{"title":"Developing a Model for Virus Contamination in Perfusion Bioreactors to Establish Rational Control Strategies for Integrated Continuous Bioprocessing.","authors":"Takao Ito,Takashi Nihei,Koichi Yamamoto,Naoto Watanabe,Yoshiyuki Tokieda,Yumiko Masuda,Masaki Okada","doi":"10.1002/bit.29021","DOIUrl":"https://doi.org/10.1002/bit.29021","url":null,"abstract":"A viral contamination model for steady-state perfusion cell culture was developed to assess how sampling frequency and volume impacted the expected downstream viral clearance factor in integrated continuous biomanufacturing processes. The model used population balance rate equations for cells and free virions, incorporating the virus infection cycle. It simulated the states of cell cultures for both endogenous viruses, which are potentially present within the cells and can be released from them, and adventitious viruses after contamination. The model also reproduced differences in virus concentration between bioreactors and harvests caused by sieving through the cell retention device. For virus risk assessments in integrated continuous biomanufacturing, the model evaluated the probability of detecting contamination based on the volume fraction of the sample tested and the downstream viral transmission by number of days after contamination. Considering the infection scenario with mouse minute virus (MVM) in 100 × 106 cells/mL cell culture for adventitious virus contamination, a notable decline in viable cell density was observed starting from Day 4. To ensure 99.999% safety of final products, the total downstream clearance achieving a log reduction value (LRV) > 15 LRV is required to remove the increased virus. If daily sampling is conducted and a downstream clearance is planned to satisfy the removal of potentially undetected MVM, a total clearance of 9 LRV is sufficient. This model enables us to simulate different scenarios for viral contamination and has the advantage of allowing assessments of virus safety strategies.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"145 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143945547","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":"Biotechnology and Bioengineering: Volume 122, Number 6, June 2025","authors":"","doi":"10.1002/bit.28746","DOIUrl":"10.1002/bit.28746","url":null,"abstract":"","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 6","pages":"1307-1311"},"PeriodicalIF":3.5,"publicationDate":"2025-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28746","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143933112","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":"Protein Engineering for Enhancing Electron Transfer in P450‐Mediated Catalysis","authors":"Yuemin Li, Hongwei Yu, Lidan Ye","doi":"10.1002/bit.29023","DOIUrl":"https://doi.org/10.1002/bit.29023","url":null,"abstract":"Cytochrome P450 enzymes (P450s) are versatile biocatalysts with applications spanning pharmaceutical development and natural product biosynthesis. A critical bottleneck in P450‐mediated reactions is the electron transfer process, which often limits catalytic efficiency and promotes uncoupling events leading to reactive oxygen species (ROS) formation. This review comprehensively examines recent protein engineering strategies aimed at enhancing electron transfer efficiency in P450 systems. We explore the design and application of different fusion constructs, which improve proximity between the P450 enzyme and its redox partners (RPs), as well as scaffold‐mediated protein assembly, enabling precise spatial organization of P450s and RPs. Furthermore, we discuss targeted modifications at the P450‐RP interaction interface and optimization of electron transfer pathways through site‐directed mutagenesis and directed evolution. These strategies enhance catalytic activity, improve coupling efficiency, and reduce ROS formation. Finally, we address the remaining challenges in understanding and engineering P450 electron transfer, and discuss the future directions, emphasizing the need for advanced computational modeling, structural characterization, and integration of synthetic and systems biology approaches.