Biotechnology and Bioengineering最新文献

{"title":"An Overview of Virus-Free Protein Expression in Insect Cells: A Mode of Rapid Manufacturing Platform for Therapeutic Protein and Virus-Like-Particles.","authors":"Jagadeesh Mahadevan, Kishalay Mitra, Lopamudra Giri","doi":"10.1002/bit.28961","DOIUrl":"https://doi.org/10.1002/bit.28961","url":null,"abstract":"<p><p>Production of therapeutic proteins, antibodies, and virus-like particles (VLP) using baculovirus expression systems (BEVS) has been explored for decades. However, we have realized an urgent need for accelerated production of recombinant proteins and VLPs to address critical situations in recent scenarios. In contrast to BEVSs, the virus-free method is significantly shorter as it bypasses the time-consuming process of infectivity monitoring and virus amplification. Moreover, in the virus-free method, complex steps of protein separation can be eliminated to ease downstream processing. Hence, we present a detailed review of the recent techniques for expressing recombinant proteins, therapeutics, and VLP in insect cells using virus-free methods. First, we focus on the specific methodologies used to optimize virus-free transfection. Here, we provide insight into the interplay between crucial factors, including concentration of transfection reagent, seeding density, and medium temperature. Secondly, we provide a structured review of the novel transfection reagents used for transient and stable transfection. Thirdly, we performed an assessment of the cell lines and plasmids used for virus-free expression and their evaluation based on corresponding protein yield. Finally, we provide the recent advancement in scaling up the transfection process from the shaker flask to the bioreactor level to achieve better yield. Various virus-free expression methodologies presented in this article are essential for evaluating the transfection processes toward improving protein yield. The readers can also use the information to design experiments and optimize process parameters for bioreactor operation.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143596134","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":"High-Efficiency PET Degradation With a Duo-Enzyme System Immobilized on Magnetic Nanoparticles","authors":"Siddhi Kotnis, Siddhant Gulati, Qing Sun","doi":"10.1002/bit.28963","DOIUrl":"https://doi.org/10.1002/bit.28963","url":null,"abstract":"The widespread consumption of PET worldwide has necessitated the search for environment-friendly methods for PET degradation and recycling. Among these methods, biodegradation stands out as a promising approach for recycling PET. The discovery of duo enzyme system PETase and MHETase in 2016, along with their engineered variants, has demonstrated significant potential in breaking down PET. Previous studies have also demonstrated that the activity of the enzyme PETase increases when it is immobilized on nanoparticles. To achieve highly efficient and complete PET depolymerization, we immobilized both FAST-PETase and MHETase at a specific ratio on magnetic nanoparticles. This immobilization resulted in a 2.5-fold increase in product release compared with free enzymes. Additionally, we achieved reusability and enhanced stability of the enzyme bioconjugates.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"49 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143582968","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":"Engineered Lactobacillus rhamnosus Producing 3-Hydroxybutyrate: A Dual-Action Therapeutic Strategy for Colon Cancer Cachexia","authors":"Xu Qiu, Zhiyun Zou, Taijie Lin, Chenyun Guo, Donghai Lin","doi":"10.1002/bit.28972","DOIUrl":"https://doi.org/10.1002/bit.28972","url":null,"abstract":"3-hydroxybutyrate (3-HB), an essential endogenous metabolite, shows significant therapeutic potential in several disease contexts. However, its clinical application has been hampered by limitations, such as adverse effects on the gut microbiota. This study introduces a genetically engineered strain of <i>Lactobacillus rhamnosus</i> GG (LGGK) that integrates the benefits of 3-HB production with the probiotic properties of LGG. Using a murine colon cancer cachexia (CAC) model, LGGK supplementation significantly improved survival, reduced tumor progression, and alleviated muscle wasting. LGGK restored gut microbial diversity, increased the abundance of beneficial bacteria, and increased the production of short-chain fatty acids while reducing harmful microbial populations. In addition, LGGK supplementation demonstrated anti-inflammatory effects, effectively reducing elevated pro-inflammatory cytokines in serum and skeletal muscle. These findings highlight LGGK as a dual-action therapeutic approach that utilizes the metabolic benefits of 3-HB and the gut-modulating properties of LGG. This innovation offers a promising strategy for the treatment of CAC and potentially other metabolic and inflammatory disorders, and highlights the potential of engineered probiotics in advanced therapeutic applications.