Biotechnology Journal最新文献

{"title":"Rheology of CHO Cell Suspensions and Its Effects on High-Density Cultivation Process and Bioreactor Design","authors":"Botao Zhang,&nbsp;Xinran Zhang,&nbsp;Qingyuan Ran,&nbsp;Weijian Zhang,&nbsp;Gance Dai,&nbsp;Liang Zhao,&nbsp;Qian Ye,&nbsp;Wen-Song Tan","doi":"10.1002/biot.70003","DOIUrl":"https://doi.org/10.1002/biot.70003","url":null,"abstract":"<div>\u0000 \u0000 <p>With the rapidly growing demand for monoclonal antibodies (mAbs) worldwide, optimizing the high-density and ultra-high-density cultivation processes of Chinese hamster ovary (CHO) cells has become crucial for enhancing production efficiency. Shear stress and mass transfer have always been the vital operating parameters for the bioreactor in creating a suitable microenvironment for cell growth and antibody production. However, researchers have not actively focused on the rheology of CHO cell suspensions and its impact on these parameters in bioreactors. The factors influencing the rheology of suspensions were first investigated in this study. The findings demonstrated that the shear-thinning behavior of the suspension was primarily affected by the cell volume fraction (<i>Φ</i>). As <i>Φ</i> increases, the shear-thinning behavior gradually weakened, and the viscosity increased. The Sisko model was used to characterize rheology, while computational fluid dynamics simulations evaluated its impact on bioreactor performance. The simulation results revealed that the rheology of the suspensions caused a multiple increase in shear stress and a 10%–40% decrease in the volumetric mass transfer coefficient (kLa) in the bioreactor. Therefore, the effects of rheology cannot be ignored while designing operating parameters. This study established empirical correlations among Pg/V, Vg, <i>Φ</i>, and kLa, thus delivering guidance for selecting appropriate operating parameters in high-density and ultra-high-density cell cultivation processes. The findings provide a scientific foundation for optimizing CHO cell cultivation processes and quantifying suitable microenvironment parameters for cell growth and production. They also offer novel ideas and strategies for scaling up and optimizing the structural parameters of bioreactors.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143646172","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":"Unraveling Cytotoxicity in HEK293 Cells During Recombinant AAV Production for Gene Therapy Applications","authors":"Pranay Ladiwala,&nbsp;Nelson Ndahiro,&nbsp;Pricila Hauk,&nbsp;Junneng Wen,&nbsp;Justin Sargunas,&nbsp;Yu-Ju Chen,&nbsp;Erik Barton,&nbsp;Michael J. Betenbaugh","doi":"10.1002/biot.202400501","DOIUrl":"https://doi.org/10.1002/biot.202400501","url":null,"abstract":"<div>\u0000 \u0000 <p>Transient transfection of HEK293 cells represents the dominant technique for the production of recombinant adeno-associated virus (AAV) vectors. However, recombinant AAV (rAAV) production is cytotoxic, potentially impacting process performance, product yields, and quality, complicating downstream processing. This study characterizes cell death response for rAAV producing HEK293 cells and explores the potential to control cytotoxicity. Initial analysis of triple transfected cells revealed caspase-mediated apoptosis as a likely mechanism of cellular death. Next, the causes of this cytotoxicity were investigated by dissecting transfection steps. Exposing cells to polyethyleneimine (PEI) alone or complexed with a blank plasmid at typical concentrations had a limited impact on cell growth. However, the inclusion of plasmid constructs containing genes to produce rAAVs triggered significant cell death, with the helper plasmid being the most toxic both independently and in combination with packaging and transgene plasmids. Additionally, apoptosis in transfected cultures could be inhibited using the pan-caspase inhibitor, <i>N</i>-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk), leading to a 65% increase in peak viable cell density (VCD). Although the rAAV genome titer remained relatively unaltered, capsid levels declined upon cell death inhibition. Consequently, the ratio of full to empty capsids, an important product quality attribute (PQA) for rAAVs increased following caspase inhibition. This study provides insights into apoptosis activation in rAAVs and uncovers avenues for its modulation to alter PQAs.