Biotechnology and Bioengineering

Bioreactor pH can significantly affect Chinese Hamster Ovary (CHO) cell metabolism, thus impacting glycoprotein titers. However, there is very limited literature on incorporating pH in mathematical models for CHO cell metabolism. To address this limitation, guided by recently published experimental data, we have curated a stoichiometric network and formulated phenotype-driven kinetic expressions to develop a Dynamic Metabolic Flux Analysis (DMFA) model. The DMFA model incorporates Critical Process Parameters (CPPs), notably bioreactor pH, basal and feed media nutrient composition, feeding times, and inoculation cell densities to predict bioreactor performance: cell growth rates, antibody titers, and nutrient and metabolite profiles. The DMFA model was trained on diverse fed-batch data of the CHO VRC01 cell line to regress the kinetic parameters. The models utility was demonstrated through experimentally validated model predictions of CHO-cell performance in intensified fed-batch cultures, perfusion cultures, and cultures with different media. Experimentally validated predictions of a culture with high initial cell density and increased feed addition (intensified fed-batch culture) showed that mAb titers similar to fed-batch culture can be achieved with shorter culture durations. Similarly, experimentally validated predictions of perfusion bioreactor performance showed that coupling historical fed-batch data with computational tools can be leveraged to predict continuous biomanufacturing performance. We thus demonstrate that the developed mathematical model can simulate culture performance outside of the training data set. This supports the predictive robustness of the framework and provides a valuable tool for bioprocess development of diverse culture modes. HighlightsO_LIExperimentally measured fed-batch cell culture data was used to curate a reaction network. This reaction network was integrated with phenotypically driven kinetic expressions to yield a dynamic metabolic flux analysis (DMFA) model. C_LIO_LIThe DMFA model can predict bioprocess performance indicators such as concentration of viable cells, mAb, amino acids, glucose, lactate, and ammonia. C_LIO_LIThe model was developed to make these predictions under various process conditions such as bioreactor pH, media concentrations, feed supplementation schedule, and initial cell densities. C_LIO_LIPredicting and experimentally validating the impact of high initial cell density and increased feed media supplementation yielded in mAb titers similar to traditional fed-batch processes with much shorter culture durations. C_LIO_LIThe application of the DMFA model trained on data from a traditional fed-batch process to predict perfusion bioreactor culture performance was successfully demonstrated and experimentally verified. C_LIO_LIThe impact of AMBIC reference media on cell culture process performance was also predicted and experimentally validated. The predictions of amino acid metabolism yielded insights into improving the media. C_LI

To comparatively evaluate cellular constraints on recombinant monoclonal antibody (mAb) production by Chinese Hamster Ovary (CHO) cells, we analysed the transcriptomes of 24 clonally derived CHO cell lines engineered with PiggyBac transposon technology to stably produce four recombinant monoclonal antibodies (mAbs) at varying specific production rates. Fed-batch cultures were sampled at exponential (day 5) and stationary (day 10) phases of culture for analysis by RNA-Seq. Recombinant mRNAs accounted for a large proportion of total mRNA across all clones, and efficient use of heavy chain (HC) mRNA to synthesise recombinant mAb (qP per HC mRNA) varied significantly with respect to both mAb product and cell line. Comparative bioinformatic analyses of CHO transcriptomes focussed on mAb specific production rate and utilised both data-driven and hypothesis-led approaches, specifically (i) production or non-production of recombinant mAb, (ii) changes in the abundance of functional groups of mRNAs abundance with mAb specific production rate and (iii) comparative analysis of informatically-mined gene subsets associated with cellular functions hypothesised to impact recombinant mAb synthesis and secretion. These analyses revealed widespread constitutive and adaptive changes in mRNA abundance associated with mAb production across a variety of cellular functions. Typically, most mechanistically consistent changes in mRNA abundance co-varying with mAb production were evident at the stationary phase sample point. These data revealed both recombinant mAb-specific limitations on cellular synthetic capacity and a generic adaptive strategy used by CHO cells to support high-level mAb production. The latter was achieved by directed and permissive regulation of endoplasmic reticulum and other processes to accommodate increased synthetic flux.

