The sixth modeling workshop (6MW) was held in June 2025 at Collegeville, Pennsylvania, USA, and supported by the Recovery of Biological Products Conference Series. The goal of the workshop was to assemble modeling practitioners to review and discuss the current state of implementation, progress since the last modeling workshop (5MW) in 2023, gaps and opportunities for development, deployment, and maintenance of models in bioprocess applications. One key observation from the 6MW was the positive impact of modeling on the development of a wide range of therapeutic modalities (proteins, vaccines, peptides) and unit operations (upstream, purification, and formulation), and across time (from discovery via molecular design/developability to commercialization). The scope of 6MW was broader than previous workshops, thus including biophysics and molecular modeling, mechanistic modeling and computational fluid dynamics, plant and sustainability modeling, artificial intelligence (AI) and big data modeling, and Highland Games 2.0. The AI and big data modeling session captured some of the initiatives across the pharmaceutical industry that have flourished with the recent development of new digital and AI technologies. The Highland Games 2.0 session benchmarked specific applications and advances of modeling tools to support developability predictions from sequence, building on the original competition in 2018. One key challenge emphasized at prior modeling workshops was the need for large data sets to support collaborative modeling development within the recovery community. This still remains a gap for the community at large (e.g., a shared reference point for academic/industrial collaborators for benchmarking). Significant progress on developing and curating large data sets within institutions to support in silico molecular and process development was reviewed in two sessions: AI and Big Data and Highland Games 2.0. A potential solution would be the use of specific descriptors in a database that would allow for detailed analyses without sharing proprietary information. The 6MW included two invited papers on building molecular understanding into the manufacturing process and summarizing the ongoing technical challenges, successes and best practices of utilizing federated learning at the National Institute for Innovation in the Manufacturing Biopharmaceuticals (NIIMBL), respectively. Advancement was reported in all of the modeling areas/disciplines discussed in sessions at the 6MW, with the greatest advancements observed in mechanistic modeling applied to mixing and filtration, AI and big data modeling, and mechanistic modeling applied to chromatography. Highlights of the workshop included significant advancements in biophysical/molecular modeling for vaccines and multi-scale applications, and mechanistic models for filtration. Nevertheless, continued research and development for all modeling approaches is still needed and should be pursued.

Anaerobic digestion is applied to organic wastes, yet a substantial fraction of carbon is released as CO2 because methanogens lack of low potential reducing equivalents. Here we show that heat-treated residues of algae Chlorella sorokiniana can be repurposed as chlorophyll-based biophotosensitizers to couple visible light harvesting to CO2 methanation by Methanosarcina barkeri (M. barkeri), providing a route supplying light-derived reducing power. Under autotrophic conditions with CO2 as the sole carbon source, residue supplementation enhanced methane formation in a chlorophyll-dependent manner and yielded 420 µmol L[-1] CH4 at 16 mg L[-1] chlorophyll after 7 d, with most of the increase occurring in the first 3 d. The residues retained functional photosystem II centers that supported charge separation and interfacial electron export, increasing the apparent electron transfer rate from 22.21 to 34.41 s[-1] and extending the fluorescence lifetime to 1.89 ns under illumination. Methane enhancement was mainly governed by photosystem II-derived electron generation and cytochrome-mediated electron entry into methanogens. Membrane-bound hydrogenases may also contribute to archaeal electron uptake at the interface, while inhibition of photosystem II or cytochrome-associated electron transfer reduced CH4 formation by approximately 70%. Illumination hyperpolarized the proton motive force of M. barkeri by 40 mV, consistent with incoming electrons and protons integrated through proton-coupled electron transfer steps that feed membrane energy-conserving modules and strengthen methanogenesis. This study demonstrates that discarded algal residues can function as renewable light-responsive inputs that introduce photogenerated reducing flux into the methanogenic network and redirect carbon flow from CO2 loss toward methane formation in anaerobic digestion.

