{"title":"CO2 Sequestration Potential and Soil Improvement Effects by Carbon-Fixing Bacteria Isolated From Degraded Soils in Shendong Coal Mining Area Located in Northwest China","authors":"Yuxin Han, Yichi Ma, Chenhao Wu, Bing Xiao, Jiahui Hu, Nengxiang Shu, Mingyu Cheng, Jianli Jia","doi":"10.1002/biot.70108","DOIUrl":"10.1002/biot.70108","url":null,"abstract":"<div>\u0000 \u0000 <p>Efficient carbon-sequestering microorganisms are crucial for enhancing soil quality and reducing carbon emissions, particularly in semiarid coal mining areas. In this study, we analyzed the soil carbon-fixing microbial taxa and pathways in the Shendong mining area via high-throughput sequencing, and concluded that their main carbon sequestering pathway is the reduced citrate cycle (rTCA cycle), which has the potential for microbial carbon sequestration. Bacterial strains with high CO<sub>2</sub> sequestration efficiency were isolated and identified, and their potential for improving soil carbon storage and quality was assessed. Through a systematic process of isolation, identification, and functional assessment, three dominant strains, <i>Streptomyces marokkonensis</i> FC1, <i>Streptomyces viridochromogenes</i> FC2, <i>and Dyadobacter endophyticus</i> FC3 with significant CO<sub>2</sub> sequestration capacity were obtained. Their adaptability and carbon fixation efficiency were confirmed through physiological, biochemical, and genetic analyses. The results from indoor <sup>13</sup>C isotope labeling experiments and 42-day soil improvement experiments demonstrated that CO<sub>2</sub> sequestration and soil improvement effects were achieved in coal mine soils under semiarid conditions. Among the strains, FC2 presented the highest carbon fixation potential and soil improvement efficiency at approximately 8% soil water content. This highlights the potential application of carbon-fixing bacteria in microbial-based strategies for the ecological restoration of degraded mining soils.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144923691","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}
Jing Wen, Lingling Huang, Danping Wang, Guoqiang Fan, Xiaojing Yang
{"title":"UV Mutagenesis Enhances DHA Biosynthesis in Schizochytrium sp. via Metabolic Reprogramming","authors":"Jing Wen, Lingling Huang, Danping Wang, Guoqiang Fan, Xiaojing Yang","doi":"10.1002/biot.70107","DOIUrl":"10.1002/biot.70107","url":null,"abstract":"<div>\u0000 \u0000 <p><i>Schizochytrium</i> sp., a marine alga prized for docosahexaenoic acid (DHA), was subjected to UV mutagenesis to boost industrial yields. The stable mutant UV1-3 achieved 5.01 g/L DHA—40.34% higher than wild-type S31. Transcriptomic and metabolomic analyses demonstrated that UV1-3 promotes docosahexaenoic acid (DHA) biosynthesis through coordinated metabolic regulation. Downregulation of key fatty acid synthase (FAS) pathway genes (<i>ACSL</i>, <i>SLC27A2</i>, <i>FabI</i>) reduced substrate competition for DHA precursors. Concurrently, RT-qPCR confirmed the upregulation of core polyketide synthase (PKS) pathway genes (<i>orfA</i>, <i>orfB</i>, <i>orfC</i>), directly enhancing DHA production. Furthermore, suppressed oxidative phosphorylation (evidenced by <i>COX</i> downregulation) and redirected carbon/nitrogen flux—achieved through diminished tricarboxylic acid (TCA) cycle activity (via downregulation of <i>HAL</i> and <i>proC</i>)—collectively favored DHA accumulation. These findings establish UV1-3 as a high-yielding industrial strain for DHA production and provide fundamental insights into metabolic flux regulation in <i>Schizochytrium</i> sp. These insights advance scalable, cost-effective microbial DHA production and deepen understanding of algal biosynthesis mechanisms, supporting sustainable omega-3 sourcing strategies.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144927513","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}
Anuradha Ramoji, Philipp Baumbach, Oleg Ryabchykov, Aikaterini Pistiki, Jan Rueger, David Vasquez Pinzon, Anja Silge, Stefanie Deinhardt-Emmer, Iwan W. Schie, Karina Weber, Charles Neu, Ute Neugebauer, Michael Kiehntopf, Thomas Bocklitz, Juergen Popp, Sina M. Coldewey
