Biology subjects

Tsukidate, T.

Publications and source records attributed to Tsukidate, T..

6 recordsLinked to original sources

bioRxiv2025

Cysteine Reactivity Profiling Illuminates Monoclonal Antibody Disulfide Bond Reduction Mechanisms in Biopharmaceutical Process Intermediates

Monoclonal antibodies (mAbs) are crucial biotherapeutics with increasing global demand, but their production can be impacted by the reduction of disulfide bonds. This study presents a chemical proteomics workflow aimed at elucidating the mechanisms underlying disulfide bond reduction in mAbs produced from Chinese hamster ovary (CHO) cells. We employed iodoacetamide-desthiobiotin (IA-DTB) and the parallel accumulation and serial fragmentation combined with data-independent acquisition (diaPASEF) methodology for cysteine reactivity profiling and successfully quantified approximately 4,500 cysteine-containing peptides from harvested cell culture fluids (HCCF). Our findings reveal that various protein disulfide oxidoreductases were active in reducing HCCF, offering critical insights into the redox environment affecting mAb stability. Notably, we quantified specific cysteine residues in enzymes such as glutaredoxin and thioredoxin domain-containing protein 12, suggesting potential links between their activity to disulfide bond dynamics. This workflow not only complements conventional abundance proteomics but also enhances the understanding of functional enzyme states in bioprocessing. Ultimately, our approach provides a promising strategy for enzymes contributing to disulfide bond reduction, paving the way for improved manufacturing processes of mAbs.

bioengineering↗

bioRxiv2025

Prolonged autophagy induction correlates with host cell protein reduction in CHO cell culture

Autophagy, a cellular recycling process regulated by the CLEAR signaling pathway, plays a pivotal role in maintaining cellular homeostasis. We hypothesize that this process may regulate and reduce HCP levels by targeting intracellular proteins and organelles for degradation. This study investigates the relationship between autophagy induction and the reduction of high-risk host cell proteins (HCPs), including polysorbate-degrading enzymes (PSDEs), to enhance the stability of therapeutic biologics such as monoclonal antibodies (mAbs). Using clonal analysis, we identified upregulation of the CLEAR pathway in one clone, correlating with a significant reduction in lipase activity and PSDE abundance. Furthermore, autophagy modulators, such as 3-methyladenine (3-MA), selectively decreased PSDE levels in both batch and fed-batch cultures. This resulted in 62% reduction in lipase activity that corresponded to a 22% improvement in polysorbate-80 stability. Additionally, 3-MA treatment increased mAb specific productivity and altered glycosylation profiles, increasing afucosylation and galactosylation levels. These findings highlight autophagy induction as a promising strategy to modulate product quality profiles and reduce high-risk HCPs in biologics production.

bioengineering↗

bioRxiv2025

Discovery of Chemical Tools for Polysorbate-Degradative Enzyme Control in Biopharmaceutical Upstream Process via Multi-Omic Profiling of Host Cell Clones

Host cell proteins are process-related impurities in biotherapeutics and can potentially pose risks to patient safety and product quality. Specifically, certain host cell-derived enzymes, including lipases, can degrade the formulation excipient polysorbate (PS) in biopharmaceutical formulations, affecting drug product stability in liquid formulations. We leveraged multi-omics approaches, including transcriptomics, proteomics, and activity-based protein profiling (ABPP), to identify mechanisms that regulate PS-degradative enzyme (PSDE) abundance and to develop strategies for their control. Comparative multiomics analysis of two monoclonal antibodies (mAb)-producing host cell clones revealed differential lipase profiles at the mRNA, protein, and enzyme activity levels and associated increased lipase activity with upregulated lipid catabolic pathways such as the fatty acid beta oxidation pathway. Further, for the first time in the literature, we identified peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) as a key regulator of PSDEs in manufacturing Chinese Hamster Ovary (CHO) cells. Downregulation of the PPAR{gamma} pathway with its antagonists resulted in selective reduction of PSDE levels and improved PS stability without compromising mAb productivity or quality. This study highlights the potential of PPAR{gamma} modulators as chemical tools for PSDE control at the gene regulation level, offering significant implications for biopharmaceutical process development and control.