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143602792","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":"Time-Resolved Hierarchical Modeling Highlights Metabolites Influencing Productivity and Cell Death in Chinese Hamster Ovary Cells","authors":"Andreas Eriksson,&nbsp;Anne Richelle,&nbsp;Johan Trygg,&nbsp;Steffi Scholze,&nbsp;Shanti Pijeaud,&nbsp;Henrik Antti,&nbsp;Christoph Zehe,&nbsp;Izabella Surowiec,&nbsp;Pär Jonsson","doi":"10.1002/biot.202400624","DOIUrl":"https://doi.org/10.1002/biot.202400624","url":null,"abstract":"<p>Biopharmaceuticals are medical compounds derived from biological sources and are often manufactured by living cells, primarily Chinese hamster ovary (CHO) cells. CHO cells display variation among cell clones, leading to growth and productivity differences that influence the product's quantity and quality. The biological and environmental factors behind these differences are not fully understood. To identify metabolites with a consistent relationship to productivity or cell death over time, we analyzed the extracellular metabolome of 11 CHO clones with different growth and productivity characteristics over 14 days. However, in bioreactor processes, metabolic profiles and process variables are both strongly time-dependent, confounding the metabolite-process variable relationship. To address this, we customized an existing hierarchical approach for handling time dependency to highlight metabolites with a consistent correlation to a process variable over a selected timeframe. We benchmarked this new method against conventional orthogonal partial least squares (OPLS) models. Our hierarchical method highlighted several metabolites consistently related to productivity or cell death that the conventional method missed. These metabolites were biologically relevant; most were known already, but some that had not been reported in CHO literature before, such as 3-methoxytyrosine and succinyladenosine, had ties to cell death in studies with other cell types. The metabolites showed an inverse relationship with the response variables: those positively correlated with productivity were typically negatively correlated with the death rate, or vice versa. For both productivity and cell death, the citrate cycle and adjacent pathways (pyruvate, glyoxylate, pantothenate) were among the most important. In summary, we have proposed a new method to analyze time-dependent omics data in bioprocess production. This approach allowed us to identify metabolites tied to cell death and productivity that were not detected with traditional models.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400624","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143594973","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":"Synthetic Osmolytes for Enhanced RNA Thermotolerance and Extended Plasmid Storage at Room Temperature","authors":"Catarina Amaral,&nbsp;Eva C. Lourenço,&nbsp;Inês Morais,&nbsp;Osvaldo Ascenso,&nbsp;Sandra I. Aguiar,&nbsp;Diana Grilo,&nbsp;M. Rita Ventura,&nbsp;Catarina Pimentel","doi":"10.1002/biot.202400608","DOIUrl":"https://doi.org/10.1002/biot.202400608","url":null,"abstract":"<div>\u0000 \u0000 <p>Stable formulations for RNA and plasmid DNA are a matter of paramount significance in several fields, ranging from medicine to biotechnology. We have investigated the potential of 15 compounds derived from natural osmolytes to enhance the thermostability and protection of both RNA and plasmid DNA. Our findings demonstrated that several compounds exhibit remarkable effects, enhancing the long-term storage of plasmid DNA at room temperature and the resilience of RNA to high-temperature stress, surpassing the performance of commercial osmolytes. Importantly, we found that one of the compounds enhanced the detection efficacy of a cost-effective RT-PCR test for COVID-19 that we had previously developed.</p>\u0000 <p>This work offers new possibilities for expanding the capabilities of molecular diagnostic assays and nucleic acid storage methods.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143581817","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":"Next-Generation Sequencing-Based In Silico Transgene Integration Profiling Tool Accelerates Cell Line Genetic Characterizations","authors":"Zhenqiu Huang,&nbsp;Hyo-Young Jeong,&nbsp;Luke Nelson,&nbsp;Caitlyn Devine,&nbsp;Guanghua (Benson) Li,&nbsp;Lin Zhang","doi":"10.1002/biot.202400675","DOIUrl":"https://doi.org/10.1002/biot.202400675","url":null,"abstract":"<div>\u0000 \u0000 <p>Genetic characterization including Southern blot analysis of recombinant Chinese hamster ovary (CHO) cell lines expressing biologics is an essential prerequisite for regulatory submissions. The transposon-based expression system for CHO cell lines enables the insertion of DNA sequences into the genome and has been widely used by the biopharmaceutical industry. The system yields clones that are both highly productive and genetically stable. However, it often incorporates target genes at multiple locations in the CHO genome, presenting challenges to profile the integration sites using Southern blotting. The