{"title":"Raman Spectroscopy Can Identify Acute and Persistent Biochemical Changes in Leukocytes From Patients With COVID-19 and Non-COVID-19-Associated Sepsis","authors":"Anuradha Ramoji, Philipp Baumbach, Oleg Ryabchykov, Aikaterini Pistiki, Jan Rueger, David Vasquez Pinzon, Anja Silge, Stefanie Deinhardt-Emmer, Iwan W. Schie, Karina Weber, Charles Neu, Ute Neugebauer, Michael Kiehntopf, Thomas Bocklitz, Juergen Popp, Sina M. Coldewey","doi":"10.1002/biot.70105","DOIUrl":"10.1002/biot.70105","url":null,"abstract":"<p>Sepsis remains a major clinical challenge, often resulting in long-term physiological and immunological disturbances. This study employed high-throughput single-cell Raman spectroscopy to analyze the biochemical profiles of peripheral blood leukocytes from patients with non-COVID-19 and COVID-19-associated sepsis. Leukocytes were assessed at multiple timepoints, including the acute phase (Days 3 and 7 after sepsis onset) and late recovery phase (6 and 12 months after sepsis onset). Raman spectroscopic profiles of leukocytes showed clear separation between healthy controls and sepsis patients during the acute phase with high balanced accuracy (BAcc: 95%–98%). Spectral differences between acute and recovery phases (BAcc: 84%–97%) and between recovery-phase leukocytes and those from healthy controls (BAcc: 81%–90%) were also observed, indicating long-lasting molecular alterations. Furthermore, distinct profiles were identified between non-COVID-19 and COVID-19-associated sepsis during the acute phase (BAcc: 65%–71%) and in the late-recovery phase (BAcc: 71%–83%). These findings demonstrate that Raman spectroscopy enables label-free, high-throughput profiling of leukocyte biochemistry across the sepsis trajectory. This suggests that Raman spectroscopy is a promising tool for high-throughput screening, offering insights into the biomolecular changes in sepsis and providing a diagnostic platform to differentiate between sepsis etiologies, a significant advancement in the field of sepsis diagnostics.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70105","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144927525","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}
Alan Foley, Nga Lao, Ciara McGuirk, Colin Clarke, Niall Barron
{"title":"Single-Cell Mitochondrial DNA Analysis of Recombinant Chinese Hamster Ovary Cells Reveals Widespread Heteroplasmy","authors":"Alan Foley, Nga Lao, Ciara McGuirk, Colin Clarke, Niall Barron","doi":"10.1002/biot.70079","DOIUrl":"10.1002/biot.70079","url":null,"abstract":"<p>Recent bulk analysis of Chinese hamster ovary (CHO) cell mitochondrial DNA revealed widespread heteroplasmy across cell lines and even within clones of the same parental host. To address this, we applied our previously developed single-cell mtDNA sequencing (scmtDNAseq) method to 84 single CHO cells. We identified widespread intercellular heteroplasmy across the CHO cell population and predicted possible phenotypic impacts. 3/11 (27%) of the most variable mutations were only identified by scmtDNAseq, indicating greater resolution when compared to bulk cell analysis. Single-cell RNAseq (scRNAseq) was also performed at the same time point and, compared to scmtDNAseq, significant differences in intercellular heteroplasmy were observed. Using an inducible mAb expression system demonstrated that short-term additional biosynthetic burden of exogenous protein production had little impact on intercellular heteroplasmy. We additionally monitored bulk heteroplasmy over 38 days, reflecting the typical timespan from vial thaw to production vessel in a Biopharmaceutical upstream cell culture process. We observed minimal change in heteroplasmy, finding no evidence that a mAb-producing CHO cell line develops impactful changes in heteroplasmy over that timeframe. This would suggest that for our cell line, the heteroplasmy profile established on Day 1 should be maintained throughout a full fed-batch bioprocess run.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70079","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144923690","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}
Jing-Yang Cai, Cun-Jin Zhang, Jia-Qi Wang, Ai-Mei Liao, Ming Hui, Long Pan, Xu-Sheng Chen
