE LIABLE FOR ANY ACT, DIRECT OR INDIRECT, INCIDENTAL, SPECIAL OR CONSEQUENTIAL DAMAGES ARISING OUT OF THE USE OF OR ACCESS TO ANY DIGITAL MATERIAL, AND/OR ANY INFORMATION CONTAINED THEREIN. CLD Platform Introduction Reporting from the Frontiers of Cell line Engineering at BPI Europe and BPI West Cheryl Scott very biomanufacturing process begins with transfection of recombinant genes into pools of cells — followed by a succession of E screenings from which will emerge (ideally) a single progenitor cell of the new production cell line. Cast aside will be those cells that do not uptake the correct genetic material, those incapable of thriving in bioprocess conditions, those that fail to produce recombinant protein at relevant levels, and those without demonstrated clonality and relative genetic stability. Over the past several years, advancing technologies for genetic engineering, screening, clone selection, analysis, and data management have HTTPS://STOCK.ADOBE.COM brought about a revolution in cell line development. Audiences at the BioProcess International (BPI) An efficient cell factory, Borth said, results from conferences have watched this field evolve as “a complex interplay of many genes and activities. presenters shared their insights and reported on their Productive cells reduce ‘unnecessary’ functions. experiences. This year’s BPI Europe and BPI West They are efficient in providing precursors and events continued in that vein with presentations on energy.” Although certain pathways do come up time technology platforms, clonality, cell line (genetic) and again, the precise genes getting regulated engineering, and development of biosimilars and differentially within those pathways will vary. other challenging product modalities. “Epigenetic regulation determines the transcriptome of each cell and cell type,” Borth The Track at BPI Europe explained. That includes different layers of The BPI Europe conference and exhibition in Vienna, regulation both for long-term “memory” and rapid Austria, this past April devoted an entire track to cell response to changing culture conditions. And this line engineering and development. Speakers came makes epigenetics the second major contributor from across the biopharmaceutical “ecosystem”: (after the genome itself) to phenotypic variation. She bioprocess suppliers, drug developers, and academia. said that the true secret to control of gene expression They discussed genetics, new technologies, and case will be to use epigenetics to determine phenotype studies from the frontiers of the industry. and behavior. “The future of phenotype control will Genomics and More: Nicole Borth (professor at require more detailed understanding of gene BOKU University and ACIB) focused on epigenetics of expression regulation. Manipulating expression of Chinese hamster ovary (CHO) cells in her talk, multiple genes simultaneously requires synthetic “Genome Scale Science for CHO.” She explained that biology and epigenetic control tools.” scientists had spent 20 years hunting for a single In “Engineering Complex Traits in the gene to boost process performance of mammalian Postgenomic Era of CHO Cell Line Development,” cell lines (whether overexpressed or knocked out/ Nathan Lewis (assistant professor in the departments down) by improving productivity, growth, and of pediatrics and bioengineering at the University of product quality all in one fell swoop. Unfortunately, California, San Diego) echoed Borth’s conclusions. she said, such a “miracle gene” is not likely to exist. “Many desirable traits require multiple gene edits,” Featured Report September 2019 17(9)i BioProcess International 1 Upcoming KNect365 Events stoichiometric gene expression. He identified four Pre-Conference Symposium on Cell Line Development categories of expression cassette design criteria: cell and Engineering at the BioProcess International productivity, quality of expressed protein products, Conference and Exhibition in Boston, MA, on design simplicity, and control and stability of protein 9 September 2019; https://lifesciences.knect365.com/ expression. bioprocessinternational/agenda/1# Focusing on design space, Brown described a Online Academy: Early Stage Upstream Process quality by design (QbD) approach to expression Development, five weeks starting 1 October 2019; https:// cassettes. Designers must answer three questions: lifesciences.knect365.com/early-stage-upstream-process- What can’t we control precisely (gene specificity)? development What rates will we keep constant (set points)? and Two-Day Course: Upstream Bioprocess Development in How will we achieve precise stoichiometries (control London, UK, on 26–27 November 2019; https://lifesciences. point)? Brown illustrated the testing phases that knect365.com/upstream-bioprocess-development follow those decisions, identifying the ultimate goal as a system that enables robust, user-defined Track on Cell Line Development and Upstream Processing manipulation of gene expression stoichiometry. at BioProcess International Asia in Kyoto, Japan, on 26–28 February 2020; https://lifesciences.knect365.com/ “Further engineering is required to achieve bpi-asia/ highly predictable, fine-tuned control,” he explained, “and to achieve the objective design criteria.” Cell Line Development and Engineering, colocated with Advancing Technologies: Anton Bauer (senior BioProcess International US West in Santa Clara, CA, on scientist at the Medical University of Vienna) 9–12 March 2020; https://lifesciences.knect365.com/ highlighted BESTcell technology from The Antibody cellline Lab in “The BEST of Both Worlds — Targeted Cell Line Development and Engineering Europe at Integration and Multiple Copies: How Can This Go BioProcess International Europe in Amsterdam, The Together for Improved Cell Line Development?” He Netherlands, on 28–30 April 2020; https://lifesciences. identified the challenge of developing highly knect365.com/bpieurope productive stable mammalian cell clones within just three weeks from transfection. he explained. “Pathway knowledge and large data Common methods for generation of stable sets can guide cell engineering.” mammalian cell lines include random integration of As an example, he highlighted the risk of virus classical plasmid constructs; including chromatin contamination of mammalian cell lines and cultures. modulators on those plasmids (for random Profiling virus-resistant cells points to regulators of integration of the plasmid constructs flanked by resistance. In a case study, knocking out “antirepressors” for chromatin modulation); and combinations of resistance repressors created virus- knock-in approaches (using highly active host-cell resistant cells. loci for transcription of a transgene). But Bauer Niall Barron (director of the National Institute for pointed out that silencing of the gene of interest