Biotechnology Journal最新文献

{"title":"Prediction of pH Gradient Elution of Ion Exchange Chromatography for Antibody Charge Variants Separation Based on Salt Gradient Elution Experiments","authors":"Yu-Xin Liao,&nbsp;Ce Shi,&nbsp;Xue-Zhao Zhong,&nbsp;Xu-Jun Chen,&nbsp;Ran Chen,&nbsp;Shan-Jing Yao,&nbsp;Dong-Qiang Lin","doi":"10.1002/biot.70029","DOIUrl":"https://doi.org/10.1002/biot.70029","url":null,"abstract":"<div>\u0000 \u0000 <p>Mechanistic modeling of ion exchange chromatography (IEC) is a promising technique to improve process development. However, when considering the pH influence, model prediction becomes challenging due to the multiple pH-dependent parameters and complex interactions. In order to more effectively predict the pH gradient elution behavior, a two-step model calibration strategy was proposed for the pH-dependent steric mass action (SMA) model with the empirical correlations of characteristic charge <i>ν</i> and equilibrium coefficient <i>k</i>_eq. The strategy was verified through a case study of monoclonal antibody charge variants purification with IEC. All nine calibration experiments were conducted using linear salt gradient elution at three fixed pH values. The average root mean square error (RMSE) was 14.28% between the model calculation and experiments. Both <i>ν</i> and ln(<i>k</i>_eq) exhibited good linear correlations with pH (<i>R</i>² &gt; 0.99). Then, the well-calibrated pH-dependent SMA model showed a satisfactory capability for predicting the pH gradient elution behaviors with an RMSE of 16.18%. Moreover, the model was used for process optimization under different elution modes, including salt gradient, pH gradient, and salt-pH dual gradient, improving the yield from 70.07% to 74.91%. The optimized linear pH gradient elution was verified by experiment (RMSE = 8.30%). Finally, a methodological framework for utilizing the simplified pH-dependent SMA model developed in this work was summarized to explore its practical applications. The two-step calibration strategy proposed significantly alleviates the workload for the pH-dependent IEC modeling. The model-based process optimization effectively enables faster pH-dependent process development with minimal experiments.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 5","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144074578","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Utilizing Stable Gene-Edited Knockout Pools for Genetic Screening and Engineering in Chinese Hamster Ovary Cells","authors":"Jannis Peter Marzluf,&nbsp;Kirchmeier Daniela,&nbsp;Jennifer Klein,&nbsp;Christoph Zehe,&nbsp;Ann-Cathrin Leroux","doi":"10.1002/biot.70033","DOIUrl":"https://doi.org/10.1002/biot.70033","url":null,"abstract":"<p>Chinese hamster ovary (CHO) cells are the primary host for biopharmaceutical production. To meet increasing demands for productivity, quality, and complex molecule expression, genetic engineering, particularly clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene knockout (KO), is widely used to optimize host cell performance. However, systematic screening of KO targets remains challenging due to the labor-intensive process of generating and evaluating individual clones. In this study, we present a robust, high-throughput CRISPR workflow using stable KO pools in CHO cells. These pools maintain genetic stability for over 6 weeks, including in multiplexed configurations targeting up to seven genes simultaneously. Compared to clonal approaches, KO pools reduce variability caused by clonal heterogeneity and better reflect the host cell population phenotype. We demonstrate the utility of this approach by reproducing the beneficial phenotypic effects of fibronectin 1 (FN1) KO, specifically prolonged culture duration and improved late-stage viability in fed-batch processes. This workflow enables efficient identification and evaluation of promising KO targets without the need to generate and test large numbers of clones. Overall, screening throughput is increased 2.5-fold and timelines are compressed from 9 to 5 weeks. This provides a scalable, efficient alternative to traditional clonal screening, accelerating discovery for CHO cell line engineering for biopharmaceutical development.