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Droplet microfluidics and split-GFP complementation enable selection of Chinese hamster ovary cells with high specific productivity of therapeutic glycoproteins
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. (Johan Rockberg)ORCID iD: 0000-0003-1096-9061
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-7875-2822
KTH, Centres, Science for Life Laboratory, SciLifeLab.
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(English)Manuscript (preprint) (Other academic)
National Category
Pharmaceutical Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-212929OAI: oai:DiVA.org:kth-212929DiVA, id: diva2:1135903
Note

QC 20170828

Available from: 2017-08-24 Created: 2017-08-24 Last updated: 2018-05-04Bibliographically approved
In thesis
1. Cell line and protein engineering tools for production and characterization of biologics
Open this publication in new window or tab >>Cell line and protein engineering tools for production and characterization of biologics
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Our increasing understanding of disease mechanisms coupled with technological advances has facilitated the generation of pharmaceutical proteins, which are able to address yet unmet medical needs. Diseases that were fatal in the past can now be treated with novel biological medications improving and prolonging life for many patients. Pharmaceutical protein production is, however, a complex undertaking, which is by no means problem-free. The demand for more complex proteins and the realization of the importance of post-translational modifications have led to an increasing use of mammalian cells for protein expression. Despite improvements in design and production, the costs required for the development of pharmaceutical proteins still are far greater than those for conventional, small molecule drugs. To render such treatments affordable for healthcare suppliers and assist in the implementation of precision medicine, further progress is needed. In five papers this thesis describes strategies and methods that can help to advance the development and manufacturing of pharmaceutical proteins. Two platforms for antibody engineering have been developed and evaluated, one of which allows for efficient screening of antibody libraries whilst the second enables the straightforward generation of bispecific antibodies. Moreover, a method for epitope mapping has been devised and applied to map the therapeutic antibody eculizumab’s epitope on its target protein. In a second step it was shown how this epitope information can be used to stratify patients and, thus, contribute to the realization of precision medicine. The fourth project focuses on the cell line development process during pharmaceutical protein production. A platform is described combining split-GFP and fluorescence-activated droplet sorting, which allows for the efficient selection of highly secreting cells from a heterogeneous cell pool. In an accompanying study, the split-GFP probe was improved to enable shorter assay times and increased sensitivity, desirable characteristics for high-throughput screening of cell pools. In summary, this thesis provides tools to improve design, development and production of future pharmaceutical proteins and as a result, it makes a contribution to the goal of implementing precision medicine through the generation of more cost-effective biopharmaceuticals for well-characterized patient groups.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. p. 91
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:16
Keywords
Pharmaceutical proteins, precision medicine, antibody engineering, epitope mapping, cell line development, split-GFP
National Category
Pharmaceutical Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-212931 (URN)978-91-7729-497-9 (ISBN)
Public defence
2017-09-29, E3, Osquars backe 14, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20170828

Available from: 2017-08-28 Created: 2017-08-24 Last updated: 2017-08-30Bibliographically approved
2. Methods for cell line and protein engineering
Open this publication in new window or tab >>Methods for cell line and protein engineering
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Therapeutic proteins are becoming increasingly important. They are desirable, as they typically possess low adverse effects and higher specificity compared to the traditional, small molecule drugs. But they are also more complex and involve different intricate and expensive development and production processes. Through new technologies in protein and cell line development, more efficient and safer drugs can be readily available and at a lower cost. This thesis gives an overview of how protein therapeutics are developed and produced. It explores strategies to improve the efficacy and safety of protein drugs and how to improve production yields. In the present investigation, two papers present new methods for high-throughput cloning and site-directed mutagenesis using solid-phase immobilization of DNA fragments. These methods were designed to generate new drug candidates with swiftness and ease. Three papers show the development of a new cell line screening system that combines droplet microfluidics and the split-GFP reporter system. This combination allows for relative quantification of secreted recombinant proteins between individual cells and provides a tool for the selection of the best-producing clones for final production from a heterologous cell pool. The final paper explores the possibility to produce proteins at a higher cell density by examining how the metabolome and proteome of a perfusion bioreactor evolve as the cell density reaches exceptionally high levels. The consistent goal of all of these studies is to expedite the development and improve the production of therapeutic proteins, to assist the discovery of new drugs and to bring down production and development costs. Engineered proteins can be used to cure previously incurable diseases or give current medications a higher efficacy. Lower production and development costs can make the treatments available to more people.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2018
Series
TRITA-CBH-FOU ; 2018:14
Keywords
Cell line development, therapeutic proteins, protein engineering, molecular cloning, mutagenesis, split-GFP
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-227195 (URN)978-91-7729-757-4 (ISBN)
Public defence
2018-05-31, Kollegiesalen, Brinellvägen 8, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20180507

Available from: 2018-05-07 Created: 2018-05-04 Last updated: 2018-05-08Bibliographically approved

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Volk, Anna-LuisaLundqvist, MagnusHammar, PetterBai, YunpengUhlén, MathiasJoensson, Haakan N.Rockberg, Johan

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