Efficient electron supply and cofactor utilization remain key bottlenecks in cytochrome P450-catalyzed oxidations. Here, we report an electro-assisted system that overcomes these limitations, using the P450 Ema1-catalyzed oxidation of avermectin B1a as a model reaction. Under a constant current of 1 mA, > 96% conversion of 2.5 mM (2.2 g/L) substrate was achieved within 4 h, whereas the non-electrified system reached only 68% conversion after 8 h. The electro-assisted system enhanced catalytic efficiency at low substrate concentrations, while the advantage diminished at higher substrate loadings (≥ 5 mM), likely attributable to H2O2 accumulation, indicating a transition between different kinetic regimes. Prolonged electrical input led to enzyme deactivation, revealing a trade-off between catalytic enhancement and enzyme stability. Notably, transient electrical exposure for 1 h produced a catalytic activity gain that was abolished by catalase, persisted after current removal, and was accompanied by minor spectroscopic changes suggesting localized structural rearrangements rather than global conformational reorganization. Finally, we establish that the NADH/NAD[+] ratio, rather than absolute NADH concentration, is the master regulator of catalytic activity, with NAD[+] acting as a high-affinity allosteric effector. Our work demonstrates a controllable strategy for enhancing P450-catalyzed reactions by integrating electrical input with cofactor regulation, providing insights for the design of electro-biocatalytic processes.

Ultrafiltration/diafiltration (UF/DF) is a key unit operation in the manufacture of antibody-drug conjugates (ADCs), commonly utilized for post-conjugation purification to remove residual Drug Linker (DL) compounds and affect buffer exchange. The clearance of residual drug linker (DL), however, is often well below that predicted by ideal diafiltration theory. This work provides the first detailed mechanistic analysis of the factors that influence DL clearance during constant volume diafiltration, with particular emphasis on antibody concentration, membrane properties, and operating conditions. Diafiltration experiments in both stirred cell and tangential flow filtration systems demonstrate that DL sieving coefficients decrease with increasing ADC concentration and with increasing filtrate flux. These trends are quantitatively described using a newly developed diafiltration model accounting for intermolecular association between the hydrophobic DL and the ADC, with the ADC concentration at the membrane surface evaluated using the classical concentration polarization analysis. Experimental data obtained over a range of conditions are well-described using this concentration polarization-binding model. The model not only explains DL clearance data, but it also provides a framework for effective process design of UF/DF processes to ensure the required levels of DL clearance during ADC purification.

Oxidosqualene cyclases (OSCs) catalyze the cyclization of 2,3-oxidosqualene into diverse triterpenoids, yet their intrinsically low catalytic efficiency restricts biosynthetic productivity. Here, we establish a mechanism-guided synergistic engineering strategy that extends beyond conventional active-site engineering by integrating distal substrate access regulation with catalytic microenvironment optimization to enhance the catalytic performance of CrAS from Catharanthus roseus. Structural modeling and mechanistic analyses revealed a conserved catalytic framework involving carbocation-mediated polycyclization and identified a surface-exposed constriction region that regulates substrate access. Guided by these insights, distal surface engineering of the constriction region was synergistically combined with active pocket optimization. The resulting combinatorial mutant, M3 (L323A/T327K/N565I), exhibited a 95.2% increase in catalytic efficiency and enhanced α-amyrin and β-amyrin by 53.2% and 49.7%, reaching 158 mg/L and 63 mg/L, respectively. Multi-scale analyses combining molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations revealed that the enhanced catalytic performance is attributable to increased flexibility of the substrate access pathway, reinforced electrostatic and cation-π interactions, and reduced reaction energy barriers. Notably, distal mutation T327K improved substrate ingress through dynamic modulation of the protein surface, while N565I optimized the catalytic microenvironment by enhancing hydrophobic packing and stabilizing key intermediates. Overall, our findings establish a generalizable framework for engineering complex cyclases and provide a foundation for the sustainable microbial production of high-value triterpenoids.

