Biotechnology and Bioengineering最新文献

{"title":"Platform‐Specific Immunoassay Development and Orthogonal Characterization for Improved Host Cell Protein Control in Vero Cell‐Derived Vaccines","authors":"Shruti Patel, Young Zhang, Holly Guan, Emily Groegler, Kuo Yin, Jessica Raffaele, Richard R. Rustandi, Alyssa Q. Stiving, Paul F. Gillespie, Yanjie Wang, Dennis Driscoll, Xuanwen Li, Fengqiang Wang","doi":"10.1002/bit.70401","DOIUrl":"https://doi.org/10.1002/bit.70401","url":null,"abstract":"Vero host cell proteins (HCPs) are critical process‐related impurities in Vero cell‐derived vaccines and require robust analytical control to support product quality, safety, and manufacturing consistency. During development of the V590 vaccine candidate, discontinuation of the original commercial Vero HCP ELISA prompted evaluation of alternative approaches for HCP monitoring and risk control. We compared first‐ and second‐generation commercial ELISA kits, assessed the impact of process‐representative HCP standards and anti‐HCP antibodies on HCP quantitation, and characterized antibody coverage by two‐dimensional differential‐in‐blot electrophoresis. Measured HCP levels in process intermediates and bulk drug substances were strongly influenced by sample composition, HCP standard, antibody reagents, and assay format. ELISA and Simple Western yielded different quantitative results even with the same standard and antibodies, underscoring the assay‐dependent nature of HCP measurement. Platform‐specific anti‐Vero HCP antibodies provided broader coverage of process‐representative HCPs than the second‐generation commercial ELISA antibodies. Orthogonal characterization by Simple Western and Liquid chromatography‐mass spectrometry (LC‐MS/MS) further enabled monitoring of fibronectin, a high‐risk Vero HCP that causes filter fouling and compromises downstream process performance. Together, these findings demonstrate that platform‐specific immunoassays combined with orthogonal characterization of process‐relevant high‐risk HCPs provide a robust analytical framework for Vero HCP control and long‐term assay lifecycle management in Vero cell‐derived vaccine manufacturing.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"326 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149087403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Next‐Generation Glutamine Synthetase Expression Vector for Therapeutic Protein Manufacturing Using CHO Cells","authors":"Yusuf B. Johari, Sophie J. Wensley, Malak A. Ammar, Avinash Srivastava, Changhavi Ragurajan, Jenish Patel, Natalie C. Elliott, Alice C. Hodgson‐Casson, Mariano Olivera Fedi, Michael Anbar, Peter M. O'Callaghan, Bernie Sweeney","doi":"10.1002/bit.70405","DOIUrl":"https://doi.org/10.1002/bit.70405","url":null,"abstract":"The glutamine synthetase expression system is widely used for therapeutic protein biomanufacturing in Chinese hamster ovary (CHO) cells. Here, we present the design of an enhanced glutamine synthetase vector for generating CHOK1SV GS‐KO stable pools that produce recombinant proteins at multigram‐per‐liter levels. We first developed a synthetic promoter, LHP‐1, for high‐level constitutive recombinant gene transcription, achieving up to 123% increase in titer across monoclonal and bispecific antibody formats compared to the viral CMV promoter. We then demonstrate, for the first time, that the bacterial origin of replication (Ori) can function as a core promoter to drive glutamine synthetase selection in CHO cells. In this novel glutamine synthetase vector, Ori and LHP‐1 each play dual roles: Ori acts as both the plasmid replication origin <jats:italic>and</jats:italic> a minimal glutamine synthetase promoter, while LHP‐1 drives product expression <jats:italic>and</jats:italic> optimal enhancement of glutamine synthetase activity without the use of methionine sulfoximine (MSX) supplementation. Together, these two elements further increased product titers by up to 73% compared to the vector harboring the SV40 promoter to drive glutamine synthetase gene expression with 50 µM MSX. Notably, the LHP‐1 Ori‐GS system effectively maintained high bulk pool titers of up to 5.7 g/L with no decline after 30 days—supporting consistent product yield and quality during culture scale‐up for toxicology material supply. Further cell line development showed that clones derived from the LHP‐1 Ori‐GS pool achieved a 23% higher median titer while maintaining long‐term expression stability compared to those generated with the SV40‐GS vector, with the top‐performing Ori‐GS clone reaching 8.3 g/L. Overall, the MSX‐free LHP‐1 Ori‐GS system offers a robust platform for both rapid, high‐yield material generation from bulk pools and efficient progression to high‐producing clonal cell lines.