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Purpose: Perfusion cultures have been extensively used in the biotechnology industry to achieve high yields of recombinant products, especially those with stability issue. The WuXiUP™ platform represents a novel intensified perfusion that can achieve ultra‐high productivity. This study describes a representative scale-down 24-deep well plate (24-DWP) cell culture model for intensified perfusion clone screening. Methods: Clonal cell lines were expanded and evaluated in 24-DWP semi-continuous culture. Cell were sampled and counted daily with the aid of an automated liquid handler and high-throughput cell counter. To mimic perfusion culture, 24-DWP plates were spun down and resuspended with fresh medium daily. Top clones were ranked based on growth profiles and productivities. The best performing clones were evaluated on bioreactors. Results: The selected clones achieved volumetric productivity (Pv) up to 5 g/L/day when expressing a monoclonal antibody, with the accumulative harvest Pv exceeding 60 g/L in a 21-day cell culture. Product quality attributes of clones cultured in 24-DWP were comparable with those from bioreactors. A high seeding strategy further shortened the clone screening timeline. Conclusion: In this study, a 24-DWP semi-continuous scale-down model was successfully developed to screen for cell lines suitable for intensified perfusion culture. [ABSTRACT FROM AUTHOR]

Single-cell selection and cloning is required for multiple bioprocessing and cell engineering workflows. Dispensing efficiency and outgrowth were optimized for multiple common suspension (CHO ES, Expi293F, and Jurkat) and adherent (MCF-7, A549, CHO-K1, and HEK293) cell lines. Single-cell sorting using a low pressure microfluidic cell sorter, the WOLF Cell Sorter, was compared with limiting dilution at 0.5 cells/well to demonstrate the increased efficiency of using flow cytometry selection of cells. In this work, there was an average single cell deposition on Day 0 of 89.1% across all the cell lines tested compared to 41.2% when using limiting dilution. After growth for 14 days, 66.7% of single-cell clones sorted with the WOLF Cell Sorter survived and only 23.8% when using limiting dilution. Using the WOLF Cell Sorter for cell line development results in higher viable single-cell colonies and the ability to select subpopulations of single-cells using multiple parameters. [ABSTRACT FROM AUTHOR]

It is widely accepted that the growing demand for recombinant therapeutic proteins has led to the expansion of the biopharmaceutical industry and the development of strategies to increase recombinant protein production in mammalian cell lines such as SP2/0 HEK and particularly Chinese hamster ovary cells. For a long time now, most investigations have been focused on increasing host cell productivity using genetic manipulating of cellular processes like cell cycle, apoptosis, cell growth, protein secretory and other pathways. In recent decades MicroRNAs beside different genetic engineering tools (e.g., TALEN, ZFN, and Crisper/Cas) have attracted further attention as a tool in the genetic engineering of host cells to increase protein expression levels. Their ability to simultaneously target multiple mRNAs involved in one or more cellular processes made them a favorable tool in this field. Accordingly, this study aimed to review the methods of selecting target miRNA for cell line engineering, miRNA gain- or loss-of-function strategies, examples of laboratory and pilot studies in this field and discussed advantages and disadvantages of this technology. [ABSTRACT FROM AUTHOR]

Objectives: To establish a high-throughput method for determination of antibodies intra- and extracellular light chain (LC) to heavy chain (HC) polypeptide ratio as screening parameter during cell line development. Results: Chinese Hamster Ovary (CHO) TurboCell pools containing different designed vectors supposed to result in different LC:HC polypeptide ratios were generated by targeted integration. Cell culture supernatants and cell lysates of a fed batch experiment were purified by combined Protein A and anti-kappa affinity batch purification in 96-well format. Capture of all antibodies and their fragments allowed the determination of the intra- and extracellular LC:HC peptide ratios by reduced SDS capillary electrophoresis. Results demonstrate that the method is suitable to show the significant impact of the vector design on the intra- and extracellular LC:HC polypeptide ratios. Conclusion: Determination of LC:HC polypeptide ratios can give important information in vector design optimization leading to CHO cell lines with optimized antibody assembly and preferred product quality. [ABSTRACT FROM AUTHOR]

At the core of a biomanufacturing process for recombinant proteins is the production cell line. It influences the productivity and product quality. Its characteristics also dictate process development, as the process is optimized to complement the producing cell to achieve the target productivity and quality. Advances in the past decade, from vector design to cell line screening, have greatly expanded our capability to attain producing cell lines with certain desired traits. Increasing availability of genomic and transcriptomic resources for industrially important cell lines coupled with advances in genome editing technology have opened new avenues for cell line development. These developments are poised to help biosimilar manufacturing, which requires targeting pre-defined product quality attributes, e.g., glycoform, to match the innovator's range. This review summarizes recent advances and discusses future possibilities in this area. [ABSTRACT FROM AUTHOR]