standard Southern blot analysis is time-consuming, as multiple rounds of screening for optimal restriction enzymes (REs) are needed to ensure the development of high-quality blots with an optimal resolution of banding patterns. In this study, we developed an innovative in silico Southern blotting tool that leverages the targeted locus amplification (TLA) data and PICRH reference genome to recapitulate only DNA fragments containing the gene of interest (GOI). The tool can plot the size and digestion pattern of these fragments using R packages, allowing for the selection of optimal REs that yield the highest-quality blots virtually, thereby eliminating the need for multiple wet-lab RE screening. The utility of this novel tool has been demonstrated across multiple cell lines and confirmed to be effective and accurate in predicting banding patterns consistent with experimental data. The tool has been implemented in our cell line development workflow which greatly streamlined our cell line genetic characterization.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143581815","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":"Investigation of the Effects of Applying Bio-Magnesium Oxide Nanoparticle Fertilizer to Moringa Oleifera Plants on the Chemical and Vegetative Properties of the Plants’ leaves","authors":"Ahmed A. Suliman,&nbsp;Camilia Y. El-Dewiny,&nbsp;Mohamed K. Y. Soliman,&nbsp;Salem S. Salem","doi":"10.1002/biot.202400536","DOIUrl":"https://doi.org/10.1002/biot.202400536","url":null,"abstract":"<div>\u0000 \u0000 <p>Magnesium (Mg) is necessary for plants as a structural element or an enzyme cofactor. It also plays a significant role in the light and dark responses of photosynthesis. Moringa plants are considered one of the most important medicinal plants that humans need for nutrition. The aim of the study is to provide Mg to plants in an easy and simple way. Therefore, it is widely available in human food due to its nutritional importance. In this experiment, the treatment was done on the Moringa plant as, one of the medicinal plants that is increasingly used in fresh form. The experiment was conducted at the research farm of the National Research Center. Magnesium oxide has been extracted and prepared in a natural way that is safe for humans, plants, and soil. <i>Trichoderma viride</i> filtrate was used as a chelating agent in an environmentally friendly process to create magnesium oxide nanoparticle (MgO-NPs). FTIR, TEM, and SEM-EDX were utilized to analyze and characterize the synthesized. Plants were treated with different doses to determine the optimal concentration for the plant. The effect of treatment on plants was to improve vegetation such, as a number of leaves (232.15), leaf area (273.97 cm²), and fresh weight (3693.17 g). It also increased dry matter in the plant (171.8 g), chemical properties, and elements.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143581862","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":"Self-Produced Brain-Like ECM From 3D-Cultured Dermal Fibroblasts Enhances Neuronal Growth and Survival","authors":"Vincent Roy,&nbsp;Isabella Bienjonetti,&nbsp;Alexandre Paquet,&nbsp;François Gros-Louis","doi":"10.1002/biot.202400594","DOIUrl":"https://doi.org/10.1002/biot.202400594","url":null,"abstract":"<p>Studying neurological disorders in vitro remains challenging due to the complexity of the human brain and the limited availability of primary neural cells. Tissue engineering enables the development of three-dimensional (3D) cell culture systems by generating a self-produced extracellular matrix (ECM) substrate. Culturing cells within this ECM substrate is known to more effectively mimic physiological conditions compared to traditional monolayer cultures. In this study, we analyzed the proteome and matrisome of 3D cultured dermal fibroblasts embedded in a self-produced ECM. Interestingly, in silico analysis predicted strong activation of neurogenesis-associated functions in this tissue-engineered 3D model. We showed that ECM proteins typically linked to neuronal development and maintenance were also expressed by dermal fibroblasts. Coculturing dermal fibroblasts with induced pluripotent stem cell (iPSC)-derived motor neurons notably enabled long-lasting culture periods while minimizing neuronal death, all without the need for costly media supplements. Furthermore, fibroblast-conditioned media enhanced neuronal survival. Although we demonstrated that the dermal fibroblast-derived ECM provided a rich matrix of proteins and signaling molecules that support neuronal growth and survival, the ECM alone seems insufficient to sustain the neuronal networks. These findings suggest that 3D cultured patient-derived dermal fibroblasts generate a neuro-supportive microenvironment and could serve as a cost-effective and less invasive alternative to brain biopsies for modeling complex neurological disorders. This approach offers a promising platform for studying such neural growth and survival and exploring therapeutic strategies for neurological diseases.