bioengineering↗

bioRxiv2024

Systematic Optimization of Activity-Based Protein Profiling for Identification of Polysorbate-Degradative Enzymes in Biotherapeutic Drug Substance Down to 10 ppb

The identification and control of high-risk host cell proteins (HCPs) in biotherapeutics development is crucial for ensuring product quality and shelf life. Specifically, HCPs with hydrolase activity can cause degradation of excipient polysorbates (PS), leading to a decrease in the shelf life of the drug product. In this study, we systematically optimized every step of an activity-based protein profiling (ABPP) workflow to identify trace amounts of active polysorbate-degradative enzymes (PSDEs) in biotherapeutics process intermediates. Evaluation of various parameters during sample preparation pinpointed optimal pH level and fluorophosphonate (FP)-biotin concentration. Moreover, the combined use of a short liquid chromatography gradient and the fast-scanning Parallel Accumulation Serial Fragmentation (PASEF) methodology increased sample throughput without compromising identification coverage. Tuning Trapped Ion Mobility Spectrometry (TIMS) parameters further enhanced sensitivity. In addition, we evaluated various data acquisition modes including PASEF combined with data-dependent acquisition (DDA PASEF), data-independent acquisition (diaPASEF), or parallel reaction monitoring (prm-PASEF). By employing the newly optimized ABPP workflow, we successfully identified PSDEs at a concentration as low as 10 parts per billion (ppb) in a drug substance sample. Finally, the new workflow enabled us to detect a PSDE that could not be detected with the original workflow during a PS degradation root-cause investigation.

bioengineering↗

bioRxiv2024

diaPASEF Enables High-throughput Proteomic Analysis of Host Cell Proteins for Biopharmaceutical Process Development

Monitoring and quantifying host cell proteins (HCPs) in biotherapeutics production processes is crucial to ensure product quality, stability, and safety. Liquid chromatography-mass spectrometry (LC-MS) analysis has emerged as an important tool for identifying and quantifying individual HCPs. However, LC-MS-based approaches face challenges due to the wide dynamic range between HCPs and the therapeutic protein, as well as laborious sample preparation and long instrument time. To address these limitations, we evaluated the application of parallel accumulation-serial fragmentation combined with data-independent acquisition (diaPASEF), to HCP analysis for biopharmaceutical process development applications. We evaluated different library generation strategies and LC methods, demonstrating the suitability of these workflows for various HCP analysis needs such as in-depth characterization and high-throughput analysis of process intermediates. Remarkably, the diaPASEF approach enabled the quantification of hundreds of HCPs that were undetectable by a standard data-dependent acquisition mode while considerably improving sample requirement, throughput, coverage, quantitative precision, and data completeness.

bioengineering↗

bioRxiv2023

N-arylpyrazole NOD2 agonists promote immune checkpoint inhibitor therapy

The characterization of microbiota mechanisms in health and disease has reinvigorated pattern recognition receptors as prominent targets for immunotherapy. Notably, our recent studies on Enterococcus species revealed peptidoglycan remodeling and activation of NOD2 as key mechanisms for microbiota enhancement of immune checkpoint inhibitor therapy. Inspired by this work and other studies of NOD2 activation, we performed in silico ligand screening and developed N-arylpyrazole dipeptides as novel NOD2 agonists. Importantly, our N-arylpyrazole NOD2 agonist is enantiomer-specific, effective at promoting immune checkpoint inhibitor therapy and requires NOD2 for activity in vivo. Given the significant functions of NOD2 in innate and adaptive immunity, these next-generation agonists afford new therapeutic leads and adjuvants for a variety of NOD2-responsive diseases.

cancer biology↗