Biomass separation represents a critical bottleneck in Komagataella phaffii-based biopharmaceutical processes, as typically high cell densities of 40 - 50 % create significant operational, technical and economic challenges for harvest operations. Yeast cell aggregation (flocculation) provides a solution to accelerate cell sedimentation by increasing particle size, thus allowing to improve biomass-supernatant separation efficiency during both natural gravity settling and (continuous) centrifugation operations. This study demonstrates successful engineering of K. phaffii strains with an inducible flocculation phenotype using CRISPR/Cas9-based genome editing to integrate the Saccharomyces cerevisiae FLO1 (ScFLO1) gene under control of various regulatory elements, including methanol-inducible and derepressible promoters. Flocculation strength could be enhanced by implementing transcriptional positive feedback circuits based on the methanol-inducible AOX1 promoter. To address methanol-free production requirements, we developed alternative systems to retrofit PAOX1-based ScFLO1 expression and exploited the derepressible PDF promoter, offering broader compatibility with biopharmaceutical manufacturing facilities. Flocculating cells cultivated in a bioreactor demonstrated significantly improved sedimentation behavior, with considerably lower supernatant turbidity after short low-speed centrifugation compared to non-flocculating controls. Crucially, cell flocculation had no negative impact on product amount and quality when expressing a multivalent NANOBODY(R) VHH molecule with pharmaceutical relevance. Thus, this work establishes the first genetically engineered flocculation system in K. phaffii compatible with recombinant protein production, providing the basis for an innovative approach to streamline harvest operations in biopharmaceutical processes.

Genomics-based safety and quality evaluation studies are advancing the bioindustry by enhancing various aspects, including viral safety, host cell protein (HCP) control, product heterogeneity control, cellular heterogeneity control, and process reproducibility. High-throughput instruments and genome-scale databases are essential in genomics, with the reference genome sequence being the most critical database. The completeness and accuracy of these genome sequences depend on DNA quality, sequencing instruments, read coverage, and assembly strategies. Significant efforts are being made to perfect genome assembly and continuously improve it. However, the quantitative impact of reference genome sequence accuracy on the safety of biologics is not yet fully understood. In this study, we compared and benchmarked six Chinese hamster genomes, including four newly sequenced genomes derived from Chinese hamster cell lines, from an industrial perspective. We also developed database assembly techniques to enhance the safety of biologics. We recommend using two or more independent reference genomes for viral safety studies. For HCP control, we suggest using protein sequences in which trypsin degradation peptides that overlap with high-risk proteins should be masked and unified. Additionally, we can predict microenvironments using single-cell transcriptome data. In bioengineering processes, any nucleotide samples have potential commercial benefits.

The suspension culture of pluripotent stem (PS) cells in stirred bioreactors poses a delicate balance between maintaining homogeneous cell dispersion and avoiding excessive shear stress that can compromise cell viability and pluripotency. In this study, we used computational fluid dynamics (CFD) coupled with a discrete particle method (DPM) to simulate iPS cell behavior in a 5 mL delta-impeller stirred tank. Our analysis revealed that upward flow at the tank bottom and downward flow at the top are critical for maintaining a stable suspension. To optimize the stirring protocol, we applied Bayesian optimization to identify a time-dependent stirring schedule that begins with a high-speed phase for resuspension, followed by a low-speed phase for sustained suspension with minimal hydrodynamic stress. The optimized schedule demonstrated improved suspension ratio and reduced slip velocity, indicating lower mechanical stress on cells. These findings provide engineering insights into scalable bioreactor operation, contributing to the design of robust iPS cell manufacturing systems.

