Improving Biopharmaceutical Production of Chinese Hamster Ovary Cells Using Targeted Genome Engineering Tools PDF Download
Are you looking for read ebook online? Search for your book and save it on your Kindle device, PC, phones or tablets. Download Improving Biopharmaceutical Production of Chinese Hamster Ovary Cells Using Targeted Genome Engineering Tools PDF full book. Access full book title Improving Biopharmaceutical Production of Chinese Hamster Ovary Cells Using Targeted Genome Engineering Tools by Kevin Kellner. Download full books in PDF and EPUB format.
Author: Kevin Kellner
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
Get Book Here
Book Description
Chinese Hamster Ovary (CHO) cells are the prominent cell line used in biopharmaceutical production. Over 70% of all therapeutically used recombinant proteins are produced by CHO cells with a market size predicted to exceed €250bn by 2021. Their favourable attributes over other cell lines are properties like resistance to viral infections, growth in chemical defined media, human like glycosylation patterns, good productivity and ease of genetic engineering. Due to the complexity of mammalian expression systems yields achieved are not phenomenal by any means. However, compared to 1986 with product concentrations of 50 mg/L, titres up to 10 g/L have been reported recently. Improvement of bioprocesses and media development contributed their part in facilitating higher titres but genetic engineering to improve host cells came to the foreground. MicroRNAs (miRNAs) have hereby been highlighted as attractive targets due to their involvement in processes like viability, secretion, productivity, product quality to mention only a few. MiRNAs are small non-coding RNAs which are about 22 nucleotides in length and were first discovered in the early 90's. A single miRNA can target 100-200 mRNAs which highlights them as key regulators for translational control. The miRNA-23 cluster was first identified as upregulated during induced hypothermic conditions. Hypothermia is a commonly used process to reduce growth and thrive CHO cells to improved productivities. Therefore, we hypothesised involvement of the miR-23 cluster or individual miRNA members in viability and productivity phenotypes. In this work we investigated the depletion of the miR-23 cluster as well as miR-23, miR-24 and miR-27 in a panel of industrially relevant cell lines expressing various recombinant products. In fact, miR-24 was identified as thriver of productivity and growth by upregulating ribosomal biogenesis, assembly of ribosomal subunits, translation as well as unfolded protein response (UPR). This was demonstrated in several cell lines and was not product specific. Furthermore, the depletion of the whole miR-23 cluster as well as miR-27 has been shown to improve productivity although in a cell line specific context. To overcome challenges of sponge technology we also implemented the recently developed CRISPR/Cas9 system to target miRNAs. When phenotypes after sponge and CRISPR/Cas9 mediated depletion of members of the miR-23 cluster were assessed, it was demonstrated that even enhanced properties were exhibited using CRISPR/Cas9 in case of miR-24 and miR-27 regarding productivity and longevity. Furthermore, we implemented a CRISPR/Cas9 library for genome wide recessive knockout screens to identify proteins involved in high productivity phenotypes or are important for survival of stress conditions i.e. hyperosmolality. Mixed populations expressing the sgRNA-library were sorted for high productivity using low temperature stains and were adapted to high salt conditions. Enrichment or depletion of sgRNAs was subsequently analyzed using Next-Generation Sequencing. SgRNA abundance analysed after low temperature stain showed enrichment of distinct populations. Functional annotation of enriched genes showed no evidence in relation to productivity. Exploiting miRNAs and genome-wide knockout studies to improve the bioprocess phenotype highlights these methods as interesting tools for further investigation regarding applications within biopharmaceutical industry.
