Cell Line Development for Therapeutic Protein Production

Noh, Soo Min; Shin, Seunghyeon; Lee, Gyun Min

doi:10.1002/9783527811410.ch2

Cited by 7 publications

(11 citation statements)

References 125 publications

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“…1 For each therapeutic product, a stable cell line with a high product titer and a desirable protein quality must be developed. 2 The conventional cell line development process is long and unpredictable, spanning several months and involving the screening of several hundred cell clones for high productivity. 3 To accelerate cell line development, more efficient and predictable methods for generating high-producing CHO cell lines are required.…”

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confidence: 99%

Multicopy Targeted Integration for Accelerated Development of High-Producing Chinese Hamster Ovary Cells

et al. 2020

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Multicopy Targeted Integration for Accelerated Development of High-Producing Chinese Hamster Ovary Cells

et al. 2020

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“…The ideal goal of a biopharmaceutical CLD workflow is to produce glycoprotein biologics or other proteins of interest in multiple gram/liter scale with desired expression stability and quality profiles (e.g., glycosylation). This development process is multifaceted and can be grouped into expression plasmid design and DNA cloning, transfection and selection of stable pools, single‐cell cloning and monoclonality assurance, cell expansion, and analysis of protein expression level and clone stability, as shown in Figure 1 10,11 . Once the expression plasmids are made, workflows generally start with transfection of host cells with one or multiple plasmids containing genes of interest.…”

Section: Introductionmentioning

confidence: 99%

“…Subsequently, the pool productivities for the recombinant protein of interest are determined. The next step involves isolating single cells from top pools, expanding them and assessing the productivity of clonal cell populations after specified expansions 11 . This isolation process is popularly known as “limited‐dilution cloning” (LDC) or “single‐cell cloning” and along with the monoclonality assurance (or proof) and productivity screening steps, form the most labor‐, time‐ and resource‐consuming steps in a manual CLD workflow.…”

Section: Introductionmentioning

confidence: 99%

“…Based on their productivity profile, clonal cells (clones) originating from single cells are ranked and top clones are expanded to test the protein expression levels and clonal stability profiles. Finally, top hits demonstrating adequate stability are grown in different media and under different process parameters to improve the productivity further and adjust certain product quality attributes (e.g., glycosylation, charge variants and aggregation) before being scaled to production levels 11,12 …”

Section: Introductionmentioning

confidence: 99%

“…Further, cell expansion and clone screening for identification of the top 10 clones (top hits) takes 4–5 weeks, and stability assessment of one or more top hits requires ∼6 weeks (or 60 generations), assuming a doubling time of ∼17 h for CHO‐S™ cells 12 . After the single‐cell cloning step, screening of several hundred clones is typically required in systems that rely on random integration of plasmid DNA into various regions of the genome, as cells transformed through such procedures vary substantially in protein expression 11 . Thus, it can take 23–27 weeks or ~6 months or more from transfection to final clone selection for stable CLD for recombinant protein production using CHO‐S™ cells and the dihydrofolate reductase/methotrexate (DHFR/MTX) system for selection 12 .…”

Section: Introductionmentioning

confidence: 99%

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High‐throughput and automation advances for accelerating single‐cell cloning, monoclonality and early phase clone screening steps in mammalian cell line development for biologics production

Tejwani

Chaudhari

Rai

et al. 2021

Biotechnology Progress

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Mammalian cell line development is a multistep process wherein timelines for developing clonal cells to be used as manufacturing cell lines for biologics production can commonly extend to 9 months when no automation or modern molecular technologies are involved in the workflow. Steps in the cell line development workflow involving single‐cell cloning, monoclonality assurance, productivity and stability screening are labor, time and resource intensive when performed manually. Introduction of automation and miniaturization in these steps has reduced the required manual labor, shortened timelines from months to weeks, and decreased the resources needed to develop manufacturing cell lines. This review summarizes the advances, benefits, comparisons and shortcomings of different automation platforms available in the market for rapid isolation of desired clonal cell lines for biologics production.

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Generation of a host cell line containing a MAR‐rich landing pad for site‐specific integration and expression of transgenes

Oliviero

Hinz

Bogen

et al. 2022

Biotechnology Progress

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In recent years, targeted gene integration (TI) has been introduced as a strategy for the generation of recombinant mammalian cell lines for the production of biotherapeutics. Besides reducing the immense heterogeneity within a pool of recombinant transfectants, TI also aims at shortening the duration of the current cell line development process. Here we describe the generation of a host cell line carrying Matrix‐Attachment Region (MAR)‐rich landing pads (LPs), which allow for the simultaneous and site‐specific integration of multiple genes of interest (GOIs). We show that several copies of each chicken lysozyme 5'MAR‐based LP containing either BxB1 wild type or mutated recombination sites, integrated at one random chromosomal locus of the host cell genome. We further demonstrate that these LP‐containing host cell lines can be used for the site‐specific integration of several GOIs and thus, generation of transgene‐expressing stable recombinant clones. Transgene expression was shown by site‐specific integration of heavy and light chain genes coding for a monospecific antibody (msAb) as well as for a bi‐specific antibody (bsAb). The genetic stability of the herein described LP‐based recombinant clones expressing msAb or bsAb was demonstrated by cultivating the recombinant clones and measuring antibody titers over 85 generations. We conclude that the host cell containing multiple copies of MAR‐rich landing pads can be successfully used for stable expression of one or several GOIs.

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Cell Line Development for Therapeutic Protein Production

Cited by 7 publications

References 125 publications

Multicopy Targeted Integration for Accelerated Development of High-Producing Chinese Hamster Ovary Cells

Multicopy Targeted Integration for Accelerated Development of High-Producing Chinese Hamster Ovary Cells

High‐throughput and automation advances for accelerating single‐cell cloning, monoclonality and early phase clone screening steps in mammalian cell line development for biologics production

Generation of a host cell line containing a MAR‐rich landing pad for site‐specific integration and expression of transgenes

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