{"title":"Advances in ε-Poly-Lysine Biosynthesis, Selection of High-Yielding Strains and Regulatory Mechanisms","authors":"Jing-Yang Cai, Cun-Jin Zhang, Jia-Qi Wang, Ai-Mei Liao, Ming Hui, Long Pan, Xu-Sheng Chen","doi":"10.1002/biot.70111","DOIUrl":"10.1002/biot.70111","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <p>ε-Poly-lysine (ε-PL) is a naturally occurring antimicrobial polypeptide that has been approved as a food preservative in several major global markets, including Japan, China, and the United States, where it is classified as Generally Recognized as Safe (GRAS). It exhibits efficacy against Gram-positive and select Gram-negative bacteria, indicating its broad potential for application in both industrial and medical sectors. The mature fermentation process of <i>Streptomyces albulus</i> has established it as the primary production strain for ε-PL. This article provides a comprehensive overview of the biosynthetic and antibacterial mechanisms of ε-PL and reviews the strategies for strain selection and breeding aimed at developing high-yield ε-PL-producing strains. Furthermore, it examines approaches to enhance ε-PL production through pathway-specific regulation and global metabolic engineering, while also identifying future research directions. This review aims to serve as a theoretical reference for future researchers focusing on high-yield strain breeding and metabolic engineering strategies to optimize ε-PL production.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Summary</h3>\u0000 \u0000 <div>\u0000 <ul>\u0000 \u0000 <li>The biosynthesis and antimicrobial mechanism of ε-PL are described, along with strategies for the selection of high-yielding strains.</li>\u0000 \u0000 <li>Regulatory mechanisms within the complex biosynthetic metabolism network are revealed.</li>\u0000 \u0000 <li>Strategies to improve the production of ε-PL through pathway-specific and global regulation are discussed.</li>\u0000 </ul>\u0000 </div>\u0000 </section>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144927524","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}
Andrés González-Techera, Gabriel Lassabe, José A. Chabalgoity, Cecilia Vallejo Garín, Julio Guarnaschelli, Gualberto González-Sapienza
{"title":"Adaptabodies: Retargeting a Predefined Monoclonal Antibody With Bispecific Nanobodies for Antibody Therapy","authors":"Andrés González-Techera, Gabriel Lassabe, José A. Chabalgoity, Cecilia Vallejo Garín, Julio Guarnaschelli, Gualberto González-Sapienza","doi":"10.1002/biot.70089","DOIUrl":"10.1002/biot.70089","url":null,"abstract":"<div>\u0000 \u0000 <p>Monoclonal antibody (MoAb) therapy is a cornerstone in treating cancer, inflammatory diseases, and infections. However, the development of new monoclonal antibodies is labor-intensive, costly, and species-specific, limiting their accessibility in veterinary medicine and slowing innovation in human therapies. In this work, we introduce adaptabodies, bispecific nanobody constructs that repurpose existing MoAbs, of irrelevant specificity, by bridging their idiotype to a new antigen. As a proof of concept, we tested this strategy using a model murine MoAb combined with a neutralizing adaptabody that redirected its binding to tetanus toxin and resulted in mice surviving at least for 8 days when challenged with 1 lethal dose 100 (LD<sub>100</sub>). Mice challenged with 20 LD<sub>100</sub> of tetanus toxin and treated with two neutralizing adaptabodies survived 8 days and increasing five-fold the amount of the MoAb increased survival to at least 20 days. This approach reduces the need to develop new monoclonal antibodies for each disease, requiring only the retargeting domain to be generated. Moreover, a single MoAb could be developed for species lacking therapeutic MoAbs for adaptabody-mediated treatment. The increasing approval of monoclonal antibodies for treatment of both humans and companion animals underscores the relevance of this flexible and scalable strategy.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 8","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144869503","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}
Marcos Fernando Basso, Arnubio Valencia-Jiménez, Fabrizio Lo Celso, Isabel Rodrigues Gerhardt, Thomas Joseph V. Higgins, Maria Fatima Grossi-de-Sa