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"53 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143575227","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":"Self-Healable Hydrogel for Regression of Liver Fibrosis","authors":"Yu-Chai Tai, Wei-Rong Yin, Kai-Yi Cheng, Xin-Yu Chou, Yong-Heng Lin, Shan-hui Hsu, Yung-Te Hou","doi":"10.1002/bit.28966","DOIUrl":"https://doi.org/10.1002/bit.28966","url":null,"abstract":"Liver fibrosis is considered as a wound healing process in the presence of chronic hepatic injury. A hydrogel (CPDP) based on chitosan–phenol that undergoes fast gelling and owns self-healing and injectable properties was investigated for the effect on regression of liver fibrosis. For the purpose, we established both in vitro and in vivo liver fibrosis models and implanted CPDP hydrogel into the injured liver. The CPDP hydrogel not only provided a suitable microenvironment for hepatocyte spheroids, but also demonstrated a potential for the hepatocyte spheroid-embedded system to mimic the liver tissue in vitro. Furthermore, the urea synthesis of injured hepatocytes cultured on hepatocyte spheroid-embedded CPDP hydrogel was 1.12 times higher than that on hepatocyte spheroid-embedded collagen hydrogel after 7 days of culture, indicating that CPDP hydrogel effectively rescued hepatic function in the injured hepatocytes. Moreover, the hepatic injury was alleviated with improved hepatic function in the liver fibrosis model in vivo. A reduction of approximately 28% in serum AST/ALT ratios and a 70% decrease in the fibrotic area suggested the regression of liver fibrosis after 2 weeks of CPDP hydrogel administration. These findings suggest that CPDP hydrogel holds promise for applications in liver tissue engineering.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"12 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143569574","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering Affibody Binders to Death Receptor 5 and Tumor Necrosis Factor Receptor 1 With Improved Stability","authors":"Gregory H. Nielsen, Jonathan N. Sachs, Benjamin J. Hackel","doi":"10.1002/bit.28954","DOIUrl":"https://doi.org/10.1002/bit.28954","url":null,"abstract":"Protein developability is an important, yet often overlooked, aspect of protein discovery campaigns that is a key driver of utility. Recent advances have improved developability screening capacity, making it an increasingly viable option in early-stage discovery. Here, we engineered one component of developability, stability, of two affibody proteins—one that targets death receptor 5 and another that targets tumor necrosis factor receptor 1—previously evolved to bind receptor and non-competitively inhibit signaling via conformational modulation. Starting from an error-prone PCR library of each affibody, variants were screened via yeast surface display binder selections, including depletion of non-specific binders, followed by developability assessment using the on-yeast protease and yeast display level assays. Multiplex deep sequencing identified variants for further evaluation. Purified variants exhibited elevated stability—8°C to 14°C increase in T_m,app—with maintained 1–2 nM affinity for the TNFR1 affibody and 30-fold improvement in the DR5 affibody affinity to 0.8 nM.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"53 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143560759","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":"Bayesian Optimization in Bioprocess Engineering—Where Do We Stand Today?","authors":"Florian Gisperg, Robert Klausser, Mohamed Elshazly, Julian Kopp, Eva Přáda Brichtová, Oliver Spadiut","doi":"10.1002/bit.28960","DOIUrl":"https://doi.org/10.1002/bit.28960","url":null,"abstract":"Bayesian optimization is a stochastic, global black-box optimization algorithm. By combining Machine Learning with decision-making, the algorithm can optimally utilize information gained during experimentation to plan further experiments—while balancing exploration and exploitation. Although Design of Experiments has traditionally been the preferred method for optimizing bioprocesses, AI-driven tools have recently drawn increasing attention to Bayesian optimization within bioprocess engineering. This review presents the principles and methodologies of Bayesian optimization and focuses on its application to various stages of bioprocess engineering in upstream and downstream processing.