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"141 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143926905","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":"Hybrid Modeling of the Reversed‐Phase Chromatographic Purification of an Oligonucleotide: Few‐Shot Learning From Differentiable Physics Solver‐in‐the‐Loop","authors":"Yu‐Cheng Chen, Ismaele Fioretti, Dong‐Qiang Lin, Mattia Sponchioni","doi":"10.1002/bit.29018","DOIUrl":"https://doi.org/10.1002/bit.29018","url":null,"abstract":"Hybrid models integrate mechanistic and data‐driven components, effectively addressing the challenges of limited process understanding and data availability typical of biopharmaceutical processes. In this study, we applied a hybrid modeling framework named differentiable physics solver‐in‐the‐loop (DP‐SOL) to describe the reversed‐phase chromatographic purification of an oligonucleotide, overcoming the mentioned limitations of purely mechanistic and data‐driven models. The framework establishes a connection between neural networks (NNs) and mechanistic models through differentiable physical operators and their gradients. We first collected a data set comprising six linear gradient elution experiments at different resin loadings and gradient slopes, split in three experiments each for training and testing, for few‐shot learning. The hyperparameters were determined through a grid search, resulting in a NN with two hidden layers and 14 nodes. Compared to a calibrated mechanistic model used for initialization of NN, the DP‐SOL hybrid model showed significant performance improvement on both training and testing sets, with 0.97 for the former. The good predictivity of DP‐SOL is attributed to the combination of mechanistic models and NNs at the solver level. As a novel and versatile hybrid modeling paradigm, DP‐SOL has the potential to significantly impact modeling approaches in the downstream processing field and the broader biopharmaceutical sector.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"44 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143930736","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}
Merve Yarıcı, Furkan Cantürk, Serdar Dursun, Hatice Nur Aydın, Muhammed Erkan Karabekmez
{"title":"RSEA: A Web Server for Pathway Enrichment Analysis of Metabolic Reaction Sets","authors":"Merve Yarıcı, Furkan Cantürk, Serdar Dursun, Hatice Nur Aydın, Muhammed Erkan Karabekmez","doi":"10.1002/bit.29020","DOIUrl":"https://doi.org/10.1002/bit.29020","url":null,"abstract":"Changes in biological pathways provide essential clues about metabolism. Genome-scale metabolic models (GEM) are network-based templates that computationally describe all stoichiometric associations and gene-protein reaction (GPR) relations found in an organism for all its metabolic genes and metabolites. Using reaction stoichiometry as input, GEMs mathematically simulate metabolic reaction fluxes occurring in an organism and predict changes in the metabolic system under the relevant condition. Multiple tools and approaches in the literature can capture fluxes sensitive to a given condition by using GEMs. However, functional enrichment analysis of these reaction lists in a systems biology perspective is not straightforward. Here, we introduce RSEA to annotate given reaction sets to significantly related metabolic pathways: Reaction Set Enrichment Analysis web server tool. RSEA converts given reaction list derived from GEMs into proper reaction identifiers and statistically analyze its enrichment in metabolic pathways. RSEA is designed to provide researchers with a practical and user-friendly platform to explore and interpret sets of reactions in biological pathways and freely available online (https://rseatool.com/).","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"39 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143926912","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}
Jia-Cong Huang, Jun-Lin Wu, Zhi-Kui Nie, Tian-Qiong Shi
{"title":"A Genetic Algorithms-Based Neural Network Model to Monitor Gibberellic Acid GA3 Fermentation Process by Fusarium fujikuroi","authors":"Jia-Cong Huang, Jun-Lin Wu, Zhi-Kui Nie, Tian-Qiong Shi","doi":"10.1002/bit.29022","DOIUrl":"https://doi.org/10.1002/bit.29022","url":null,"abstract":"A genetic algorithm-optimized neural network (ANN-GA) was developed for real-time monitoring of gibberellin (GA3) production during <i>Fusarium fujikuroi</i> fermentation. This model addresses the limitations of traditional off-line detection methods, such as contamination risks and delayed feedback, by integrating six critical inputs—initial glucose concentration, fermentation time, temperature, pH, dissolved oxygen, and rotational speed—to predict glucose consumption and GA3 synthesis with an accuracy of 99.41%. During the implementation phase, by dynamically controlling the temperature (28°C–32°C) and pH, the biomass accumulation rate increased by 84% within 48 h, while the GA3 accumulation rate improved by 66.7% compared to constant-temperature fermentation at 28°C. The ANN-GA framework enables dynamic adjustment of glucose supply based on real-time predictions, thereby optimizing carbon source utilization and enhancing process stability. This data-driven approach effectively overcomes the drawbacks of costly sensors and labor-intensive manual sampling, showcasing significant potential for industrial-scale fermentation optimization. With its high accuracy and adaptability, the model holds substantial application value in advancing intelligent biological process control for secondary metabolite production.