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Cell line and protein engineering tools for production and characterization of biologics
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. (Johan Rockberg)ORCID iD: 0000-0003-1096-9061
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 [en]
Pharmaceutical proteins, precision medicine, antibody engineering, epitope mapping, cell line development, split-GFP
National Category
Pharmaceutical Biotechnology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-212931ISBN: 978-91-7729-497-9 (print)OAI: oai:DiVA.org:kth-212931DiVA, id: diva2:1135929
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
List of papers
1. Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders
Open this publication in new window or tab >>Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders
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2017 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347Article in journal (Refereed) Epub ahead of print
Abstract [en]

Surface display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various display systems available, phage display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive bacterial host for display of a library of human antibody genes which, when combined with phage display, provides ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying low nanomolar affinity scFv fragments towards human epidermal growth factor receptor 2 (HER2). The ranking and performance of the scFv isolated by flow sorting in surface-immobilised form was retained when expressed as soluble scFv and analysed by biolayer interferometry, as well as after expression as full-length antibodies in mammalian cells. We also demonstrate the possibility of using Gram-positive bacterial display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. This combined approach has the potential for a more complete scan of the antibody repertoire and for affinity maturation of human antibody formats.

Place, publisher, year, edition, pages
Elsevier, 2017
Keywords
Affinity maturation, Antibody, Cell-surface display, Flow cytometry, HER2, Phage display, S. carnosus, Binders, Bins, Cell membranes, Display devices, Genes, Libraries, Mammals, Cell surface displays, Antibodies
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-213019 (URN)10.1016/j.nbt.2017.07.011 (DOI)000441913800011 ()2-s2.0-85027243712 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20170828

Available from: 2017-08-28 Created: 2017-08-28 Last updated: 2020-01-10Bibliographically approved
2. Bi-specific antibody molecule inhibits tumor cell proliferation more efficiently than the two-molecule combination
Open this publication in new window or tab >>Bi-specific antibody molecule inhibits tumor cell proliferation more efficiently than the two-molecule combination
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(English)Manuscript (preprint) (Other academic)
Keywords
Bispecific antibody, Her2, EGFRm gastric cancer, trastuzumab, AffiMab
National Category
Pharmaceutical Biotechnology
Identifiers
urn:nbn:se:kth:diva-212926 (URN)
Note

QC 20170828

Available from: 2017-08-24 Created: 2017-08-24 Last updated: 2020-01-10Bibliographically approved
3. Stratification of responders towards eculizumab using a structural epitope mapping strategy
Open this publication in new window or tab >>Stratification of responders towards eculizumab using a structural epitope mapping strategy
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 31365Article in journal (Refereed) Published
Abstract [en]

The complement component 5 (C5)-binding antibody eculizumab is used to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome (aHUS). As recently reported there is a need for a precise classification of eculizumab responsive patients to allow for a safe and cost-effective treatment. To allow for such stratification, knowledge of the precise binding site of the drug on its target is crucial. Using a structural epitope mapping strategy based on bacterial surface display, flow cytometric sorting and validation via haemolytic activity testing, we identified six residues essential for binding of eculizumab to C5. This epitope co-localizes with the contact area recently identified by crystallography and includes positions in C5 mutated in non-responders. The identified epitope also includes residue W917, which is unique for human C5 and explains the observed lack of cross-reactivity for eculizumab with other primates. We could demonstrate that Ornithodorus moubata complement inhibitor (OmCI), in contrast to eculizumab, maintained anti-haemolytic function for mutations in any of the six epitope residues, thus representing a possible alternative treatment for patients non-responsive to eculizumab. The method for stratification of patients described here allows for precision medicine and should be applicable to several other diseases and therapeutics.

Place, publisher, year, edition, pages
Nature Publishing Group, 2016
Keywords
Paroxysmal-Nocturnal Hemoglobinuria, Hemolytic-Uremic Syndrome, Catastrophic Antiphospholipid Syndrome, Antibody-Mediated Rejection, Bacterial Surface Display, Tick Ornithodoros-Moubata, Optica Spectrum Disorders, Complement Inhibitor, Transplant Recipients, Renal-Transplantation
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-191745 (URN)10.1038/srep31365 (DOI)000381185300001 ()27509843 (PubMedID)2-s2.0-84982176744 (Scopus ID)
External cooperation:
Funder
Novo NordiskScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20160915

Available from: 2016-09-15 Created: 2016-09-02 Last updated: 2020-01-10Bibliographically approved
4. Droplet microfluidics and split-GFP complementation enable selection of Chinese hamster ovary cells with high specific productivity of therapeutic glycoproteins
Open this publication in new window or tab >>Droplet microfluidics and split-GFP complementation enable selection of Chinese hamster ovary cells with high specific productivity of therapeutic glycoproteins
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(English)Manuscript (preprint) (Other academic)
National Category
Pharmaceutical Biotechnology
Identifiers
urn:nbn:se:kth:diva-212929 (URN)
Note

QC 20170828

Available from: 2017-08-24 Created: 2017-08-24 Last updated: 2020-01-10Bibliographically approved
5. Chromophore prematuration for improved speed and sensitivity of split-GFP in vitro applications
Open this publication in new window or tab >>Chromophore prematuration for improved speed and sensitivity of split-GFP in vitro applications
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-212930 (URN)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20170828

Available from: 2017-08-24 Created: 2017-08-24 Last updated: 2020-01-10Bibliographically approved

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