In the face of rising energy demands, environmental degradation, and growing water scarcity, Microbial Fuel Cells (MFCs) offer a promising solution by harnessing waste to generate bioelectricity and treat wastewater. Among these, photosynthetic MFCs, particularly single-chamber systems, offer distinct advantages due to their compact design, sustainability, and dual functionality for power generation and pollution mitigation. These devices convert chemical energy from organic substrates directly into electrical energy via bacterial processes, while also enabling pollutant breakdown and carbon dioxide sequestration. Recently, there has been increased interest in integrating photosynthetic organisms, such as microalgae, into MFCs. Microalgae-assisted MFCs (MA-MFCs) can enhance power production by leveraging sunlight during photosynthesis, which simultaneously produces oxygen, acts as an electron acceptor, and facilitates pollutant degradation. Furthermore, microalgal biomass can serve as an anode substrate for bioelectricity generation. MA-MFCs are notable for their potential to improve Coulombic efficiency and eliminate the need for external aeration. Despite these advancements, challenges persist, including low energy yields and the complexity of upscaling. However, innovative approaches, such as modular MFC stacking, optimized photobioreactor integration, and the usage cost-effective materials, are showing considerable promise. This review examines recent developments, scalability barriers, and future directions in single-chamber photosynthetic microbial fuel cells, emphasizing their applicability in wastewater treatment, sustainable bioelectricity production, and pollutant remediation.

Recent discoveries of merbecoviruses such as HKU5, NeoCoV, and HKU5-CoV-2 that use angiotensin-converting enzyme 2 (ACE2) as their entry receptor underscore the potential for future spillover to humans. To combat these ACE2-utilizing merbecoviruses, we developed virus-like particle (VLP) vaccines displaying their receptor-binding domain and subdomain 1 (RBD-SD1) antigens fused to SpyTags. These antigens were then conjugated to SpyCatcher-mi3 nanoparticles. Immunization of mice with each RBD-SD1-mi3 vaccine produced robust IgG responses against homologous antigens and measurable but reduced binding to heterologous RBD-SD1 proteins. Antigenic cartography showed that antigenic relationships broadly reflected sequence identity with HKU5 positioned between NeoCoV and HKU5-CoV-2. Despite HKU5 and HKU5-CoV-2 sharing the greatest genetic similarity, cross-reactive titers between these two groups were not substantially higher than those observed with NeoCoV. In a surrogate virus neutralization test (sVNT), sera from mice vaccinated with NeoCoV RBD-SD1-mi3 or HKU5-CoV-2 RBD-SD1-mi3 showed strong inhibition of receptor binding by their respective Fc-fused RBD-SD1 proteins. Sera from mice vaccinated with HKU5 RBD-SD1-mi3 exhibited some cross-inhibition activity against both NeoCoV and HKU5-CoV-2 Fc-fused RBD-SD1. Together, these findings show that the RBD-SD1-displaying nanoparticle vaccine can elicit cross-reactive antibodies against ACE2-binding merbecoviruses and that antigenic relationships are shaped by more than sequence similarity.