For protein drug purification, packed-bed chromatography often remains both the predominant method and a bottleneck for cost and scalability. Accordingly, extensive efforts have been made to develop alternatives, such as precipitation and liquid-liquid extraction. Despite decades of development, such methods have been slow to see adoption in commercial processes. To diagnose the key barriers to implementation and guide future work, we have systematically reviewed studies of protein precipitation and liquid-liquid extraction. We classify the products, methods, and results of 168 publications representing 290 unique purification operations and analyze these operations in terms of both process economics and purification performance. Whereas it is generally assumed that precipitation and extraction methods will have lower costs than chromatography, we find that this is only the case under specific process conditions such as at a large manufacturing scale and low initial sample purity. Furthermore, we find that only a small number of the many precipitation and extraction methods reported to date have shown readiness for implementation in protein drug purification processes. Finally, we identify key factors governing both the economic and purification performance of this class of methods: first, that operating costs are almost entirely predictable by the ratio between the mass of phase-forming materials used and the mass of product protein yielded; second, that use of modern optimization techniques such as Design of Experiments is associated with significantly better purification performance and cost-effectiveness. HighlightsO_LIAlternative separation purification methods are not always cheaper than chromatography C_LIO_LIThe use of a combination of phase separating agents remains largely underexplored/underutilized C_LIO_LILower initial purity and increasing production scale favor phase-separation over chromatography C_LIO_LIThe direct material usage rate is an important predictor of alternative separation cost-effectiveness C_LIO_LICurrent alternative separation method development has largely ignored optimization of direct material usage rate C_LI

Bispecific antibodies are a growing class of therapeutics that simultaneously engage two targets. However, their complex molecular structures pose challenges for production in Chinese hamster ovary cells, the current industry standard for biologics manufacturing. Here we present a case study of three IgG-scFv format bsAbs expressed in CHO cells, in which one candidate exhibited markedly lower titers despite high sequence homology to the other two. Using multi-omics analysis (RNA sequencing, splicing prediction, codon optimization assessment, and motif screening) to investigate potential causes, we identified several likely mechanisms for poor expression, including aberrant splicing motifs, ribosome pausing sites, and suboptimal codon usage. Through targeted protein and DNA sequence engineering, we generated a revised variant with an 11-fold increase in stable expression titers. This work demonstrates that integrating sequence-level bioinformatic and synthetic biology diagnostics can directly improve manufacturability, providing a generalizable framework for resolving hidden expression liabilities in complex biologics.

The Coalition for Epidemic Preparedness Innovations 100-day moonshot aspires to launch a new vaccine within 100 days of pathogen identification. Here, we describe work to optimize adenovirus vector manufacturing for rapid response, by minimizing time to clinical trial and first large-scale supply, and maximizing the output from the available manufacturing footprint. We describe a rapid viral seed expansion workflow that allows vaccine release to clinical trials within 60 days of antigen sequence identification, followed by vaccine release from globally distributed sites within a further 40 days. We also describe a new perfusion-based upstream production process, designed to maximize output while retaining simplicity and suitability for existing manufacturing facilities. This improves upstream volumetric productivity of ChAdOx1 nCoV-19 by around four-fold and remains compatible with the existing downstream process, yielding drug substance sufficient for 10000 doses from each liter of bioreactor capacity. Transition to a new production process across a large manufacturing network is a major task. In the short term, the rapid seed generation workflow could be used with the existing production process. We also use techno-economic modelling to show that, if linear scale-up were achieved, a single cleanroom containing two 2000 L bioreactors running our new perfusion-based process could supply bulk drug substance for around 120 million doses each month, costing <0.20 EUR/dose. We estimate that a manufacturing network with 32000 L of bioreactor capacity could release around 1 billion doses of a new vaccine within 130 days of genomic sequencing of a new pathogen, in a hypothetical surge campaign with suitable prior preparation and resources, including adequate fill-and-finish capacity. This accelerated manufacturing process, along with other advantages such as thermal stability, supports the ongoing value of adenovirus-vectored vaccines as a rapidly adaptable and deployable platform for emergency response.

