Hofreither, Dominik

Hofer, Larissa

Rauter, Thomas

Liesinger, Laura

Bednárová Schäpertöns, Veronika

Fortelny, Nikolaus

Schittmayer-Schantl, Matthias

Borth, Nicole

Birner-Grünberger, Ruth

2025-01-03T11:39:18Z

2025-01-03T11:39:18Z

2024-09-23

<div class="csl-bib-body"> <div class="csl-entry">Hofreither, D., Hofer, L., Rauter, T., Liesinger, L., Bednárová Schäpertöns, V., Fortelny, N., Schittmayer-Schantl, M., Borth, N., &#38; Birner-Grünberger, R. (2024, September 23). <i>Time-Resolved Mass Spectrometry-Based Multi-Omics Characterisation of the NISTCHO Bioprocess</i> [Poster Presentation]. APMRS/CEEPC APMRS-CEEPC 2024, Wien, Austria. http://hdl.handle.net/20.500.12708/207806</div> </div>

http://hdl.handle.net/20.500.12708/207806

Introduction To meet the growing demand for recombinant biotherapeutics in use today, producer cell lines are under constant development to improve their productivity, stability and product quality. Enhancing our understanding of biological responses during cultivation through host-cell omics, complementing conventional bioprocess monitoring and medium-based measurements as proxies for cellular function, may enable immediate adaptation of critical process parameters (CPPs) to ensure efficient and quality-assured production. Methods The design and evaluation of a fast, feasible and representative multi-omics sampling and downstream analysis approach for suspension cultures utilising state-of-the-art methodology and instrumentation were conduced. The resulting workflow allows for analysis of the host-cell proteome, phosphoproteome, metabolome, and secretome. To demonstrate applicability, a fed-batch culture of the openly available, monoclonal antibody-producing cell line NISTCHO was consequently examined for the impact of feeding on cellular processes as indicated by changes in the proteome and reversible phosphorylation of proteins. Sampling was performed 60 min prior to feeding, as well as 15, 60, 90, 120 and 240 min post-feed, in both exponential and stationary growth phases to reflect multiple stages of the bioprocess. Parallel monitoring of nutrient consumption and metabolic excretions was deployed. To fully leverage the high-resolution LC-MS/MS data generated on the Bruker timsTOF pro, curation of the Chinese hamster (Cricetulus griseus) reference proteome, reducing redundancy and improving annotation and coverage, was performed. Additionally, to utilise the statistical power of time-course data, an in-house R package was developed to identify and cluster significant temporal dynamics of features. Results and Discussion A bioprocess-integrated multi-omics sampling and analysis workflow, addressing multiple key challenges, was designed and implemented. Further evaluation included investigation of a NISTCHO fed-batch culture. This work provides comprehensive temporal profiling of protein and protein phosphorylation dynamics in response to changes in nutrient availability around feeding windows distinct to different phases of the bioprocess. Key results range from master regulators of redox homeostasis and metabolism to pinpointing individual regulatory sites of DNA repair enzymes. We emphasise that mass spectrometry can provide an in-depth analysis of cellular function in various product and process-relevant biological settings. The advanced characterisation of an academically available producer cell line and the generation of high-quality bioprocess reference data as part of our DigiTherapeutX project will fuel research efforts towards integrated digitalised production and predictable quality of protein therapeutics. Innovative aspects • Design and implementation of a bioprocess-integrated multi-omics sampling and analysis workflow • Data processing and analysis pipeline includes curated Cricetulus griseus reference proteome and in-house software to handle time-course data • Comprehensive temporal profiling of more than 7000 proteins and 3000 phosphorylation sites in response to feeding distinct to different phases of the NISTCHO fed-batch bioprocess

FWF - Österr. Wissenschaftsfonds

en

multi-omics

CHO host cells

monoclonal antibody production

Time-Resolved Mass Spectrometry-Based Multi-Omics Characterisation of the NISTCHO Bioprocess

Presentation

Vortrag

BOKU University, Austria

University of Salzburg, Austria

BOKU University, Austria

FG 1200-N

Poster Presentation

Digitalized Production of Therapeutics

M6

Biological and Bioactive Materials

100

E164-01-3 - Forschungsgruppe Bioanalytik

0009-0006-2194-706X

0009-0004-5578-3628

0000-0001-8076-3187

0000-0003-4025-9968

0000-0003-3249-655X

0000-0001-6324-9338

0000-0003-3950-0312

APMRS/CEEPC APMRS-CEEPC 2024

23-09-2024

25-09-2024

On Site

Event for scientific audience

Wien

AT

APMA

Hofreither, Dominik

Chemie

Industrielle Biotechnologie

1040

2090

50

50

conference poster not in proceedings

en

Publications

no Fulltext

none

http://purl.org/coar/resource_type/c_18co

FWF - Österr. Wissenschaftsfonds

FG 1200-N

E164-01-3 - Forschungsgruppe Bioanalytik

BOKU University

University of Salzburg

E164-01-3 - Forschungsgruppe Bioanalytik

E164-01-3 - Forschungsgruppe Bioanalytik

BOKU University

E164-01 - Forschungsbereich Imaging und Instrumentelle Analytische Chemie

0009-0006-2194-706X

0009-0004-5578-3628

0000-0001-8076-3187

0000-0003-4025-9968

0000-0003-3249-655X

0000-0001-6324-9338

0000-0003-3950-0312

E164-01 - Forschungsbereich Imaging und Instrumentelle Analytische Chemie

E164-01 - Forschungsbereich Imaging und Instrumentelle Analytische Chemie

E164-01 - Forschungsbereich Imaging und Instrumentelle Analytische Chemie

E164 - Institut für Chemische Technologien und Analytik