This volume explores the latest engineering methods of mammalian cells that are useful for controlling the performance of engineered mammalian cells for future cell-based therapeutics and for better understanding of complex biological systems. The chapters in this book are organized into five parts. Part One described methods to engineer mammalian cells to sense biologically relevant inputs, such as cell contacts and soluble proteins. Part Two looks at techniques to engineer mammalian cells to sense artificial inputs, such as light and ultrasound. Part Three provides cutting-edge CRISPR-Cas-based methods to carry out highly multiplexed genome editing and spatiotemporally controlled genome editing. Part Four discusses ways to control and engineer biological events in mammalian cells in combination with chemical compounds and systems. Part Five explores techniques to engineer specific mammalian cells in targeted manners. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and authoritative, Mammalian Cell Engineering: Methods and Protocols is a valuable resource that allows scientists to successfully carry out their research, thus ultimately contributing to the future advancement of this field.

Offers a comprehensive overview of cell culture engineering, providing insight into cell engineering, systems biology approaches and processing technology In Cell Culture Engineering: Recombinant Protein Production, editors Gyun Min Lee and Helene Faustrup Kildegaard assemble top class authors to present expert coverage of topics such as: cell line development for therapeutic protein production; development of a transient gene expression upstream platform; and CHO synthetic biology. They provide readers with everything they need to know about enhancing product and bioprocess attributes using genome-scale models of CHO metabolism; omics data and mammalian systems biotechnology; perfusion culture; and much more. This all-new, up-to-date reference covers all of the important aspects of cell culture engineering, including cell engineering, system biology approaches, and processing technology. It describes the challenges in cell line development and cell engineering, e.g. via gene editing tools like CRISPR/Cas9 and with the aim to engineer glycosylation patterns. Furthermore, it gives an overview about synthetic biology approaches applied to cell culture engineering and elaborates the use of CHO cells as common cell line for protein production. In addition, the book discusses the most important aspects of production processes, including cell culture media, batch, fed-batch, and perfusion processes as well as process analytical technology, quality by design, and scale down models. -Covers key elements of cell culture engineering applied to the production of recombinant proteins for therapeutic use -Focuses on mammalian and animal cells to help highlight synthetic and systems biology approaches to cell culture engineering, exemplified by the widely used CHO cell line -Part of the renowned "Advanced Biotechnology" book series Cell Culture Engineering: Recombinant Protein Production will appeal to biotechnologists, bioengineers, life scientists, chemical engineers, and PhD students in the life sciences.

Mammalian cell lines command an effective monopoly for the production of therapeutic proteins that require post-translational modifications. This unique advantage outweighs the costs associated with mammalian cell culture, which are far grater in terms of development time and manufacturing when compared to microbial culture. The development of cell lines has undergone several advances over the years, essentially to meet the requirement to cut the time and costs associated with using such a complex hosts as production platforms. This book provides a comprehensive guide to the methodology involved in the development of cell lines and the cell engineering approach that can be employed to enhance productivity, improve cell function, glycosylation and secretion and control apoptosis. It presents an overall picture of the current topics central to expression engineering including such topics as epigenetics and the use of technologies to overcome positional dependent inactivation, the use of promoter and enhancer sequences for expression of various transgenes, site directed engineering of defined chromosomal sites, and examination of the role of eukaryotic nucleus as the controller of expression of genes that are introduced for production of a desired product. It includes a review of selection methods for high producers and an application developed by a major biopharmaceutical industry to expedite the cell line development process. The potential of cell engineering approch to enhance cell lines through the manipulation of single genes that play important roles in key metabolic and regulatory pathways is also explored throughout.

The completion of the Human Genome Project and the rapid progress in cell bi- ogy and biochemical engineering, are major forces driving the steady increase of approved biotech products, especially biopharmaceuticals, in the market. Today mammalian cell products (“products from cells”), primarily monoclonals, cytokines, recombinant glycoproteins, and, increasingly, vaccines, dominate the biopharmaceutical industry. Moreover, a small number of products consisting of in vitro cultivated cells (“cells as product”) for regenerative medicine have also been introduced in the market. Their efficient production requires comprehensive knowledge of biological as well as biochemical mammalian cell culture fundamentals (e.g., cell characteristics and metabolism, cell line establishment, culture medium optimization) and related engineering principles (e.g., bioreactor design, process scale-up and optimization). In addition, new developments focusing on cell line development, animal-free c- ture media, disposables and the implications of changing processes (multi-purpo- facilities) have to be taken into account. While a number of excellent books treating the basic methods and applications of mammalian cell culture technology have been published, only little attention has been afforded to their engineering aspects. The aim of this book is to make a contribution to closing this gap; it particularly focuses on the interactions between biological and biochemical and engineering principles in processes derived from cell cultures. It is not intended to give a c- prehensive overview of the literature. This has been done extensively elsewhere.