The Ebola virus (EBOV) disease has caused serious and recurrent epidemics in recent years, resulting in a fatality rate of nearly 50%. The most effective experimental therapy against the EBOV is the use of monoclonal antibodies (mAbs). In this work, we describe the development of HEK293T cells engineered for the transient and stable expression of mAb13C6, a neutralizing anti-EBOV monoclonal antibody. We transfected the HEK293T cells with a tricistronic vector to produce the heavy and the light chain of the antibody 13C6 and intracellular Green Fluorescent Protein (GFP) using Lipofectamine 3000. We then selected the transfected cells using puromycin pressure, dilution cloning, and cloning disks. This integrated strategy generated mAb-producing cells in 7 days with a transient expression of [~]1 mg/L. Stable pools were produced after 4 weeks, with expression levels of [~]0.8 mg/L. Stable clones with expression levels of [~]1.8 mg/L were obtained within 10 weeks. The produced antibodies exhibited the expected functionality; they recognized the GP glycoprotein of the Ebola virus in both ELISA assays and cell binding experiments using HEK293T cells engineered to express the EBOV GP at their membrane surface. By the combined use of GFP and the set of selection techniques here described, we drastically reduced the time from transfection to stable clone generation without resorting to costly equipment. In outbreaks or emergencies, this platform can significantly shorten the development of new biopharmaceuticals and vaccines.

Proaerolysin is a bacterial toxin produced by Aeromonas hydrophila that specifically binds to GPI-anchored proteins on the plasma membrane, creating transmembrane pores that lead to cell death within a few hours. Leveraging this unique property, proaerolysin is widely used in diagnostic tests for paroxysmal nocturnal hemoglobinuria (PNH), a disease caused by somatic mutations in the PIGA gene, which is involved in the biosynthesis of GPI anchors. Additionally, proaerolysin serves as a counter-selection agent in genetic manipulations. Although bacterial expression and purification of proaerolysin have been previously reported, yields were low due to the absence of internal disulfide bonds, which are crucial for protein stability. Here, we demonstrate that using the Shuffle E. coli strain, which facilitates the formation of disulfide bonds in the cytoplasm, significantly improves the solubility and proper folding of proaerolysin. We achieved a high yield of proaerolysin, approximately 3 mg from a 50 ml bacterial culture, with a purity of over 99%. The functionality of recombinant proaerolysin was confirmed by testing in mouse embryonic stem cells (mESCs), demonstrating that this high-yield production method offers a reliable and cost-effective source of functional proaerolysin for a wide range of biotechnological applications.

Bispecific antibodies are at the forefront of biopharmaceutical drug development. With over 100 different molecular architectures combined with diverse individual subunit sequences, choosing the most suitable structure and predicting the ideal subunit expression ratios for successful heterodimerization is a significant challenge. In this paper, we demonstrate that the recently described cell line development paradigm shift (Rajendran et al. 2021), enabled by the Leap-In transposon platform, can be extended to the development of bispecific monoclonal antibody-producing cell substrates (stable clones and pools). The key features are 1) Parental pools reliably predict the derivative clonal productivity and clonal heterodimer fractions. 2) Clonal productivity and clonal heterodimer fraction remained stable for at least 60 population doublings. 3) Depending on the products biophysicochemical properties, the stable pools exhibit variable productivity stability. 4) Heterodimer fractions remain stable in the Leap-In mediated stable pools independently of the productivity stability of the pools. 5) Structures and subunit ratios can be triaged at stable pool level, and 6) Due to the homogeneous clonal productivity distribution, only a small number ([~]50) of clones need to be isolated and characterized.