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"23 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149087401","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Microneedle Based Platforms for Glucose Monitoring: Current Status and Future Directions","authors":"V. Sujan, Jobin Jose, Prachi Dwivedi, Naidulekha Aswani, Eyman M. Eltayib","doi":"10.1002/bit.70379","DOIUrl":"https://doi.org/10.1002/bit.70379","url":null,"abstract":"Diabetes mellitus is a metabolic disease that requires continuous glucose monitoring to achieve glycemic control. However, current glucose monitoring technologies have many limitations and are often associated with adherence issues. Therefore, in this review, we considered some recent developments in biosensors using microneedles due to their non‐invasive character and interstitial fluid measurement for real‐time monitoring. The review highlights the use of nanomaterials in nanotechnology, the microfabrication methods, enzyme immobilization methods, and surface modification methods that lead to increased sensitivity and biocompatibility of the sensors. The review also emphasized the advances in clinical studies and the artifacts related to microneedle sensors, such as biofouling and signal drift. Moreover, the review specifically targeted the use of emerging technologies such as non‐enzymatic sensing, multiplex biomarker detection, AI‐based analysis, health monitoring, and a closed‐loop insulin release system. The current review includes more than 100 peer‐reviewed articles. It provides a thorough analysis of how microneedle‐based biosensors can be considered a new approach for glucose detection that improves the quality of patients’ lives. Collectively, all the recent advancements in microneedle‐based biosensors have made them a new emerging platform for next‐generation personalized medicine, along with a digital healthcare system.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"13 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149087370","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering the α‐Helix Dynamics of Hydroxysteroid Dehydrogenase by Computational Design to Balance Catalytic Activity and Stability","authors":"Shu‐Fang Li, Ting Zou, Sen‐Yu Fu, Jin‐Chuan Li, Yuan Li, Ya‐Ru Shen, Ya‐Ping Xue, Yu‐Guo Zheng","doi":"10.1002/bit.70399","DOIUrl":"https://doi.org/10.1002/bit.70399","url":null,"abstract":"The stability‐activity trade‐off remains a major bottleneck in engineering robust biocatalysts for industrial applications. Here, we developed an integrated computational strategy to simultaneously enhance the catalytic activity and thermostability of 7α‐hydroxysteroid dehydrogenase (7α‐HSDH) by engineering highly flexible regions, with a particular focus on α‐helices. Evolutionary conservation analysis combined with multi‐temperature molecular dynamics (MD) simulations was first applied to identify weakly conserved and flexible α‐helices. Subsequently, computational tools (PROSS and Pythia) were utilized to generate mutations within these flexible regions. Through modular assembly of synergistic mutations, the optimal mutant M9 (D12K/V15I/V50E/I56R/D66A/T76R/A126I/A132H/S158A/S161A/A203P/H208Q) was obtained. Compared with the WT, M9 exhibited a 10.5°C increase in melting temperature ( <jats:italic>T</jats:italic> <jats:sub> <jats:italic>m</jats:italic> </jats:sub> ), a 2.3‐fold prolongation of half‐life ( <jats:italic>t</jats:italic> <jats:sub>1/2</jats:sub> ) at 40°C (reaching 75.3 h), a 3.14‐fold improvement in catalytic efficiency ( <jats:italic>k</jats:italic> <jats:sub>cat</jats:sub> <jats:italic>/K</jats:italic> <jats:sub> <jats:italic>m</jats:italic> </jats:sub> ) to 29,032.73 mM <jats:sup>−1</jats:sup> s <jats:sup>−1</jats:sup> , and enhanced substrate tolerance. Structural and molecular dynamics analyses indicated that the enhanced performance of M9 was associated with increased rigidity of key α‐helix regions, redistribution of surface electrostatic potential, and remodeling of the substrate‐binding pocket. This study thus offers a novel design framework for improving the catalytic activity, thermostability, and substrate tolerance, balancing the stability‐activity trade‐off.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"64 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149087402","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation and Characterization of Random Citrate Synthase Variants That Increase Acetate Generation by Escherichia coli","authors":"Jeffrey K. Dodelin, Ronnie L. Fulton, Abigail E. Rose, Mark A. Eiteman","doi":"10.1002/bit.70403","DOIUrl":"https://doi.org/10.1002/bit.70403","url":null,"abstract":"A method to select <jats:italic>Escherichia coli</jats:italic> citrate synthase variants having reduced activity was developed based initially on the increased generation of acid, observed visually by prolonged color change in the pH indicator bromocresol purple. Five hundred fifty‐two potential variants were isolated and each were cultured on glucose as sole carbon source. Of this library, 41 variants showed increased duration of the color change, indicating elevated acid formation. These strains were then examined in single shake flasks, and 16 demonstrated at least 2.5× greater acetate production than the wild‐type citrate synthase parent strain. These 16 strains were selected for sequencing and quantitative studies. In shake flask studies, an inverse correlation was observed between