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Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 18016Article in journal (Refereed) Published
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Text
Abstract [en]

Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals. In contrast to the highly optimized production processes for monoclonal antibody (mAb)-based biopharmaceuticals, improving productivity of non-mAb therapeutic glycoproteins is more likely to reduce production costs significantly. The aim of this study was to establish a versatile target gene screening platform for improving productivity for primarily non-mAb glycoproteins with complete interchangeability of model proteins and target genes using transient expression. The platform consists of four techniques compatible with 96-well microplates: lipid-based transient transfection, cell cultivation in microplates, cell counting and antibody-independent product titer determination based on split-GFP complementation. We were able to demonstrate growth profiles and volumetric productivity of CHO cells in 96-half-deepwell microplates comparable with those obtained in shake flasks. In addition, we demonstrate that split-GFP complementation can be used to accurately measure relative titers of therapeutic glycoproteins. Using this platform, we were able to detect target gene-specific increase in titer and specific productivity of two non-mAb glycoproteins. In conclusion, the platform provides a novel miniaturized and parallelisable solution for screening target genes and holds the potential to unravel genes that can enhance the secretory capacity of CHO cells.

Place, publisher, year, edition, pages
Nature Publishing Group, 2015. Vol. 5, article id 18016
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-180214DOI: 10.1038/srep18016ISI: 000366190500001PubMedID: 26657798Scopus ID: 2-s2.0-84949656277OAI: oai:DiVA.org:kth-180214DiVA, id: diva2:895659
Note

QC 20160119

Available from: 2016-01-19 Created: 2016-01-08 Last updated: 2018-05-04Bibliographically approved
In thesis
1. 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)
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Supervisors
Note

QC 20180507

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

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Lundqvist, Magnus

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