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"48 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143926436","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}
Tomáš Podzimek, Terezie Cisarová, Michal Dvořák, Barbora Vokatá, Christina Karmann, Jaroslav Hanuš, Martin Balouch, Matěj Malý, Jana Hajšlová, Vojtěch Kouba, Jan Bartáček, František Štěpánek, Petra Lipovová
{"title":"Isolation of Anammoxosomes From the Aggregate Culture of Ca. Brocadia Sapporoensis and Assembly of Ladderane Liposomes","authors":"Tomáš Podzimek, Terezie Cisarová, Michal Dvořák, Barbora Vokatá, Christina Karmann, Jaroslav Hanuš, Martin Balouch, Matěj Malý, Jana Hajšlová, Vojtěch Kouba, Jan Bartáček, František Štěpánek, Petra Lipovová","doi":"10.1002/bit.29011","DOIUrl":"https://doi.org/10.1002/bit.29011","url":null,"abstract":"Anammox bacteria wield an energy‐efficient nitrogen metabolism enveloped in anammoxosome organelle composed of unique ladderane lipids. Thus, waste anammox biomass seems to be an attractive target for the isolation of ladderanes and subsequent production of artificial vesicles for drug delivery. This study proposed a novel method to isolate ladderane‐rich anammoxosomes from aggregate mixed culture of <jats:italic>Ca</jats:italic>. Brocadia sapporoensis. Compared to conventional isolation protocols, the protocol was simplified by omitting the prepurification of anammox cells, replacing Percoll® with a sucrose gradient and prolonging the application of EDTA. This enhanced and simplified procedure efficiently removed EPS and other debris, thus yielding the layer of anammoxosomes as confirmed by control experiments and TEM. For the first time, the resulting ladderane isolates were used for the preparation of liposomes, both with and without the addition of pure dipalmitoylphosphatidylcholine (DPPC). Vesicles were successfully created, characterised by TEM and DLS, and anammox‐based ladderanes were incorporated into their walls. These liposomes had interesting functional properties such as increased colloid stability at elevated concentrations, meaning a reduced tendency to form aggregates compared to model liposomes made solely of DPPC. Overall, this study offers insights into converting waste anammox biomass into a valuable resource for drug delivery.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"35 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143920046","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}
Eun Jeong Sim, Quynh‐Giao Tran, Yu Rim Lee, Trang Thi Le, Hyang Ran Yoon, Dong‐Yun Choi, Dae‐Hyun Cho, Jin‐Ho Yun, Hong Il Choi, Hee‐Sik Kim, Yong Jae Lee
{"title":"Cell‐Penetrating Peptide‐Based Triple Nanocomplex Enables Efficient Nuclear Gene Delivery in Chlamydomonas reinhardtii","authors":"Eun Jeong Sim, Quynh‐Giao Tran, Yu Rim Lee, Trang Thi Le, Hyang Ran Yoon, Dong‐Yun Choi, Dae‐Hyun Cho, Jin‐Ho Yun, Hong Il Choi, Hee‐Sik Kim, Yong Jae Lee","doi":"10.1002/bit.29019","DOIUrl":"https://doi.org/10.1002/bit.29019","url":null,"abstract":"Microalgae are a promising solution for mitigating climate change due to their ability to capture greenhouse gases and produce renewable materials. However, their effective application is often hindered by barriers that necessitate advances in genetic engineering to improve photosynthesis and productivity. One major obstacle is the microalgal cell wall, which complicates the delivery of genetic material into these organisms. To address these challenges, we developed a novel triple nanocomplex system integrating cell‐penetrating peptides (CPPs), nuclear localization signal (NLS) peptides, and plasmid DNA. This system allows simple preparation while achieving efficient nuclear translocation of plasmid DNA. We evaluated two CPPs, pVEC‐ORI and pVEC‐R6A, for their efficacy in facilitating intracellular transfer of DNA into wild‐type <jats:italic>Chlamydomonas reinhardtii</jats:italic> cells. Notably, pVEC‐R6A demonstrated a 6.88‐fold increase in efficiency compared to pVEC‐ORI, despite the presence of thick cell walls. The optimal CPP:DNA ratio for stable nanocomplex formation was determined to be 5:1 for pVEC‐ORI and 10:1 for pVEC‐R6A. By incorporating the simian virus 40 (SV40) NLS into CPP/DNA nanocomplexes, we successfully directed the localization of plasmid DNA into the nucleus. Our findings indicate that this simple and efficient DNA delivery system has significant potential as a tool to advance microalgal synthetic biology.