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 5","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70033","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144074577","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of Physiological Integrity in Six-Gene-Edited Bama Miniature Pigs as a Model for Xenotransplantation","authors":"Wen Chuan Peng,&nbsp;Yuan Yuan Zhai,&nbsp;Meng Ke Li,&nbsp;Chu Xiong Zhang,&nbsp;Jia Xiang Du,&nbsp;Yan Yan Jiang,&nbsp;Deng Ke Pan,&nbsp;Jiang Wei Wu","doi":"10.1002/biot.70030","DOIUrl":"https://doi.org/10.1002/biot.70030","url":null,"abstract":"<div>\u0000 \u0000 <p>Genetically engineered pigs exhibit significant potential as a solution to organ scarcity in xenotransplantation. Nonetheless, a formidable challenge lies in overcoming the rejection of porcine organs by the human immune system. In this study, we generated a six-gene-edited pig model by simultaneously knocking out three major xenoantigens, GGTA1, CMAH, and β4GalNT2, along with the incorporation of coagulation regulatory factor THBD, and two complement regulatory proteins, hCD55 and hCD46, in Bama miniature pigs. These pigs exhibit genetic modifications designed to reduce xenograft rejection while maintaining normal physiology. Assessments of vital organ structure and function, including the heart, liver, spleen, lungs, and kidneys in the gene-edited pigs, showed no abnormalities. The pigs with human transgene knock-in THBD and complement genes exhibited no significant alterations in coagulation function and immune performance. These healthy features hold promise for further xenotransplantation. In conclusion, we successfully constructed six-gene-edited miniature pigs. This work serves as a valuable reference for gene editing strategies for xenotransplantation pig models.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 5","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143950096","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Numerical Simulation and Comparison of Flow Field in Different Dynamic Co-Culture Conditions","authors":"Liying Li,&nbsp;Xinyue Liu,&nbsp;Huamao Sun,&nbsp;Hailin Ma,&nbsp;Yuen Yee Cheng,&nbsp;Xiangqin Li,&nbsp;Zhilin Jia,&nbsp;Jiaquan Zhao,&nbsp;Kedong Song","doi":"10.1002/biot.70039","DOIUrl":"https://doi.org/10.1002/biot.70039","url":null,"abstract":"<div>\u0000 \u0000 <p>Bioreactor technology facilitates the gradual automation of cell expansion and the development of biofunctional synthetic alternatives. However, it is difficult to fully understand the flow field and force field environments formed in it by experimental means. Computational fluid dynamics (CFD) offers a robust framework for analyzing and understanding the impacts of fluid flow, material diffusion, and fluid shear stress (FSS) on in vitro cell and tissue regeneration dynamics. In this study, the FLUENT software is used to simulate and calculate the flow field environment of the rotary cell culture system (RCCS) and spinner flask (SF), including dynamic pressure, shear stress, and velocity distribution. Particles of two diameters for three-dimensional cell culture were randomly arranged in different radial/axial positions, and the FSS on the particles in RCCS and SF at different rotational speeds was also analyzed. It is expected to visualize the flow field distribution of the bioreactor and local hydrodynamic changes near the particles, and provide positive assistance for the dynamic culture/co-culture of different cells-microcarriers complex.\u0000\u0000 </p><ul>\u0000 \u0000 <li>The distribution of FSS on randomly arranged L and S particles was analyzed in detail to evaluate and screen the suitable operating conditions of these two bioreactors.</li>\u0000 \u0000 <li>Visually understanding the flow field distribution and local hydrodynamic changes within the bioreactor is expected to provide positive assistance for dynamic culture.</li>\u0000 \u0000 <li>The particles may periodically contact the fresh oxygenated medium during rotation with the fluid.</li>\u0000 \u0000 <li>Two fluid circulations in SF were generated in the upper/lower area of the blade, and a relatively static fluid circulation area was formed at the bottom with low velocity and pressure in the center, which was not conducive to material exchange.</li>\u0000 \u0000 <li>Rotary bioreactors may be more suitable than spinner flasks as a dynamic culture tool for some types of cells or other constructs.