Identification Of Novel Modulators Towards High Cell Density And High Producing Chinese Hamster Ovary Suspension Cell Cultures As Well As Their Application In Biopharmaceutical Protein Production

Amongst the mammalian producer cell lines, the Chinese hamster ovary (CHO) cell lines are of predominant importance in biopharmaceutical production. Thus, novel factors increasing overall productivity are sought and bear the potential to reduce the unit costs of a production process. Furthermore, the current patent situation for several therapeutic proteins demands innovative tools to at least maintain or preferentially increase the cost-effectiveness of their production processes. In this thesis, hitherto unknown factors were revealed by next generation sequencing of chemically mutated and selected CHO-K1 suspension cell lines. Two factors were proven to improve CHO-based production processes: cgrSnord78 and cgrTtc36. The Cricetulus griseus Ttc36 increases the integral as well as the maximal viable cell density and abolishes the cell-cell aggregation whilst cgrSnord78 improves the specific as well as volumetric productivity without significant impact on cell growth. Based on the present results and discussion, foundations for future research on these functionally unrevealed factors are laid. Hence, this work represents the first step towards the application of the genuine biomolecules cgrTtc36 and cgrSnord78 in biopharmaceutical protein production.

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 ability to detect and quantify apoptosis, to understand its biochemistry, and to identify its regulatory genes and proteins is crucial to biomedical research. In Apoptosis: Methods and Protocols, Second Edition, expert researchers describe the techniques to best investigate the critical steps involved in the apoptotic process. Presented from several different research perspectives, the volume contains sections covering detection of apoptosis, detection of non-apoptotic cell death, modifications of apoptotic proteins during apoptosis, the analysis of its major regulators, as well as analysis of apoptosis in different organs and in model organisms. Written in the highly successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Comprehensive and cutting-edge, Apoptosis: Methods and Protocols, Second Edition constitutes a key technical reference to the significant methodologies used in the field, and offers beginners and experienced researchers powerful tools to illuminate the phenomena of programmed cell death.

The editors have enlisted a broad range of experts, including microbial ecologists, physiologists, geneticists, biochemists, molecular biologists, and biochemical engineers, who offer practical experience not found in texts and journals. This comprehensive perspective makes MIMB a valuable "how to" resource, the structure of which resembles the sequence of operation involved in the development of a commercial biological process and product.

This report considers the biological and behavioral mechanisms that may underlie the pathogenicity of tobacco smoke. Many Surgeon General's reports have considered research findings on mechanisms in assessing the biological plausibility of associations observed in epidemiologic studies. Mechanisms of disease are important because they may provide plausibility, which is one of the guideline criteria for assessing evidence on causation. This report specifically reviews the evidence on the potential mechanisms by which smoking causes diseases and considers whether a mechanism is likely to be operative in the production of human disease by tobacco smoke. This evidence is relevant to understanding how smoking causes disease, to identifying those who may be particularly susceptible, and to assessing the potential risks of tobacco products.

This book is a monography about perfusion cell cultures for the production of biopharmaceuticals, such as therapeutic proteins (i.e. biomolecules like monoclonal antibodies), and describes the fundamentals, design and operation of these processes. Context is given in the first chapters to understand the state-of-the-art of the technology. We then give an overview of the challenges and objectives in operating mammalian cell perfusion cultures and provide guidelines for the design and setup of lab-scale bioreactor systems, and the required control structure to achieve stable operation. Scale-down devices and PAT tools are described in the context of continuous manufacturing and guidelines for process optimization are given using a variety of case studies to illustrate different approaches. Scale-up is also adressed with a strong focus on bioreactor aeration and mixing, shear stress and cell retention device. Finally, a general introduction for the application of mechanistic and statistic models in bioreactor process development and optimization is given in the last chapter.

In this practice-oriented two volume handbook, professionals from some of the largest biopharmaceutical companies and top academic researchers address the key concepts and challenges in the development of protein pharmaceuticals for medicinal chemists and drug developers of all trades. Following an introduction tracing the rapid development of the protein therapeutics market over the last decade, all currently used therapeutic protein scaffolds are surveyed, from human and non-human antibodies to antibody mimetics, bispecific antibodies and antibody-drug conjugates. This ready reference then goes on to review other key aspects such as pharmacokinetics, safety and immunogenicity, manufacture, formulation and delivery. The handbook then takes a look at current key clinical applications for protein therapeutics, from respiratory and inflammation to oncology and immune-oncology, infectious diseases and rescue therapy. Finally, several exciting prospects for the future of protein therapeutics are highlighted and discussed.