Temperature exerts a critical influence upon coffee fermentation, driving microbial metabolic pathways, acidification kinetics, and post-harvest bean quality. This study presents an integrated computational fluid dynamics (CFD)-experimental validation framework to analyze a temperature-controlled coffee fermentation bioreactor utilizing an external water-jacket system. To establish a computationally efficient engineering design tool, transient CFD simulations were executed utilizing a specialized macro-scale bulk fluid domain. Spatial discretization integrity was verified via a rigorous grid convergence index study, yielding a low numerical uncertainty (GCI21 = 0.47%). Large-scale (25 kg) experimental trials conducted across three distinct thermal boundaries (17°C-20°C, 23°C-26°C, and 32°C-35°C) confirmed excellent thermal control stability, maintaining coefficients of variation below 5% and uniformity indices above 0.95. Macro-scale energy modeling reproduced the mean measured reactor temperature with a mean absolute percentage error (MAPE) of 8.38%, a mean absolute error (MAE) of 2.16°C, and a root mean square error (RMSE) of 2.24°C. It should be emphasized that the experimental validation was limited to the volume-averaged (bulk) reactor temperature at two operating conditions; the predicted spatial temperature distribution and transient flow field were not directly validated. This indicates that a simplified bulk-domain approach can approximate macro-scale thermal performance while avoiding costly multi-phase porous media overhead. Fermentation temperature strongly influenced processing kinetics and physical bean characteristics. Operating at 32°C-35°C maximized physical acceleration, driving a rapid pH reduction (to 4.14 within 12.0 h) and lowering residual mucilage to 11.2%. These effects, together with the significantly faster fermentation duration, were statistically significant (one-way ANOVA, p < 0.001). Conversely, the 23°C-26°C range produced a statistically significant non-monotonic acidification response (p < 0.001); we hypothesize that this reflects a shift in the dominant microbial pathway within the spontaneous mixed culture, although this interpretation was not confirmed by microbiological analysis. Bulk density and bean weight did not differ significantly among treatments (p = 0.25 and p = 0.91, respectively). These findings define a heat-transfer design space for temperature-regulated agricultural bioreactors that balances computational efficiency with physical processing predictability, within the bulk-thermal scope validated here.

Resolvin E2 (RvE2; 5S,18R-dihydroxyeicosapentaenoic acid) and 17R-resolvin D5 (17R-RvD5; 7S,17R-dihydroxydocosahexaenoic acid) are specialized pro-resolving mediators (SPMs) that actively promote the resolution of inflammation. However, their efficient biocatalytic production remains challenging because of the limited catalytic performance and poor stability of previously reported 5S-lipoxygenases (5S-LOXs). Here, we identified and characterized a novel double-oxygenating 15S-LOX from Pseudobdellovibrionaceae bacterium exhibiting 5S-LOX activity and evaluated its potential as a whole-cell biocatalyst for SPM production. Recombinant Escherichia coli cells expressing this enzyme stereoselectively converted 18R-hydroxyeicosapentaenoic acid (18R-HEPE) and 17R-hydroxydocosahexaenoic acid (17R-HDHA), prepared using engineered 18R- and 15R-LOXs from Sorangium cellulosum, into RvE2 and 17R-RvD5, respectively. Product identities were confirmed by chiral-phase HPLC and LC-MS/MS analyses. Reaction conditions, including pH, temperature, and DMSO, cell, and substrate concentrations, were optimized to enhance biotransformation performance. Under optimized conditions, 4.0 mM 18R-HEPE and 17R-HDHA were converted into 0.57 mM (190.61 mg/L) RvE2 and 1.48 mM (533.5 mg/L) 17R-RvD5 within 60 and 30 min, respectively. RvE2 production was 2.4-fold higher than that obtained using a previously reported purified 5S-LOX, and this study provides the first quantitative report of 17R-RvD5 production. These findings establish a double-oxygenating 15S-LOX with 5S-LOX activity as an efficient whole-cell platform for SPM production.

The plant-mediated biosynthesis of metal oxide nanoparticles (MONPs) has gained significant attention as a green, cost-effective, and sustainable alternative to conventional chemical and physical synthesis methods. By utilizing bioactive phytochemicals such as phenolics, flavonoids, alkaloids, terpenoids, and proteins, plant extracts act as natural reducing, capping, and stabilizing agents during nanoparticle formation. This eco-friendly approach avoids toxic reagents and harsh conditions, enabling controlled synthesis of nanoparticles with tunable size, shape, and surface properties. Various metal oxides, including zinc oxide (ZnO), titanium dioxide (TiO2), copper oxide (CuO), nickel oxide (NiO), silver oxide (Ag2O), and iron oxide (Fe2O3/Fe3O4), have been successfully synthesized using extracts derived from different plant parts such as leaves, roots, flowers, barks, and fruits. Characterization of these biosynthesized MONPs typically involves a range of instrumental characterization techniques. Synthesis parameters like pH, temperature, and extract concentration strongly influence the physicochemical attributes of the nanoparticles. This review presents a comprehensive overview of eco-friendly strategies for synthesizing MONPs, with a particular focus on their green synthesis from different plant parts through rapid and sustainable approaches. It discusses various synthesis methodologies, characterization techniques, and the resulting functional properties of the biosynthesized MONPs, supported by notable proof-of-concept studies. Furthermore, the review highlights the wide range of biomedical applications of plant-mediated MONPs, emphasizing their remarkable antimicrobial, antioxidant, anticancer, and anti-inflammatory activities. However, challenges remain in achieving reproducibility, large-scale synthesis, and mechanistic understanding. Considering the limited available data and the increasing industrial interest in plant-mediated MONPs, this review seeks to encourage researchers from diverse scientific fields to further investigate the potential of naturally synthesized MONPs and their emerging applications, thereby fostering progress in sustainable materials science and green nanotechnology.