Since the introduction of recombinant human growth hormone and insulin a quarter century ago, protein therapeutics has greatly broadened the ho- zon of health care. Many patients suffering with life-threatening diseases or chronic dysfunctions, which were medically untreatable not long ago, can attest to the wonder these drugs have achieved. Although the ?rst generation of p- tein therapeutics was produced in recombinant Escherichia coli, most recent products use mammalian cells as production hosts. Not long after the ?rst p- duction of recombinant proteins in E. coli, it was realized that the complex tasks of most post-translational modi?cations on proteins could only be ef?ciently carried out in mammalian cells. In the 1990s, we witnessed a rapid expansion of mammalian-cell-derived protein therapeutics, chie?y antibodies. In fact, it has been nearly a decade since the market value of mammalian-cell-derived protein therapeutics surpassed that of those produced from E. coli. A common characteristic of recent antibody products is the relatively large dose required for effective therapy, demanding larger quantities for the treatment of a given disease. This, coupled with the broadening repertoire of protein drugs, has rapidly expanded the quantity needed for clinical applications. The increasing demand for protein therapeutics has not been met exclusively by construction of new manufacturing plants and increasing total volume capacity. More - portantly the productivity of cell culture processes has been driven upward by an order of magnitude in the past decade.

The application of CRISPR/Cas systems for programmable DNA manipulation has revolutionized the field of genome engineering. In addition to their use for cutting DNA, nuclease-dead versions of Cas proteins fused to different effectors are a powerful tool for cell engineering and synthetic biology. In this thesis, I will discuss two projects from my Ph.D., both demonstrating how Cas12a can be used for constructing next-generation genetic circuits in human cells. For my first project I developed a split Cas12a protein, which spontaneously re-assembles into a functional gene-activating effector when all components are present. Therapeutic cell engineering, in which cells are programmed to sense and respond to complex disease states, requires integration of information about the cellular environment and execution of a functional response only in response to the specified combination of inputs. By linking the expression of individual components of the split Cas12a system to different input signals, it forms the functional core of multi-input, multi-output logic circuits in mammalian cells. The system is highly programmable and can generate expandable AND gates, as well as incorporate NOT logic by using anti-CRISPR proteins as an OFF switch. For my second project I established a platform for parallel signal recording using a hyper-efficient Cas12a base editor. Biological signal recording enables the study of molecular inputs experienced throughout cellular history, by encoding a transient signal as a permanent, targeted DNA mutation that can be read out at a later time by sequencing. However, current methods are often limited in their ability to scale up beyond a single biologically relevant signal in mammalian contexts. This novel implementation of Cas12a for signal recording provides a powerful platform for scalable recording, demonstrated by the recording of up to four signals in parallel in human cells. This method is plug-and-play with diverse inputs in different cell types, including tumor antigen recording in CAR-T cells. It can also be easily extended for more sophisticated applications, such as time-delimited recording and dual recorder-effector cells. Overall, these two projects demonstrate the utility of Cas-based effectors in complex genetic circuits, for both understand-ing fundamental biological processes as well as constructing smarter cellular therapeutics.

Transcriptome Analysis, by Frank Stahl, Bernd Hitzmann, Kai Mutz, Daniel Landgrebe, Miriam Lübbecke, Cornelia Kasper, Johanna Walter und Thomas Scheper Transcriptome Data Analysis for Cell Culture Processes, by Marlene Castro-Melchor, Huong Le und Wei-Shou Hu Modeling Metabolic Networks for Mammalian Cell Systems: General Considerations, Modeling Strategies, and Available Tools, by Ziomara P. Gerdtzen Metabolic Flux Analysis in Systems Biology of Mammalian Cells, by Jens Niklas und Elmar Heinzle Advancing Biopharmaceutical Process Development by System-Level Data Analysis and Integration of Omics Data, by Jochen Schaub, Christoph Clemens, Hitto Kaufmann und Torsten W. Schulz Protein Glycosylation and Its Impact on Biotechnology, by Markus Berger, Matthias Kaup und Véronique Blanchard Protein Glycosylation Control in Mammalian Cell Culture: Past Precedents and Contemporary Prospects, by Patrick Hossler Modeling of Intracellular Transport and Compartmentation, by Uwe Jandt und An-Ping Zeng Genetic Aspects of Cell Line Development from a Synthetic Biology Perspective, by L. Botezatu, S. Sievers, L. Gama-Norton, R. Schucht, H. Hauser und D. Wirth.