This study explores the potential of utilizing {beta}-cells, exemplified with R7T1 {beta}-cell pseudoislets, as a transplantable cell factory for on-demand recombinant protein therapeutic delivery. While mammalian cell lines are widely used for in vitro protein production, the commonly utilized constitutive secretion pathway poses challenges to in vivo cell therapy, especially for delivering proteins requiring precise exposure kinetics. The proposed approach capitalizes on unique aspects of {beta}-cells, including substantial vesicular protein storage capacity and electrochemically-regulated protein release, to facilitate timely and titratable in vivo therapeutic delivery. Examining a variety of strategies to acheive {beta}-cell glucagon or glucagon-like peptide 1 (GLP-1) storage and secretion, we devised a flexible {beta}-cell-based expression platform for efficient cellular peptide production and on-demand release. This platform utilizes the preproinsulin coding sequence as a template, wherein therapeutic peptides of interest (glucagon or GLP-1) are substituted for C-peptide while the A- and B-peptide insulin chains are mutated to prevent bio-active insulin production. This approach overcomes the challenge of efficient bio-active peptide expression by leveraging the endogenous {beta}-cell peptide expression, translation, processing, storage and secretion machinery. Furthermore, {beta}-cells provide a mechanism for scalable electyrochemnically-triggered peptide delivery. This transformative strategy, which may be extended to other proteins and peptide expression cassettes, holds significant promise for targeted and temporally controlled in vivo production and release of recombinant protein therapeutics. The study suggests potential applications in addressing challenges in metabolic disorders, blood disorders, and oncology. Future refinements may focus on optimizing vector design, peptide production, and in vivo adaptation.

There is a growing interest in continuous processing of the biopharmaceutical industry. However, the technology transfer from traditional batch-based processes is considered a challenge as protocol and tools still remain to be established for their usage at the manufacturing scale. Here, we present a model-based approach to design optimized perfusion cultures of CHO cells using only the knowledge captured during small-scale fed-batch experiments. The novelty of the proposed model lies in the simplicity of its structure. Thanks to the introduction of a new catch-all variable representing a bulk of by-products secreted by the cells during their cultivation, the model was able to successfully predict cellular behavior under different operating modes without changes in its formalism. To our knowledge, this is the first experimentally validated model capable, with a single set of parameters, to capture culture dynamic under different operating modes and at different scales.

SARS-CoV-2 Spike is a key protein that mediates viral entry into cells and elicits antibody responses. Its importance in infection, diagnostics, and vaccinations has created a large demand for purified Spike for clinical and research applications. Spike is difficult to express, prompting modifications to the protein and expression platforms to improve yields. Alternatively, Spike receptor binding domain (RBD) is commonly expressed with higher titers, though it has lower sensitivity in serological assays. Here, we improve transient Spike expression in Chinese hamster ovary (CHO) cells. We demonstrate that Spike titers increase significantly over the expression period, maximizing at 14 mg/L at day 7. In comparison, RBD titers peak at 54 mg/L at day 3. Next, we develop 8 Spike truncations (T1-T8) in pursuit of a truncation with high expression and antibody binding. The truncations T1 and T4 express at 130 mg/L and 73 mg/L, respectively, which are higher than our RBD titers. Purified proteins were evaluated for binding to antibodies raised against full-length Spike. T1 has similar sensitivity as Spike against a monoclonal antibody and even outperforms Spike for a polyclonal antibody. These results suggest T1 is a promising Spike alternative for use in various applications.

The need for protein production has been growing over the years in various industries. We here present a high-throughput screening strategy to isolate high producer budding yeast clones from a mutagenized cell population using gel microdrop (GMD) technology. We use a microfluidic water-in-oil (W/O) emulsion method to produce monodisperse GMDs and a microfluidic cell sorter for damage-free sorting of GMDs by fluorescently quantifying secreted proteins. As a result, this high-throughput GMD screening method effectively selects high producer clones and improves protein production up to five-fold. We speculate that this screening strategy can be applied, in principle, to select any types of high producer cells (bacterial, fungal, mammalian, etc.) which produce arbitrary target protein as it does not depend on enzymes to be produced.