{"title":"Exploring Plant α-Amylase Inhibitors: Mechanisms and Potential Application for Insect Pest Control","authors":"Marcos Fernando Basso, Arnubio Valencia-Jiménez, Fabrizio Lo Celso, Isabel Rodrigues Gerhardt, Thomas Joseph V. Higgins, Maria Fatima Grossi-de-Sa","doi":"10.1002/biot.70098","DOIUrl":"10.1002/biot.70098","url":null,"abstract":"<p><i>α</i>-Amylases are found in microbes, plants, and animals, including insect pests. They play crucial roles in catalyzing the hydrolysis of <i>α</i>-1,4-glucan bonds within starch, glycogen, and related carbohydrates, forming shorter oligomers. In green plants, these enzymes are pivotal for starch degradation during photosynthesis and seed germination, whereas in phytophagous insect pests, they predominantly facilitate seed parasitism by degrading raw starch granules. Amylase inhibitors in plants appear to function as part of their defense against pests and pathogens. In the context of insect pests, some of these amylase inhibitors can target <i>α</i>-amylases in the digestive system of certain insects. Both mono- and dicotyledonous plants harbor multiple genes encoding proteinaceous <i>α</i>-amylase inhibitors. Previous studies have demonstrated that <i>α</i>-amylase inhibitors, whether produced in vitro or overexpressed in transgenic plants, can exhibit entomotoxic activity against certain insect pests. Field trials involving transgenic plants that overexpress <i>α</i>-amylase inhibitors have been conducted, laying the foundation for the potential commercialization of crops engineered with these genes. Herein, this review explores the molecular interactions between plant <i>α</i>-amylase inhibitors and insect <i>α</i>-amylases, shedding light on the underlying mechanisms of action, structural diversity, and assessing the broader biotechnological applications of this promising strategy.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 8","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70098","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144869504","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":"Uncovering Growth-Inhibitory Metabolic Byproducts in HEK293 Fed-Batch Cultures and Strategies for Their Control","authors":"Cameron Harrington, Bhanu Chandra Mulukutla","doi":"10.1002/biot.70087","DOIUrl":"10.1002/biot.70087","url":null,"abstract":"<div>\u0000 \u0000 <p>Human Embryonic Kidney 293 (HEK293) cells are currently one of the preferred host cell lines for the production of biologics, specifically, AAV-based viral vectors. These fast-growing cells consume significant amounts of nutrients and often convert them into byproducts such as lactate and ammonia. In fed-batch cultures, accumulation of lactate and ammonia to high levels can inhibit cell proliferation. In this study, we demonstrate that lactate and ammonia accumulation alone doesn't fully explain the growth inhibition observed in HEK293 fed-batch cultures. Growth inhibition was noted even when the residual levels of these byproducts were well controlled. Instead, we show that several previously unknown compounds accumulate in HEK293 cell fed-batch cultures, some of which can inhibit HEK293 cell growth either individually or synergistically. Many of these newly identified compounds are intermediates or byproducts of amino acid catabolism. When residual levels of the source amino acids for these novel byproducts were controlled in the low concentration range (∼1 mM) in HEK293 fed-batch cultures, lactate accumulated to higher levels, causing growth inhibition. This prompted the use of High-end pH Delivery of Glucose (HIPDOG), a control strategy that limits lactate production by keeping low residual concentrations of glucose. In HIPDOG cultures, controlling the source amino acids at low concentrations resulted in lower accumulations of the corresponding growth-inhibitory byproducts when compared to the control HIPDOG conditions with typical levels of amino acids. This led to higher viable cell densities (VCD) and viabilities in low amino acid conditions. Strategies that reduce byproduct accumulation, whether classical or novel byproducts, in HEK293 fed-batch processes can result in enhanced VCDs, potentially leading to higher volumetric productivities.