Food security and environmental sustainability have emerged as major global challenges, driving the search for alternative protein production systems that can meet growing demand while reducing the environmental burden of conventional livestock farming. Among emerging alternatives, cultivated meat has gained considerable attention due to its potential to produce animal-derived protein without the need for large-scale animal rearing and slaughter. Although cultivated meat remains absent from most commercial markets owing to technical, economic, and regulatory challenges, significant milestones have been achieved, including regulatory approval in Singapore in 2020, completion of the U.S. Food and Drug Administration pre-market safety consultation in 2022, and authorization for commercial sale by the U.S. Department of Agriculture in 2023. Substantial progress has been made in cell sourcing, cell-line engineering, serum-free culture media development, scaffold fabrication, and bioprocess optimization. However, large-scale production of cultivated meat with desirable texture, flavor, nutritional quality, and economic viability remains challenging. Key bottlenecks include the development of cost-effective animal-component-free media, scalable bioreactor systems, edible scaffold materials, and efficient downstream processing strategies. This review provides an updated assessment of recent advances in cultivated meat technology, encompassing cell-line development, scaffold engineering, bioprocessing, post-processing approaches, sustainability considerations, and regulatory frameworks. Furthermore, current technological limitations, commercialization challenges, and future research priorities are discussed to evaluate the potential role of cultivated meat within sustainable and resilient future food systems.

Adeno-associated virus (AAV) has emerged as a leading platform for gene therapy. However, to unlock the full potential, their manufacturing yields, stability, and efficacy must all be improved. Rational design is limited by poor predictability and the potential impact of mutations on multiple important vector properties. Directed evolution requires little knowledge or predictability, but requires large libraries, and lacks appropriate selection methods to ensure that all properties needed for successful translation are achieved. Here we bring semi-rational design to AAV capsid engineering, combining co-evolutionary coupling and consensus-based design. We designed 110 mutants at sites with different degrees of coupling and mutated them either back to consensus or with various residue similarities according to BLOSUM62. Seven were selected based on their yields and transduction efficiencies before and after heat treatment. Our results demonstrate that mutations at sites within coupled networks exhibited the highest success rates. The BLOSUM62 selection strategy and consensus design approaches had similar success rates. K-means clustering of mutants further revealed that five of the successful mutants clustered in regions with moderate coupling scores and positive BLOSUM62 values. This study highlights the potential of using evolutionary coupling analysis to guide AAV capsid engineering, suggesting that conservative mutations informed by evolutionary data can improve AAV stability and efficacy.

Recombinant humanized type III collagen has attracted increasing interest for biomedical and tissue engineering applications due to its roles in extracellular matrix remodeling and tissue repair. However, the production of long-continuous, structurally stable type III collagen fragment in Pichia pastoris is limited by host-derived proteolysis of the recombinant α1 chain, resulting in fragmentation and loss of structural integrity. In this study, a long-continuous type III collagen fragment was produced in P. pastoris by proteolytic selection and identified as a continuous 585-amino acid sequence (N611-P1195) by LC-MS/MS. Secretion efficiency was improved by signal peptide optimization, enabling the development of high-producing strains (6.22 g/L). rColIII was purified to > 90% purity with low endotoxin levels (< 10 EU/mg) using multimodal chromatography and exhibited enhanced thermal stability. Although CD analysis indicated the absence of a canonical triple-helix conformation, FITR spectroscopy confirmed preservation of key peptide backbone structures. Biological assays demonstrated favorable cytocompatibility, with rColIII promoting HSF cell viability, adhesion, and migration. This study provides a scalable strategy for producing long, continuous type III collagen fragment with improved stability in P. pastoris, and supports their potential applications in cosmetic, tissue engineering, regenerative medicine, and other biomedical fields.