Author: Kevin Kellner
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
Get Book Here
Book Description
Chinese Hamster Ovary (CHO) cells are the prominent cell line used in biopharmaceutical production. Over 70% of all therapeutically used recombinant proteins are produced by CHO cells with a market size predicted to exceed €250bn by 2021. Their favourable attributes over other cell lines are properties like resistance to viral infections, growth in chemical defined media, human like glycosylation patterns, good productivity and ease of genetic engineering. Due to the complexity of mammalian expression systems yields achieved are not phenomenal by any means. However, compared to 1986 with product concentrations of 50 mg/L, titres up to 10 g/L have been reported recently. Improvement of bioprocesses and media development contributed their part in facilitating higher titres but genetic engineering to improve host cells came to the foreground. MicroRNAs (miRNAs) have hereby been highlighted as attractive targets due to their involvement in processes like viability, secretion, productivity, product quality to mention only a few. MiRNAs are small non-coding RNAs which are about 22 nucleotides in length and were first discovered in the early 90's. A single miRNA can target 100-200 mRNAs which highlights them as key regulators for translational control. The miRNA-23 cluster was first identified as upregulated during induced hypothermic conditions. Hypothermia is a commonly used process to reduce growth and thrive CHO cells to improved productivities. Therefore, we hypothesised involvement of the miR-23 cluster or individual miRNA members in viability and productivity phenotypes. In this work we investigated the depletion of the miR-23 cluster as well as miR-23, miR-24 and miR-27 in a panel of industrially relevant cell lines expressing various recombinant products. In fact, miR-24 was identified as thriver of productivity and growth by upregulating ribosomal biogenesis, assembly of ribosomal subunits, translation as well as unfolded protein response (UPR). This was demonstrated in several cell lines and was not product specific. Furthermore, the depletion of the whole miR-23 cluster as well as miR-27 has been shown to improve productivity although in a cell line specific context. To overcome challenges of sponge technology we also implemented the recently developed CRISPR/Cas9 system to target miRNAs. When phenotypes after sponge and CRISPR/Cas9 mediated depletion of members of the miR-23 cluster were assessed, it was demonstrated that even enhanced properties were exhibited using CRISPR/Cas9 in case of miR-24 and miR-27 regarding productivity and longevity. Furthermore, we implemented a CRISPR/Cas9 library for genome wide recessive knockout screens to identify proteins involved in high productivity phenotypes or are important for survival of stress conditions i.e. hyperosmolality. Mixed populations expressing the sgRNA-library were sorted for high productivity using low temperature stains and were adapted to high salt conditions. Enrichment or depletion of sgRNAs was subsequently analyzed using Next-Generation Sequencing. SgRNA abundance analysed after low temperature stain showed enrichment of distinct populations. Functional annotation of enriched genes showed no evidence in relation to productivity. Exploiting miRNAs and genome-wide knockout studies to improve the bioprocess phenotype highlights these methods as interesting tools for further investigation regarding applications within biopharmaceutical industry.
Author: Gyun Min Lee
Publisher: John Wiley & Sons
ISBN: 3527343342
Category : Science
Languages : en
Pages : 436
Get Book Here
Book Description
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.
Author: Jonathan Joseph Cacciatore
Publisher:
ISBN:
Category :
Languages : en
Pages :
Get Book Here
Book Description
Site-specific recombination (SSR) technology was utilized to target the transgene to this location which was deemed capable of amplifying a transgene at a high rate. The second project also utilizes SSR technology to integrate many copies of a transgene into many recombination sites in the CHO genome. A cell line containing several thousand integration sites was isolated, however only about twenty of these sites successfully integrated a transgene after optimizing cell transfection conditions. Efforts towards engineering an improved recombinase for this purpose has led to the result that DNA sequences flanking recombination sites have the ability to greatly improve this integration process. Potential future experiments are described which may isolate such sequences and ultimately increase the number of transgenes integrated into the CHO cell genome. Overall, these improvements to CHO cells have the ability to ultimately isolate a higher producing cell line faster, thus decreasing the time to get a potential drug candidate to market.