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 8","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144869933","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":"A Miniaturized Centrifugal Pump ECMO System Enhances Hemocompatibility in Small Animal Models","authors":"Zhen Yang, Yiai Li, Jingyi Peng, Danhe Jia, Youpeng Zhang, Wenxing Huo, Zhigang Liu, Xian Huang","doi":"10.1002/biot.70103","DOIUrl":"10.1002/biot.70103","url":null,"abstract":"<div>\u0000 \u0000 <p>Extracorporeal membrane oxygenation (ECMO) is a fundamental treatment for cardiovascular and severe pulmonary diseases, with small animal models providing critical insights into organ protection during cardiopulmonary bypass and ECMO therapy. Conventional roller pumps, however, induce hemolysis and organ injury through repetitive compression-shear exposure, severely limiting their utility. While centrifugal pumps reduce shear stress and are effective in large animal ECMO, their flow range and priming volume are unsuitable for small animals. Here, we present a miniaturized, fully integrated ECMO system with an optimized centrifugal pump tailored for small animal models. The system reduces shear stress, minimizes blood damage, and enhances organ protection. Integrated multi-parameter sensors enable real-time monitoring of blood flow, pressure, and temperature, thereby streamlining setup and improving perioperative support. In a 6-h experiment, the system demonstrated significant hemolysis suppression, improved renal function (with reduced levels of Neutrophil Gelatinase-Associated Lipocalin, or NGAL), and stable hemodynamics. This innovation offers safer extracorporeal support for small animal studies on cardiovascular diseases, organ recovery, and ECMO mechanisms, furthering research into therapeutic interventions. By addressing key limitations of existing pumps, the system provides a reliable platform for exploring hemodynamic and pathophysiological processes in ECMO treatment, establishing a foundation for future preclinical investigations.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 8","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144861865","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":"Evaluation of “Difficult-to-Express” Monoclonal Antibodies in a CHO-Based Hybrid Site-Specific Integration System Under Industrially Relevant Conditions","authors":"Alana C. Szkodny, Kelvin H. Lee","doi":"10.1002/biot.70102","DOIUrl":"10.1002/biot.70102","url":null,"abstract":"<p>Variation in the primary sequence of monoclonal antibodies (mAbs) can negatively affect their behavior in biopharmaceutical manufacturing platforms, and efforts to identify mAbs with poor “developability” characteristics lack robust methods for assessing mAb expression from an industrially relevant platform. Recent advancements in site-specific integration-based (SSI) platforms in Chinese hamster ovary (CHO) cells can mitigate the high transcriptional variation observed with random integration and the low industrial relevance of transient expression by providing a flexible platform for mAb expression from a consistent clonal background. This work applies a novel SSI-based expression system capable of generating isogenic cell pools in less than 1 month to systematically compare the expression of ten sequence variants of two therapeutically relevant mAbs from two genomic loci under industrially relevant culture conditions. Eight single amino acid mutations in trastuzumab resulted in reduced productivity compared to the wild-type mAb in batch cultures, and three mutations maintained a low-expressing phenotype in fed-batch cultures. The mutations resulted in variant-specific patterns of decreased domain stability and increased ER stress. The application of industrially relevant SSI systems in developability workflows could strengthen the understanding of the sequence determinants of mAb expression to improve mAb design, candidate selection, and process development decisions.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 8","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70102","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144861866","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}