maximum specific growth rate and acetate yield, with lowered growth rate being an indication of mutation severity. The wild‐type citrate synthase and two variant enzymes were purified and their kinetic parameters determined. These three strains were also compared under carbon‐limited, steady‐state conditions at a growth rate of 0.20 h <jats:sup>−1</jats:sup> , where acetate yield and specific glucose uptake were correlated with severity of mutation. The results are explained mathematically in terms of an enzyme partitioning ratio using the relative kinetics of the competing enzymes (modified citrate synthase and phosphotransacetylase). The results demonstrate how partitioning of glycolytic flux between the tricarboxylic acid and other competitive pathways to products derived from acetyl‐CoA can be modulated by decreasing citrate synthase activity, a method which complements other methods including those using promoter strength and expression level.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"25 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149086862","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"GlycoMAC: A Multiscale Metabolic‐Glycosylation Framework for Predicting Glycosylation Across Conditions in Mammalian Cell Cultures","authors":"Yuming Zeng, Sarah W. Harcum, Jinxiang Pei, Wei Xie","doi":"10.1002/bit.70393","DOIUrl":"https://doi.org/10.1002/bit.70393","url":null,"abstract":"Antibody productivity and glycosylation quality in CHO cell cultures emerge from a dynamically evolving metabolic environment, yet existing models often work in isolation or at a single scale. Here, we present a multiscale mechanistic framework linking molecular, cellular, and macroscopic scales to predict how inputs shape bioprocess trajectories. The framework combines a single‐cell kinetic model of metabolism and glycosylation with a stochastic population model that captures environment‐dependent transitions among growth, production, and decline states. To characterize metabolic adaptation, we introduce the cumulative variation in oxygen uptake rate, a trajectory‐based biomarker that quantifies the total metabolic adjustment experienced during culture. Unlike population‐averaged approaches, the model propagates cell‐resolved metabolic states—including ammonia‐regulated Golgi pH, nucleotide sugar availability, manganese cofactors, and synthesis rates—into glycan processing. The framework was evaluated using CHO‐K1 fed‐batch cultures producing VRC01 IgG1 under targeted ammonia stress, matched control conditions, and a pyramid‐feeding strategy with tighter control. It accurately reproduced trajectories of cell growth, metabolites, productivity, and harvest glycosylation, including increased G0F abundance and reduced galactosylation under ammonia stress. By mechanistically linking process conditions to cell‐state dynamics and glycosylation outcomes, the framework provides a unified foundation for digital bioprocessing, predictive biomanufacturing, and advanced process control.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"23 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149086861","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Growth Model for Continuous Culture of a Hydrogen-Oxidizing Bacterium, Hydrogenophilus thermoluteolus Strain TH-1.","authors":"Tokihiro Kurita, Naoya Ohmura, Shin-Ichi Hirano, Thomas R Glass","doi":"10.1002/bit.70292","DOIUrl":"10.1002/bit.70292","url":null,"abstract":"<p><p>Hydrogenophilus thermoluteolus strain TH-1 is a thermophilic hydrogen-oxidizing bacterium that can autotrophically grow using H₂, CO_2, and O₂ with the highest rate among autotrophs. However, despite its significant potential for use in biomanufacturing from CO₂, no model to date has accurately described strain TH-1 growth under both H₂- and O₂-limiting conditions. Here, we report on a growth model for continuous culture. Theoretical equations were established for cell yields from the substrate consumption, overall mass transfer coefficients of H₂ and O₂, cell densities, and dilution rates. These equations were globally fitted to a series of cell culture profiles. Global fitting showed that the model closely reproduced cell production in continuous culture under H₂-limiting and O₂-limiting conditions. This model will be useful for understanding culture profiles and optimizing production.