Human pluripotent stem cells (hPSCs) hold significant promise for regenerative medicine, yet optimizing their expansion in three-dimensional bioreactor systems remains challenging due to complex interactions between mechanical forces, metabolic constraints, and aggregate formation dynamics. This study developed and validated a mechanistic mathematical model to predict hPSC proliferation dynamics in vertical-wheel bioreactor (VWBR) systems, incorporating the effects of shear stress and energy dissipation rate (EDR) on cell growth and aggregate dynamics. Seven model variants employing different kinetic formulations for shear stress and energy dissipation rate effects were systematically evaluated through model selection, identifiability analyses, and experimental validation. Experimental data from six bioreactor conditions varying in initial cell density (2 x 104-15 x 104 cells/mL), agitation rate (30-60 RPM), and working volume (100-500 mL) were used for model calibration and selection. Bayesian Information Criterion analysis identified a model combining Michaelis-Menten kinetics for shear stress inhibition with a EDR-mediated aggregate detachment formulation as the best-performing variant, achieving a Mean Relative Prediction Error of 13.97%, comparable to the experimental variability of 16.29%. Independent validation experiments using leave-out data gathered under different media exchange schedules confirmed model accuracy with prediction errors below 14%, consistent with observed experimental variability around 12%. The validated model was used to optimize the media exchange protocol, leading to a 37.5% reduction in media consumption with only a 13.5% reduction in final cell yield, demonstrating its utility for prospective, quantitative bioprocess design in VWBR systems.

PurposeLarge quantities of human pluripotent stem cells (hPSCs) are required for clinical applications. 3D suspension cultures are suitable for large scale manufacturing of hPSCs but yield, viability and quality are affected by the hydrodynamic environment. This paper characterizes the hydrodynamic environment inside vertical wheel bioreactors (VWBRs) as a function of size and agitation rates, measures its effect on cell aggregation and proliferation, and proposes the use of Lagrangian-based shear stress and energy dissipation rate (EDR) exposures to support scale-up. MethodsIn silico: Transient, 3D, turbulent flow simulations are conducted for two VWBR sizes (100, 500 mL) at five agitation rates between 20 and 80 rpm. Trajectories of cell aggregates of sizes from 200 to 1,000 microns are calculated, and shear stress and EDR exposures are collected along these trajectories. In vitro: ESI-017 hPSCs were cultured in VWBRs for 6 days. Aggregation efficiency and daily fold ratios were calculated based on cell counts and initial inoculation density. ResultsAggregate size, agitation rate and bioreactor size modulate cell aggregate exposures to EDR and shear stress, which significantly depart from maximum or volume average metrics used for scale-up. Combined in vitro/in silico results show EDR affects aggregation efficiency, cell counts and aggregate size, and has a small effect on daily fold ratios but a significant effect on total fold ratio. ConclusionHistory of trajectory-based cell aggregate exposures to EDRs provide a better scale-up basis for VWBRs than volume-averaged EDR. Shear stress does not significantly affect hPSC aggregation, proliferation and expansion in VWBRs under the tested conditions.

Global shortages of human plasma-derived immunoglobulin G (IgG) remain a major challenge for treating primary immunodeficiencies, especially in low- and middle-income countries. Ensuring virus safety is essential, and nanofiltration provides robust removal of small, non-enveloped viruses. We examined whether removing immunoglobulin A (IgA) and immunoglobulin M (IgM) by anion-exchange chromatography improves the performance of 20-nm nanofiltration applied to small-pool caprylic acid-purified IgG. Cryo-poor plasma was treated with 5% caprylic acid at pH 5.5, concentrated by ultrafiltration, and processed on Fractogel TMAE to deplete IgA and IgM. The IgG flow-through was filtered sequentially through Planova 35N and 20N (or S20N) filters. Direct nanofiltration of caprylic acid-treated IgG with residual IgA and IgM led to rapid membrane clogging and low throughput. Depletion of IgA and IgM increased filtration capacity more than threefold and stabilized flux. Dynamic light scattering confirmed the predominance of monomeric IgG and absence of aggregates after chromatography and nanofiltration. Overall, this process combines two complementary virus reduction steps, caprylic acid treatment and nanofiltration, and provides a practical option for LMICs to convert available domestic plasma into IgG; it could also be adapted to the manufacture of hyperimmune or convalescent IgG preparations.