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400594","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143581816","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":"Issue Information: Biotechnology Journal 3/2025","authors":"","doi":"10.1002/biot.70004","DOIUrl":"https://doi.org/10.1002/biot.70004","url":null,"abstract":"","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70004","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565222","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":"Overexpression of Geranylgeranyl Diphosphate Synthase and Cyclase Enhances Pleuromutilin Production in Clitopilus Passeckerianus T6","authors":"Bochun Wen,&nbsp;Huiwen Huang,&nbsp;Li Lu,&nbsp;Tiangang Liu,&nbsp;Ran Liu","doi":"10.1002/biot.202500004","DOIUrl":"https://doi.org/10.1002/biot.202500004","url":null,"abstract":"<div>\u0000 \u0000 <p>Pleuromutilin, a tricyclic diterpene compound with significant inhibitory activity against gram-positive bacteria and mycoplasmas, serves as a precursor for various veterinary and human medicines. Previous efforts have primarily focused on strain screening and fermentation process optimization to enhance pleuromutilin production in native pleuromutilin-producing strains, with the absence of genetic engineering strategies. In this study, we performed whole-genome sequencing of the pleuromutilin-producing strain <i>Clitopilus passeckerianus</i> T6 to identify the biosynthetic genes related to pleuromutilin production. Transcriptomic and metabolomic data were collected during the fermentation of <i>C. passeckerianus</i> T6, and gene transcription and metabolite accumulation in the pleuromutilin biosynthetic pathway were analyzed to identify the rate-limiting steps in pleuromutilin biosynthesis. Overexpression of the key genes <i>ple-ggpps</i> and <i>ple-cyc</i> increased pleuromutilin production by 50%, achieving a titer of 6.9 g/L. This study is the first to employ metabolic engineering to enhance pleuromutilin production in a native strain, providing a strategy for efficient pleuromutilin production.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 2","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143497273","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":"Enzymatically Crafted Bacterial Cellulose Nanoparticles Functionalized With Antimicrobial Peptides: Toward Sustainable Antimicrobial Formulations","authors":"Martina Schibeci,&nbsp;Rosa Gaglione,&nbsp;Noemi Russo,&nbsp;Raffaele Velotta,&nbsp;Bartolomeo Della Ventura,&nbsp;Angela Arciello","doi":"10.1002/biot.202400573","DOIUrl":"https://doi.org/10.1002/biot.202400573","url":null,"abstract":"<p>Although natural antimicrobial peptides (AMPs) are endowed with excellent antimicrobial properties, only a few of them have been successfully translated to the market so far. This is mainly due to their short half-life, to their high susceptibility to protease degradation, and to the lack of appropriate strategies for their efficient targeted delivery. Hence, the development of an effective system to deliver AMPs to the site of infection is urgent. The system here selected is represented by bacterial cellulose nanoparticles (BCNPs). Nanocellulose has recently emerged as one of the most promising “green” materials, attracting great attention due to its unique features, including biodegradability, sustainability, biocompatibility, and special physicochemical properties. To produce BCNPs, <i>Komagataeibacter xylinus</i> has been selected as host producing strain. Once obtained BC macrofibers, the production of BCNPs was set up by enzymatic hydrolysis using a commercial mixture of cellulases from <i>Trichoderma reesei</i> to develop a sustainable green biotechnological process. The storage stability of produced BCNPs has been also evaluated. Obtained BCNPs have been functionalized through non-covalent bindings with an antimicrobial peptide previously identified in human apolipoprotein B and found to be endowed with strong antimicrobial properties in in vitro analyses and with good biocompatibility profiles when analyzed on human skin cells. This opens interesting perspectives to the applicability of the developed system in several biotechnological fields.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 2","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400573","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143475359","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}