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":" ","pages":"2747-2757"},"PeriodicalIF":3.9,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13576768/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148359427","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mechanistic Digital Model for Real-Time Monitoring and Optimization of Adenovirus Production.","authors":"Xingge Xu, Daniel Rüdiger, Jeongbin Shin, Shawninder Chahal, Amine Kamen","doi":"10.1002/bit.70395","DOIUrl":"https://doi.org/10.1002/bit.70395","url":null,"abstract":"<p><p>Adenoviral vectors are a critical vaccine platform, with billions of vaccine doses administered during the COVID-19 pandemic. In addition, they are promising therapeutics for cancer treatment. To advance their manufacturing, real-time monitoring of adenovirus production is essential for process optimization, yield improvement, and product quality assurance. However, despite modern bioreactors being equipped with advanced sensors, comprehensive monitoring of viral production in real-time remains limited. In this work, we investigated potential indicators of the cell density effect (CDE), which imposes a trade-off between high cell density and specific productivity, thereby hindering process optimization. Glucose availability (expressed as glucose per cell) at time of infection was identified as the most effective proxy indicator. We developed an age-structured mechanistic model linking measurable process variables, such as glucose and viable cell density to viral production while capturing the CDE. This framework enables the prediction of viral particle titers in both batch and fed-batch operation modes, achieving an RMSE of 0.39 in log₁₀ IVP/mL for infectious viral particles and 0.20 in log₁₀ VP/mL for total viral particles, corresponding to approximately 2.6 and 2.0 times the measurement standard deviation of the respective assays. The proposed model and calibration method lay the foundation for a digital twin of adenovirus production, bridging real-time sensing with model-based simulation to support adaptive control and accelerate the transition toward Biopharma 4.0.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2026-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149079745","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Immobilization of Alginate Microbeads With Whole-Cells Expressing Nitrilase for Continuous Nicotinic Acid Production in Chip Millireactor.","authors":"Jonathan Bassut, Emina Mehić, Luana Cuvillier, Carine Vergne-Vaxelaire, Jean-Louis Petit, Maïté Michaud, Zoé Anxionnaz-Minvielle, Anne Zaparucha, Guillaume Nonglaton","doi":"10.1002/bit.70391","DOIUrl":"https://doi.org/10.1002/bit.70391","url":null,"abstract":"<p><p>The biocatalytic production of nicotinic acid using nitrilases has emerged as a promising alternative to conventional chemical synthesis, offering improved selectivity and environmental sustainability. In this study, we developed a continuous-flow millireactor system employing Escherichia coli cells expressing a nitrilase from Paraburkholderia phymatum (Nit_Phym) for the continuous production of nicotinic acid. To enhance catalytic efficiency and stability, whole cells were immobilized using two different strategies: covalent attachment to the reactor walls and encapsulation in alginate microbeads. The performance of these immobilization techniques was evaluated in millireactors with two different geometries: pillar and zigzag configurations. Encapsulation of Nit_Phym-expressing E. coli cells in alginate microbeads was achieved through a centrifuge-assisted microencapsulation method, yielding beads with diameters of up to 350 µm. Continuous-flow reactions demonstrated that the microencapsulated cells maintained their activity over at least 7 days, achieving stable conversion rates of approximately 30% in both pillar and zigzag millireactors. Compared to direct covalent immobilization, microencapsulation significantly reduced cell leaching and contamination of the reaction product. These findings highlight the potential of whole-cell microencapsulation for enhancing the stability and efficiency of biocatalytic processes in continuous-flow millireactors.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2026-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149079810","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Loop-Centric Engineering of Terminal Deoxynucleotidyl Transferase for Enhanced Catalysis of 3'-Modified Nucleotides.","authors":"Kun Shi, An-Na Li, Jian-He Xu, Hui-Lei Yu","doi":"10.1002/bit.70397","DOIUrl":"https://doi.org/10.1002/bit.70397","url":null,"abstract":"<p><p>Efficient enzymatic DNA synthesis requires terminal deoxynucleotidyl transferases (TdTs) that can accept reversibly blocked nucleotides, but improving wild-type enzymes has traditionally depended on labor-intensive screening. Here, we combine deep learning-based structure prediction with molecular dynamics simulations to guide loop-centric engineering of a TdT from Crocodylus porosus. The resulting variant M3 (V253E/L256M/N338R) showed a 26-fold increase in specific activity toward 3'-ONH₂-dCTP while maintaining expression and thermostability. Simulations indicated that the mutations reorganized Loop1 dynamics and reproducibly biased the incoming nucleotide toward a more favorable in-line attack orientation. The key N338R change also improved a second TdT scaffold, supporting N338 as a potentially transferable engineering hotspot. These results provide a practical computational strategy for improving terminal deoxynucleotidyl transferases for incorporation of 3'-modified nucleotides, reducing reliance on large-scale screening.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2026-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149079740","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}