Author: Ankur Solanki
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
Get Book Here
Book Description
Chinese Hamster Ovary (CHO) cells are the biopharmaceutical industry's "mini biofactories" for the production of complex, post-translationally modified therapeutic proteins. In order to address the ever-growing market need for these recombinant proteins, various genetic engineering tools have been employed. Here, we describe genetic approaches to improve CHO cell culture longevity, with a view to increasing overall process yield via the manipulation of two miRNAs: Let-7a and miR-7. Previous miRNA profiling studies in our laboratory and in the published literature helped in the identification of these miRNAs, which have shown to be disregulated in various tumor types and are key regulators of the cell cycle. Therefore, this stimulated the interest of our research group to manipulate these miRNAs in CHO cells with a view to positively impact bioprocess-relevant CHO cell phenotypes. In the first approach, we used a Let-7 sponge decoy vector to deplete endogenous Let-7 levels with a view to increasing culture longevity and productivity of CHO-K1 SEAP expressing cells. Despite let-7 having a recognised role in deregulated cell growth no improvement was observed in stable, sponge-transfected clones. Out of a panel of 40 clones, we observed only two with improved cellular viability in 24 well plate format, however, the results were not reproduced in a 5 mL scale-up batch study. In the second approach, we targeted a previously verified miRNA for improved CHO cell growth and productivity i.e. miR-7, using a bacterial genome-editing tool, CRISPR-Cas9. A considerable amount of optimisation work was performed to establish the CRISPR system for use in the lab, initially using eGFP as model target gene in CHO cells. Finally we designed single guide RNAs to target Cas9 to the miR-7a-5p genomic locus to disrupt miR-7 in order to enhance growth of a CHO-K1 cell line producing an IgG-1. We estimated ~ 40% targeting efficiency of miRNAs using this approach. After an extensive screen, one stable clone was identified with what appeared to be a heterozygous deletion of one miR-7a copy. We demonstrate that CRISPR-Cas9 can be successfully used to target miRNA loci in the CHO genome but that functional knockout may be more difficult compared to protein coding genes.
Author: Hooman Hefzi
Publisher:
ISBN:
Category :
Languages : en
Pages : 136
Get Book Here
Book Description
Chinese hamster ovary (CHO) cells are the dominant host for producing biotherapeutic proteins. Despite a long history of use, the techniques used to generate high quantities of recombinant drugs from this cell line remain largely unchanged. This is--at least partly--due to the complexity of mammalian cell lines. Historically--in contrast to microbial systems--CHO cells have been viewed as a bioprocessing black box, a system to optimize around rather than engineer. In this dissertation, advancements in gene editing tools and the availability of a genomic sequence are used to establish a framework to move away from that perspective and enable rational engineering of CHO cells for desirable phenotypes. Specifically, a genome-scale model of CHO cell metabolism, iCHO1766, was reconstructed from the genome sequence. iCHO1766 contains the biochemical basis for growth and protein production in CHO cells and predicts growth rates as well as CHO-specific amino acid auxotrophies. This model was also deployed to identify strategies that efficiently redirect resources from growth to protein production. Using the recently characterized CRISPR-Cas9 system, we were able to eliminate the Warburg effect in CHO cells by simultaneously knocking out lactate dehydrogenase and regulators involved in a negative feedback loop that typically inhibits pyruvate conversion to acetyl-CoA. In contrast to long-standing assumptions about the role of aerobic glycolysis, Warburg-null cells maintain the same growth rate as wildtype cells while consuming less glucose without increasing oxygen uptake to compensate for lost glycolytic ATP. The cells produce negligible lactate--allowing prolonged growth to higher cell densities--and remain viable for generating recombinant protein producing cell lines. Introducing this phenotype into a CHO cell line already producing a biotherapeutic antibody maintained protein production and improved glycan galactosylation. Thus, by leveraging accessible gene editing techniques, an improved CHO cell line has been generated. With newly available systems biology models, the tools for further rational engineering toward better and more affordable biotherapeutics are now available.
