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High cell density perfusion culture has a maintained exoproteome and metabolome
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Chinese hamster ovary (CHO) cells are the workhorse to produce recombinant proteins in the biopharmaceutical industry using mammalian cells and are commonly cultured in either fed-batch or perfusion mode. The optimization of the complex biological systems used in such processes is extremely challenging. Multi-omics approaches can reveal otherwise unknown characteristics of these systems and identify culture parameters that can be manipulated to optimize the cultivation process. Here we have ap- plied both metabolomic and proteomic profiling to a monoclonal antibody (mAb) production operated in perfusion mode to explore how cell biology and reactor environment change as the cell density reaches ≥ 200 x 106 cells/mL. The extracellular metabolic composition obtained in perfusion mode was also com- pared to fed-batch, which showed a more stable profile for perfusion despite a far larger range of viable cell densities. The proteomics data showed an increase of structural proteins as the cell density increased, and both the proteomic and metabolic results showed signs of oxidative stress and changes in glutathione metabolism at very high cell densities. The methodology presented herein could be a powerful tool for optimizing cultivation processes and recombinant protein production.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:kth:diva-227183OAI: oai:DiVA.org:kth-227183DiVA, id: diva2:1203697
Note

QC 20180523

Available from: 2018-05-04 Created: 2018-05-04 Last updated: 2018-05-23Bibliographically 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)
Opponent
Supervisors
Note

QC 20180507

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

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CiteExportLink to record
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Citation style
  • apa
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Output format
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