</li>\u0000 </ul>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 5","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143950211","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sustainable Pesticide Degradation Using Esterase and Coimmobilized Cells in Agriculture","authors":"Vinutsada Pongsupasa,&nbsp;Pratchaya Watthaisong,&nbsp;Nidar Treesukkasem,&nbsp;Apisit Naramittanakul,&nbsp;Charndanai Tirapanampai,&nbsp;Nopphon Weeranoppanant,&nbsp;Pimchai Chaiyen,&nbsp;Thanyaporn Wongnate","doi":"10.1002/biot.70034","DOIUrl":"https://doi.org/10.1002/biot.70034","url":null,"abstract":"<div>\u0000 \u0000 <p>This study presents an enzymatic approach to mitigate the environmental and health impacts of organophosphate pesticides (OPs) in agriculture. Using esterase enzymes from the <i>Sphingobium fuliginis</i> strain ATCC 27551 (Opd), we developed a bioremediation system capable of degrading OPs under both buffered and unbuffered conditions. Enzyme activity was evaluated across pH and temperature ranges, with optimal performance observed at pH 8.5–10 and sustained stability for over 28 days. A key innovation was the coimmobilization of <i>Escherichia coli</i> cells expressing Opd and flavin-dependent monooxygenase (HadA) in calcium alginate, enabling the transformation of toxic OPs into less harmful benzoquinones. The system demonstrated high degradation efficiency, achieving 100% degradation of ethyl parathion, along with substantial degradation of methyl parathion (98%), fenitrothion (91%), ethyl chlorpyrifos (83%), and profenofos (62%). Validation in flow cells and column-based setups confirmed the practical applicability of this approach for treating OP-contaminated soil and water. These findings highlight the potential of enzyme-based, cell-immobilized systems for sustainable pesticide remediation. This method offers a practical, eco-friendly solution for reducing pesticide residues in agricultural environments and supports the advancement of greener farming practices.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 5","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143950097","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Approaches for Cannabinoid Glycosylation Catalyzed by CrUGT74AN3 and BlCGTase","authors":"Christina Schmidt,&nbsp;Astrid Maria Imann,&nbsp;Nikolay Vasilev,&nbsp;Oliver Kayser","doi":"10.1002/biot.70007","DOIUrl":"https://doi.org/10.1002/biot.70007","url":null,"abstract":"<div>\u0000 \u0000 <p>Phytocannabinoids are natural products with highly promising pharmaceutical potential, mainly known from the plant <i>Cannabis sativa</i>. However, their bioavailability is limited due to their high lipophilicity. Modification through glycosylation is known to improve the water solubility and stability of molecules. Enzymatic glycosylation requires specific enzymes with high catalytic activity in combination with efficient production systems. To date, only a few glycosyltransferases with activity toward cannabinoids have been described. In this study, we explore the substrate spectrum of the promiscuous UDP-glycosyltransferase <i>Cr</i>UGT74AN3 from <i>Catharanthus roseus</i> and demonstrate activity towards a broad range of cannabinoids and their biosynthetic intermediates. The highest activity was observed using cannabidiol (CBD) as an acceptor molecule. In addition, we show efficient biotransformation of CBD in an engineered <i>Saccharomyces cerevisiae</i> strain. We investigate the influence of the hydrolytic activity of endogenous glucosidases and identify the UDP-glucose supply as a limiting factor in the yeast system. The co-expression of <i>Cr</i>UGT74AN3 and a cyclodextrin glycosyltransferase from <i>Bacillus licheniformis</i> in the engineered yeast strain led to the production of CBD-glycosides with up to six glucose moieties from CBD and cyclodextrin in vivo. Finally, we confirm the applicability of the engineered yeast systems to other cannabinoids using cannabigerol and cannabinol.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 5","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143944571","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Issue Information: Biotechnology Journal 5/2025","authors":"","doi":"10.1002/biot.70035","DOIUrl":"https://doi.org/10.1002/biot.70035","url":null,"abstract":"","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 5","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70035","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143930425","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development and Characterization of the NISTCHO Reference Cell Line","authors":"Hussain Dahodwala,&nbsp;Irfan Hodzic,&nbsp;Alexei Slesarev,&nbsp;Benjamin Cutak,&nbsp;Alexander Kuzin,&nbsp;Rahul Lal,&nbsp;Jiajian Liu,&nbsp;James Mahon,&nbsp;Rajagopalan Lakshmi Narasimhan,&nbsp;Jaya Onuska,&nbsp;James Ravellette,&nbsp;Kelsey Reger,&nbsp;Sadie Sakurada,&nbsp;Floy Stewart,&nbsp;Trissa Borgschulte,&nbsp;Colette Cote,&nbsp;Kelvin H. Lee,&nbsp;William B. O'Dell,&nbsp;Zvi Kelman,&nbsp;Britta A. Anderson","doi":"10.1002/biot.70012","DOIUrl":"https://doi.org/10.1002/biot.70012","url":null,"abstract":"<p>Well-characterized