Biology subjects

Sargunas, J.

Publications and source records attributed to Sargunas, J..

3 recordsLinked to original sources

bioRxiv2026

Exhaustive Isotope Tracing Reveals the Butterfly Effect of Mammalian Producer Cell Line Selection for Biomanufacturing

Chinese hamster ovary (CHO) cells are the dominant platform for recombinant biotherapeutics, yet the impact of producer cell line selection on mammalian cell metabolism remains poorly understood. Here, we performed 41 independent 13C-tracer experiments using uniformly labeled glucose or individual amino acids to comprehensively map carbon utilization in the two principal CHO production platforms: methotrexate-selected CHO-K1 and glutamine synthetase-selected CHO-GS cells. Time-resolved GC-MS analysis revealed distinct metabolic phenotypes spanning central carbon metabolism, amino acid interconversion, lipid biosynthesis, and one-carbon metabolism. CHO-K1 cells exhibited extensive reductive carboxylation and pyruvate carboxylase-mediated anaplerosis, whereas CHO-GS cells redirected glutamate toward glutamine synthesis and relied on asparagine and aspartate to support TCA cycle activity. Isotopomer analysis uncovered substantial intracellular-extracellular cycling of alanine, glycine, glutamate, and serine despite contrasting uptake profiles and quantified differential amino acid contributions to fatty acids and cholesterol. Serine, glycine, and methionine labeling revealed active folate-cycle interconversion in CHO-K1 and enhanced methionine-cycle activity in CHO-GS. Aspartate is identified as a key redox exchange factor and uniquely informative tracer for pathway characterization following glutamine depletion. Together, this exhaustive isotope-tracing framework establishes how producer cell line selection rewires mammalian metabolism and provides a foundation for cell engineering, media optimization, metabolic modeling, and next-generation biomanufacturing.

cell biology↗

bioRxiv2026

Tailored Cell Cycle Modulation Enhances AAV Manufacturing: Balancing Arrest with Adaptive Stress Responses

Recombinant adeno-associated virus (rAAV) vectors show therapeutic potential, but their biomanufacturing is limited by low yields and high costs. Host cell-cycle modulation is emerging as a promising strategy to enhance rAAV production. Two G2/M phase-arresting small molecules, ABT-751, a microtubule inhibitor, and helenalin, a thiol-reactive sesquiterpene lactone, were applied post-transfection in HEK293 cells to evaluate how cell-cycle arrest and stress pathways influence rAAV yields. ABT-751 induced G2/M arrest with minimal cytotoxicity, leading to a near five-fold increase in rAAV vector genomes across multiple serotypes and production platforms. Helenalin caused G2/M arrest, yet suppressed rAAV production. Comparative transcriptomic profiling (RNA-Seq) revealed that helenalin altered expression of a widespread set of genes (4,579) compared to control, characterized by rampant p53, ferroptosis, and endoplasmic reticulum dysregulation that overflowed into unfolded protein response with CHOP induction and apoptosis. ABT-751 elicited a more moderate, targeted response (1,895 differentially expressed genes) in a similar subset of pathways, including compensatory mechanisms mitigating oxidative stress. Together, these findings indicate that cell-cycle arrest alone is insufficient to improve rAAV yield. Indeed, tailored cell-cycle modulation, coupled with balanced activation of cellular stress pathways, can enhance rAAV manufacturing efficiency, facilitating more scalable and cost-effective gene therapy production strategies for the future.

bioengineering↗

bioRxiv2026

A continuous viral vaccine biomanufacturing platform utilizing multiple bioreactor configurations

Scalable, continuous biomanufacturing processes have grown in importance to meet demand for smaller bioreactor sizes, lowered production costs, and improved quality attributes. The Sf9/recombinant baculovirus (rBV) expression system demonstrates promise for virus-like particle (VLP) vaccine and gene therapy production. Here, we present a continuous rBV platform integrating an infection plug flow reactor (PFR) between stirred tank growth (gCSTR) and production (pCSTR) bioreactors. Cell expansion in the gCSTR included a ramp-up stage followed by continuous growth, reaching a steady state of 5x106 cells/mL and >90% viability. Peclet number-fit tracer studies confirmed near-ideal plug flow in the PFR, yielding a 10 h residence time and progressive infection as measured by gp64 signaling. Finally, a pCSTR with a residence time of 48 h exhibited sustained recombinant protein production. An integrated pilot cascade incorporating all reactors ran continuously for 5 days, maintaining stable CSTR cell densities and a measurable increase in infected cell diameter from 14.5 m to 16.1 m. Western blotting and EM of [~]100 nm VLPs in pCSTR effluent demonstrated platform success. Digital twin mechanistic models across four distinct stages of bioreactor operation and Hill-type relationships for rBV infection kinetics predicted cell growth and death for a 7-day run, demonstrating promise for designing continuous systems in silico and building a quantitative framework for scale-up and optimization. Our multi-stage reactor configuration represents a cell host- and product-agnostic production scheme, particularly for processes prone to product heterogeneity, and paves the way towards a true end-to-end continuous platform for myriad modalities in the future.

bioengineering↗