Virus-like particles (VLPs) are a promising modality with extensive applications in prophylactic and therapeutic vaccine design. Human Papillomavirus (HPV) vaccines are one such example where VLP vaccine applications have led to the successful reduction in HPV-associated diseases, such as cervical cancer. Current purification approaches utilize hydroxyapatite (HA) chromatography in bind-and-elute mode to purify HPV VLPs from host-cell impurities such as proteins and nucleic acids, though binding capacity for VLPs is low due to mass transfer limitations related to their large particle size. The intent of this work was to utilize VLP disassembly prior to HA chromatography in order to increase column yield and capacity. Since L1 protein subunits demonstrated weaker binding avidity for HA resin compared to VLPs, this enabled flow-through purification of L1 protein from strongly adsorbed host cell impurities. Mobile phase conditions were evaluated in batch partition screening experiments to determine the optimal conditions for L1 protein recovery with selective clearance of nucleic acid. Dynamic loading studies showed that while nucleic acid removal was equivalent to the VLP purification process, the flow-through process with disassembled L1 protein achieved higher L1 yield and column capacity. Optimal conditions were applied to HA flow-through purification for multiple HPV types with recombinant L1 protein expressed in yeast, demonstrating > 35% increased yield for six of eight types. The purified, disassembled proteins were subsequently reassembled and had comparable product attributes to the VLP purification process. The implementation of disassembly prior to purification also eliminates the requirement to perform a distinct final disassembly/reassembly process after isolating crude VLPs during purification.

Mixotrophic growth of microalgae involves the simultaneous assimilation of inorganic and organic carbon sources, but the quantitative contributions of photoautotrophic and photoheterotrophic metabolisms remain poorly resolved. In this study, a stoichiometric and data reconciliation framework was developed to partition mixotrophic metabolism of the microalga Scenedesmus obliquus into photoautotrophic and photoheterotrophic fluxes using macroscopic measurements of carbon, nitrogen and oxygen exchanges. Continuous cultures were conducted under photoautotrophic, photoheterotrophic and mixo-trophic conditions in a controlled photobioreactor. Elemental (C, N) and redox balances were applied to reconcile the experimental measurements before estimating metabolically consistent flux distributions. Under the specific experimental conditions investigated, mixotrophic biomass productivity was consistent with the sum of the photoautotrophic and photoheterotrophic productivities. This observation did not provide evidence of a positive macroscopic synergistic effect, although run-to-run biological variability could not be assessed because each trophic mode was investigated in a single independent steady-state experiment. Based on external fluxes, photoautotrophy was the dominant contributor to biomass formation under these conditions, accounting for approximately 73% of carbon assimilation. Under photoheterotrophic conditions, near-zero net CO 2 and O 2 fluxes were observed, suggesting efficient intracellular coupling between respiratory metabolism and photosynthetic carbon fixation. Light availability emerged as a major determinant of biomass productivity in both photoautotrophic and mixotrophic cultures. Although differences in light attenuation between trophic modes affected the comparison of biomass productivities, the radiative-transfer analysis showed that these differences alone could not explain the approximately additive relationship observed between mixotrophic, photoautotrophic and photoheterotrophic productivities. Overall, this work provides a quantitative framework for analyzing mixotrophic metabolism from macroscopic fluxes and offers new insights into carbon and oxygen partitioning in microalgal cultures, with implications for wastewater treatment and the optimization of photobioprocesses.