reference materials enable successful collaborations within the scientific community by establishing common reagents for benchmarking studies and reducing the barriers to sharing materials and information. Here, we report the development of NISTCHO, a recombinant Chinese hamster ovary cell line expressing a nonoriginator version of the NISTmAb IgG1. We evaluated candidate clonal cell lines in a fed-batch cell culture model to assess growth and productivity of the cell lines and protein quality attributes of the recombinant IgG produced, which demonstrated suitability of multiple candidates. Selection of a preferred candidate was accomplished through sequencing-based analysis of the transgene integration sites, and a base-pair resolution map of the transgene integration site was developed and verified using PCR-based methods. Lastly, a validation study performed by an independent laboratory confirmed the robustness of the preferred candidate, which has been selected for further development as the NISTCHO reference cell line. Together, these results describe the origin of this new reference material and will serve as the foundation for future interlaboratory studies using the NISTCHO cell line.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 5","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70012","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143930426","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Allosteric Effectors Outcompete Transcript Levels and Substrate Concentration in Regulating Central Carbon Flux During the Crabtree Effect Transition","authors":"Min Chen,&nbsp;Junfeng Jiang,&nbsp;Tingting Xie,&nbsp;Yingping Zhuang,&nbsp;Jianye Xia","doi":"10.1002/biot.70024","DOIUrl":"https://doi.org/10.1002/biot.70024","url":null,"abstract":"<div>\u0000 \u0000 <p>While the metabolic shift in <i>Saccharomyces cerevisiae</i> across the Crabtree effect is well-documented, the role of allosteric regulation in this transition remains unclear. Here, we investigated allosteric regulation by inducing a growth rate shift from 0.2 to 0.35 h. Our results revealed 35 regulatory interactions across 23 central carbon metabolism reactions, with allosteric effectors explaining 29% of flux changes—surpassing the contributions of transcript abundance and substrate concentration. Additionally, key Crabtree-responsive reactions' flux changes were co-regulated by allosteric effectors, transcript abundance, and enzyme turnover numbers. These underscore the significance of allosteric regulation in metabolic adaptation during growth rate transitions in <i>Saccharomyces cerevisiae</i>.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 4","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143879868","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimization of Isoamylase Production in Escherichia coli Under Different Nutrient Limitation Conditions","authors":"Dapeng Wang,&nbsp;Yuqing Bai,&nbsp;Min Chen,&nbsp;Jianye Xia","doi":"10.1002/biot.70009","DOIUrl":"https://doi.org/10.1002/biot.70009","url":null,"abstract":"<div>\u0000 \u0000 <p>Isoamylase (IA), a starch debranching enzyme that cleaves α-1,6-<span>d</span>-glycosidic bonds in amylose, is used in various industries. However, its low expression in <i>Escherichia coli</i> BL21 (DE3) limits its application, necessitating fermentation optimization. Limiting carbon (C) and other nutrients can optimize bacterial growth for better IA expression. Herein, using <i>E. coli</i> expressing IA-mCherry as the research object, we explored whether low concentrations of the nutrients nitrogen (N), phosphorus (P), magnesium (Mg), and sulfur (S) have a limiting effect on bacterial growth. We subsequently investigated differences in expression under restricted nutrient conditions and performed batch fermentation experiments under various conditions to determine optimal nutrient limitation conditions. Low concentrations of N and P had a limiting effect on bacterial growth. IA expression in the P-limited group was increased by ∼10% but limiting N had no influence. Combinations of C and P restriction during fed-batch fermentation showed that the total yield was 73.73% higher under C excess and P-limited conditions than under other combinations, while corresponding expression per unit biomass was 24.42% higher. The results revealed that P restriction plays a key role in increasing IA production in <i>E. coli</i>.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 4","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143879861","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}