The virus-based immunosorbent nanoparticle is a nascent technology being developed to serve as a simple and efficacious agent in biosensing and therapeutic antibody purification. There has been particular emphasis on the use of plant virions as immunosorbent nanoparticle chassis for their diverse morphologies and accessible, high yield manufacturing via crop cultivation. To date, studies in this area have focused on proof-of-concept immunosorbent functionality in biosensing and purification contexts. Here we consolidate a previously reported pro-vector system into a single Agrobacterium tumefaciens vector to investigate and expand the utility of virus-based immunosorbent nanoparticle technology for therapeutic protein purification. We demonstrate the use of this technology for Fc-fusion protein purification, characterize key nanomaterial properties including binding capacity, stability, reusability, and particle integrity, and present an optimized processing scheme with reduced complexity and increased purity. Furthermore, we present a coupling of virus-based immunosorbent nanoparticles with magnetic particles as a strategy to overcome limitations of the immunosorbent nanoparticle sedimentation-based affinity capture methodology. We report magnetic separation results which exceed the binding capacity of current industry standards by an order of magnitude.

Efficient and robust bacterial cellulose production is essential for advancing the sustainable bioeconomy. In this study, we investigated the impacts of metabolism of organic acids, mainly gluconic acid (GA), on nanofibrillated bacterial cellulose (NFBC) production by Komagataeibacter intermedius NEDO-01 under various culture conditions in aerated stirred-tank reactors. In cultures of the wild-type strain in a standard medium, rapid GA production decreased the medium pH and depleted glucose, inhibiting cell growth and reducing the NFBC yield. However, proper pH control and continuous feeding reversed these effects, resulting in a 3-fold increase in NFBC yield (from 2.45 to 7.59 g/L). In cultures of a glucose dehydrogenase gene-deficient ({Delta}gcd) strain, lack of a pH drop and glucose depletion facilitated better cell growth, yielding 1.85-times more NFBC than that in wild-type cultures under pH-uncontrolled no-feed conditions (4.53 g/L). Notably, GA supplementation accelerated cell growth but significantly inhibited NFBC synthesis, suggesting that GA uptake redirects the carbon flux toward central metabolism. In the corn steep liquor (Csl)-based medium, cell growth was significantly enhanced, and NFBC yield was equivalent to or higher than that obtained with the Hestrin-Shramm medium. GA accumulation was markedly reduced, suppressing pH fluctuation. Under these optimized conditions, three molasses types were tested with Csl, yielding relatively high NFBC. Structural analysis of NFBC produced using these alternative media revealed slight differences in the fiber width distribution, with crystallinity and fiber width remaining constant. Overall, NFBC of consistent quality can be produced in stirred-tank reactors using Komagataeibacter spp. from various agricultural by-products. ImportanceIn this study, we investigated the interplay between organic acid metabolism and nanofibrillated bacterial cellulose (NFBC) production in stirred-tank reactor (STR) cultures of Komagataeibacter intermedius NEDO-01. While it is well known that gluconic acid production competes with cellulose biosynthesis in Komagataeibacter, the quantitative relationship between these pathways under varying culture conditions has not been fully elucidated. By applying optimized feeding strategies and employing a glucose dehydrogenase knockout mutant, we demonstrated that suppressing gluconic acid accumulation significantly enhances NFBC yield. Furthermore, we explored the use of agro-industrial by-products, including molasses and corn steep liquor, as alternative, low-cost feedstocks. Structural characterization confirmed that NFBCs produced under these conditions maintained consistent quality. These findings contribute to the development of scalable, cost-effective microbial production processes for nanocellulose, which is essential for advancing the sustainable bioeconomy. Key PointsO_LIGA accumulation inhibited growth and cellulose production by Komagataeibacter C_LIO_LIEnhanced central metabolism elevated NFBC yield but reduced its production rate C_LIO_LIConsistent NFBC properties were achieved in STRs using various by-product sources C_LI