Biology subjects

Borth, N.

Publications and source records attributed to Borth, N..

5 recordsLinked to original sources

bioRxiv2026

MCBO: Mammalian Cell Bioprocessing Ontology, A Hub-and-Spoke, IOF-Anchored Application Ontology

Mammalian cell-based biopharmaceutical manufacturing generates vast, heterogeneous datasets that remain fragmented due to the lack of a standardized metadata framework. A key challenge in biologics manufacturing is linking bioreactor conditions, cell line characteristics and recombinant product production. A datahub for mammalian cell bioprocessing, integrated by semantic technologies, will serve as a tool to understand and query the connections between these complex datasets. While existing ontologies cover general biological and experimental concepts, they often lack the operational specificity required to harmonize bioreactor conditions, cell line engineering, and product quality metrics. To address this specific gap, we present the Mammalian Cell Bioprocessing Ontology (MCBO), a hub-and-spoke application ontology built on Basic Formal Ontology (BFO) foundations and anchored to the Industrial Ontology Foundry (IOF) Core. MCBO formalizes the process-participant-quality modeling pattern, enabling precise tracking of culture environmental conditions as qualities of the physical culture system. We demonstrate the utility of MCBO through a central datahub populated with 723 curated cell culture process instances and 325 unique bioprocess samples from published studies. The framework is validated against eight competency questions implemented via SPARQL, demonstrating efficient cross-study querying of culture optimization, cell line engineering, and multi-omics integration. By providing a stable, schema-independent substrate for data harmonization, MCBO enables AI agent-powered, human-in-the-loop workflows and facilitates LLM-assisted extraction of structured metadata from legacy records. MCBO is open-source and designed for deployment behind institutional firewalls to support interoperable biomanufacturing intelligence while maintaining intellectual property sensitivity. MCBO is supported by the International Biomanufacturing Network (IBioNe), which aims to accelerate discoveries and developments by providing a network of biomanufacturing training and workforce development to educate the next generation of biomanufacturing experts. MCBO is evaluated using over 700 curated cell culture processes, validated against eight competency questions, and quality-controlled using automated ontology checks. Evaluation results and formal reasoning validation are provided in the Supplementary Materials. Availability: https://github.com/lewiscelllabs/mcbo

bioinformatics↗

bioRxiv2025

Overcoming lentiviral delivery limitations in hard-to-transduce suspension cells for genome-wide CRISPR screening

Lentiviral vectors are a cornerstone delivery modality of biomedical research, renowned for their ability to stably integrate genetic material into the host genome, enabling sustained transgene expression and long-term genetic manipulation. These properties make them indispensable tools in functional genomics and genome engineering, particularly for delivering molecular components in high-throughput CRISPR screening, a powerful approach for uncovering the genetic basis of complex cellular mechanisms and phenotypes. However, challenges such as lentiviral-induced recombination, unpredictable integration profiles, and variable susceptibility of target cells to transduction can introduce noise and compromise experimental outcomes. In this study, we selected two suspension-adapted mammalian cell lines, Chinese Hamster Ovary cells CHO-K1 and Human Embryonic Kidney cells HEK293-6E, due to their widespread use in recombinant protein production. Recognizing the influence of intrinsic cell line properties and transduction methodology, we compared two distinct procedures: spinoculation and static transduction. By implementing a two-step static transduction protocol, we achieved significantly higher transduction efficiencies while minimizing cellular stress, streamlining workflows, and eliminating scalability limitations inherent to large-scale lentiviral applications like genome-wide CRISPR screens. To further characterize the variation in lentiviral integration, we used droplet digital PCR (ddPCR) to quantify copy number variation (CNV) both at the pooled population level and within individual clonal isolates. This comprehensive analysis underscores the robustness of our optimized protocol in enhancing transduction efficiency in difficult-to-transduce suspension cell lines. It further emphasizes the importance of carefully modulating infection rates to limit multiple integrations, ensuring the accuracy and consistency required for large-scale functional genomics applications.

molecular biology↗

bioRxiv2025

A genome-scale CRISPR deletion screen in Chinese Hamster Ovary cells reveals essential regions of the coding and non-coding genome

The biopharmaceutical sector relies on CHO cells to investigate biological processes and as the preferred host for production of biotherapeutics. Simultaneously, advancements in CHO cell genome assembly have provided insights for developing sophisticated genetic engineering strategies. While the majority of these efforts have focused on coding genes, with some interest in transcribed non-coding RNAs (e.g., microRNAs and lncRNAs), there remains a lack of genome-wide systematic studies that precisely examine the remaining 90% of the genome. This unannotated "dark matter" includes regulatory elements and other, poorly understood or characterized functionality of the genome that may be potentially critical for cell survival. In this study, we deployed a genome-scale CRISPR screening platform with 112,272 paired guide RNAs targeting 14,034 genomic regions for complete deletion of 150 kb long sections. This platform enabled the execution of a negative screen that selectively identified dying cells to determine regions essential for cell survival. By using paired gRNAs, we overcame the intrinsic limitations of traditional frameshift strategies, which will likely have little or no effect on the non-coding genome. This study revealed 427 regions essential for CHO survival, many of which currently lack gene annotation or known function. For these regions we present annotation status, transcriptional activity as well as annotated chromatin states such as enhancers. Selected regions, specifically those that were negative for all the above, were individually deleted for confirmation. This work sheds a novel light on a substantial part of the mammalian genome which is traditionally difficult to investigate and therefore, neglected.

cell biology↗

bioRxiv2020

Inclusion of maintenance energy improves the intracellular flux predictions of CHO

Chinese hamster ovary (CHO) cells are the leading platform for the production of biopharmaceuticals with human-like glycosylation. The standard practice for cell line generation relies on trial and error approaches such as adaptive evolution and high-throughput screening, which typically take several months. Metabolic modeling could aid in designing better producer cell lines and thus shorten development times. The genome-scale metabolic model (GSMM) of CHO can accurately predict growth rates. However, in order to predict rational engineering strategies it also needs to accurately predict intracellular fluxes. In this work we evaluated the agreement between the fluxes predicted by pFBA using the CHO GSMM and a wide range of 13C metabolic flux data from literature. While glycolytic fluxes were predicted relatively well, the fluxes of tricarboxylic acid (TCA) cycle were vastly underestimated due to too low energy demand. Inclusion of computationally estimated maintenance energy significantly improved the overall accuracy of intracellular flux predictions. Maintenance energy was therefore determined experimentally by running continuous cultures at different growth rates and evaluating their respective energy consumption. The experimentally and computationally determined maintenance energy were in good agreement. Additionally, we compared alternative objective functions (minimization of uptake rates of seven nonessential metabolites) to the biomass objective. While the predictions of the uptake rates were quite inaccurate for most objectives, the predictions of the intracellular fluxes were comparable to the biomass objective function.

systems biology↗

bioRxiv2020

A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes

Over the past decades, optimization of media formulation and feeding strategies have fueled a many-fold improvement in CHO-based biopharmaceutical production. While Design of Experiments (DOE) and media screens have led to many advances, genome editing offers another avenue for enhancing cell metabolism and bioproduction. However the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we developed a comprehensive pooled CRISPR screen for CHO cell metabolism, including [~]16,000 gRNAs against [~]2500 metabolic enzymes and regulators. We demonstrated the value of this screen by identifying a glutamine response network in CHO cells. Glutamine is particularly important since it is often substantially over-fed to drive increased TCA cycle flux but can lead to accumulation of toxic ammonia. Within the glutamine-response network, the deletion of a novel and poorly characterized lipase, Abhd11, was found to substantially increase growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait.

bioengineering↗