Author: Shangzhong Li
Publisher:
ISBN:
Category :
Languages : en
Pages : 108
Get Book Here
Book Description
For 60 years, Chinese hamster ovary (CHO) cells have been invaluable for biomedical research and fundamental to the study of several biological processes, such as glycosylation and DNA repair. In addition, for >30 years, they have been the host cell of choice for the production of most biotherapeutics because of its easiness to overexpress target genes and its similarity to the human cell system. Drug production in CHO-based cell lines has been improved by over 100-fold during the past 3 decades. However, due to the absence of genomic resources, efforts in improving the production predominantly rely on media and process optimization. With the decrease in the price of high throughput sequencing technology and some CHO and hamster genome assemblies published after 2011, new opportunities of optimizing CHO cell lines are arising rapidly. However, the draft nature of these genome sequences and therefore the non-perfect genome annotations still pose challenges for many applications. The new Chinese hamster genome assembled using Pacbio and illumina hybrid strategy in 2018 removes large number of obstacles for applying cutting-edge technologies for cell line development and engineering. In this doctoral dissertation, high throughput sequencing guided cell line development strategy and high quality genomics information of CHO was provided to boost the development of the CHO field. First, Ribosome Profiling, a next generation sequencing (NGS) technology which provides systematic view of protein translation was applied to CHO cell. Using the information we identified the unnecessary highly translated gene, knocking it down improves the production and growth rate. Second, we quantified the improvements in the new Chinese hamster genome compared to the RefSeq one. And found the genes and mutations that would be missed if we use the old genome. Finally, proteogenomics method was utilized to generate a high quality genome annotation through combining RNA-Seq and proteomics data from multiple hamster tissues.
Author: Mohamed Al-Rubeai
Publisher: Springer Science & Business Media
ISBN: 9048122457
Category : Medical
Languages : en
Pages : 259
Get Book Here
Book Description
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.
Author: Wei-Shu Hu
Publisher: Springer
ISBN: 3540340076
Category : Science
Languages : en
Pages : 179
Get Book Here
Book Description
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.
Author: Christoph Wittmann
Publisher: Springer Science & Business Media
ISBN: 9400745346
Category : Medical
Languages : en
Pages : 391
Get Book Here
Book Description
Systems Metabolic Engineering is changing the way microbial cell factories are designed and optimized for industrial production. Integrating systems biology and biotechnology with new concepts from synthetic biology enables the global analysis and engineering of microorganisms and bioprocesses at super efficiency and versatility otherwise not accessible. Without doubt, systems metabolic engineering is a major driver towards bio-based production of chemicals, materials and fuels from renewables and thus one of the core technologies of global green growth. In this book, Christoph Wittmann and Sang-Yup Lee have assembled the world leaders on systems metabolic engineering and cover the full story – from genomes and networks via discovery and design to industrial implementation practises. This book is a comprehensive resource for students and researchers from academia and industry interested in systems metabolic engineering. It provides us with the fundaments to targeted engineering of microbial cells for sustainable bio-production and stimulates those who are interested to enter this exiting research field.
Author: Alexandra Castilho
Publisher: Humana
ISBN: 9781493927593
Category : Science
Languages : en
Pages : 0
Get Book Here
Book Description
Conceived with the intention of providing an array of strategies and technologies currently in use for glyco-engineering distinct living organisms, this book contains a wide range of methods being developed to control the composition of carbohydrates and the properties of proteins through manipulations on the production host rather than in the protein itself. The first five sections deal with host-specific glyco-engineering and contain chapters that provide protocols for modifications of the glycosylation pathway in bacteria, yeast, insect, plants and mammalian cells, while the last two sections explore alternative approaches to host glyco-engineering and selected protocols for the analysis of the N-glycans and glyco-profiling by mass spectrometry. Written for the highly successful Methods in Molecular Biology series, 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. Authoritative and extensive, Glyco-Engineering: Methods and Protocols offers vast options to help researchers to choose the expression system and approach that best suits their intended protein research or applications.