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"31 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143926906","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":"A Comprehensive Model for Separating Systematic Bias and Noise in Metabolomic Timecourse Data—A Nonlinear B‐Spline Mixed‐Effects Approach","authors":"Kathy Sharon Isaac, Stanislav Sokolenko","doi":"10.1002/bit.29008","DOIUrl":"https://doi.org/10.1002/bit.29008","url":null,"abstract":"The simultaneous detection of tens to hundreds of metabolites in a single metabolomic timecourse sample offers a unique but often unrealized opportunity for quantification validation. An individual timecourse fit for each metabolite fundamentally convolutes measurement noise with systematic sample bias (stemming from, for example, variable sample dilution, extraction, and normalization). However, since systematic bias, by its definition, influences all metabolites within a sample in a similar fashion, it can be identified and corrected through the simultaneous fit of all detected metabolites in a single timecourse model. This study presents a nonlinear B‐spline mixed‐effects model as a convenient formulation capable of estimating and correcting such bias. The proposed model was successfully applied to real cell culture data and validated using simulated timecourse data perturbed with varying degrees of random noise and systematic bias. The model was able to accurately correct systematic bias of 3%–10% to within 0.5% on average for typical data. An R package for the correction model has been developed to facilitate model adoption and use. The proposed nonlinear B‐spline mixed‐effects formulation is general enough for application to a broad range of research areas beyond just cell culture metabolomics.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"3 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143920233","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}
Yanan Du, Majid Ghodousi, Hao Qi, Nikhil Haas, Wenqian Xiao, Ali Khademhosseini
{"title":"Correction to “Sequential Assembly of Cell-Laden Hydrogel Constructs to Engineer Vascular-Like Microchannels”","authors":"Yanan Du, Majid Ghodousi, Hao Qi, Nikhil Haas, Wenqian Xiao, Ali Khademhosseini","doi":"10.1002/bit.28953","DOIUrl":"https://doi.org/10.1002/bit.28953","url":null,"abstract":"<p>In the published article, the images in Figure 4B,D were found to be from the same sample that was sequentially exposed to different conditions and analyzed. The authors have data from an independent experiment in which the gels were not sequentially analyzed (Figure 4 below). The updated figure does not alter the interpretation of the data or the conclusions of the study. We apologize for any inconvenience.</p>\u0000<figure><picture>\u0000<source media=\"(min-width: 1650px)\" srcset=\"/cms/asset/25b14313-1fa2-4a8c-849a-b81db280adb8/bit28953-fig-0001-m.jpg\"/><img alt=\"Details are in the caption following the image\" data-lg-src=\"/cms/asset/25b14313-1fa2-4a8c-849a-b81db280adb8/bit28953-fig-0001-m.jpg\" loading=\"lazy\" src=\"/cms/asset/ebdbe2d9-5e5a-4e08-bb2a-ae050edd4204/bit28953-fig-0001-m.png\" title=\"Details are in the caption following the image\"/></picture><figcaption>\u0000<div><strong>Figure 4<span style=\"font-weight:normal\"></span></strong><div>Open in figure viewer<i aria-hidden=\"true\"></i><span>PowerPoint</span></div>\u0000</div>\u0000<div>Viability tests for the cell-laden tubular hydrogels as a function of the steps in the assembly process. The sequential assembly procedure was shown to be cell-friendly as demonstrated by viability tests for: (A) 3T3 fibroblast-laden microgels. (B) Microgels washed three times with photoinitiator (PI) (1% in DPBS). (C) Microgels exposed to oil. (D) Microgels exposed to both PI and oil. (E) Microgel assembly after 2 days in culture medium. (F) Quantification of samples (<i>n</i> = 3) after each fabrication step showed that the assembly process did not result in a significant loss of cell viability. Scale bar: 500 µm.</div>\u0000</figcaption>\u0000</figure>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"57 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143910854","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}