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"1 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545986","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":"Current Advances and Future Prospects of Bulk and Microfluidic-Enabled Electroporation Systems","authors":"Fei Li, Cheng Dong, Tianlan Chen, Siming Yu, Chunzhao Chen","doi":"10.1002/bit.28965","DOIUrl":"https://doi.org/10.1002/bit.28965","url":null,"abstract":"Reversible electroporation (EP) is a pivotal biophysical technology that leverages pulsed electric fields to enhance the permeability of cell membranes, thereby facilitating the introduction of foreign material into cells. In this review, we provide an overview of bulk electroporators and microfluidic-enabled EP systems, focusing on their controversial points of mechanisms, architectures, and parameter settings. Bulk electroporators have been extensively commercialized with settled form including pulse generator and accessories (i.e., EP cuvette and plates). Researchers have made efforts to increase the throughput and simplify the operation of bulk EP systems. Additionally, microfluidics has emerged as a promising technology for optimizing EP parameters and enhancing the performance. Given the significant structural differences between these two types of EP systems, their operating conditions such as temperature, voltage, and pulse parameters are discussed. Research tend to operate single cells under more concentrated electric field induced by low voltage, enabling a quantitative exogenous materials delivery and numerical simulation. However, due to cost constraints and properties of materials utilized in laboratories, the commercialization of laboratory prototypes has been impeded. Furthermore, the technological limitations, current commercialization status, and development trends have been examined.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"16 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143546070","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":"MAT2A Knockdown Enhances Recombinant Protein Expression in Transgenic CHO Cells Through Regulation of Cell Cycle.","authors":"Yan-Ping Gao, Jiang-Tao Lu, Hui-Jie Zhang, Zhao-Ming Cui, Yang Guo, Xi Zhang, Wen Wang, Le-Le Qiu, Xiao-Yin Wang, Tian-Yun Wang, Yan-Long Jia","doi":"10.1002/bit.28962","DOIUrl":"https://doi.org/10.1002/bit.28962","url":null,"abstract":"<p><p>Chinese hamster ovary (CHO) cells represent the most widely utilized host system for industrial production of high-quality recombinant protein therapeutics. Novel CHO cell line development is achieved through genetic and cellular engineering approaches, effectively addressing limitations such as clonal variation and productivity loss during culture. Previous studies have established that MAT2A inhibition in tumor cells promotes expression of the cyclin-dependent kinase inhibitor p21, inducing antitumor activity. Notably, p21 induction has been shown to enhance recombinant protein expression in CHO cells by triggering cell cycle arrest. In this study, we identified MAT2A as a potential regulatory target, showing significant differential expression in transfected CHO cells with elevated versus diminished recombinant protein production. To investigate this phenomenon, we generated CHO cells with MAT2A knockdown (shMAT2A) and evaluated their recombinant protein output. Results demonstrated that MAT2A silencing enhanced recombiant protein/antibody production by 1.73-/1.70-fold through suppression of CyclinD1, thereby activating p21 and inducing G1 phase arrest. Furthermore, pharmacological inhibition of MAT2A using small molecules increased cell volume, boosted metabolic activity, and improved specific antibody productivity of recombiant protein/antibody production by 1.88-/2.16-fold in transfected CHO cells. These findings advance our understanding of MAT2A-mediated regulatory mechanisms and provide a strategic framework for developing high-efficiency CHO cell expression systems.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143514625","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":"Use of Whole Cells and Cell-Free Extracts of Catalase-Deficient E. coli for Peroxygenase-Catalyzed Reactions","authors":"Ana C. Ebrecht, U. Joost Luelf, Kamini Govender, Diederik J. Opperman, Vlada B. Urlacher, Martha S. Smit","doi":"10.1002/bit.28959","DOIUrl":"https://doi.org/10.1002/bit.28959","url":null,"abstract":"Unspecific peroxygenases (UPOs) and cytochrome P450 monooxygenases (CYPs) with peroxygenase activity are becoming the preferred biocatalysts for oxyfunctionalization reactions. While whole cells (WCs) or cell-free extracts (CFEs) of <i>Escherichia coli</i> are often preferred for cofactor-dependent monooxygenase reactions, hydrogen peroxide (H₂O₂) driven peroxygenase reactions are generally performed with purified enzymes, because the catalases produced by <i>E. coli</i> are expected to quickly degrade H₂O₂. We used the CRISPR/Cas system to delete the catalase encoding chromosomal genes, <i>katG</i>, and <i>katE</i>, from <i>E. coli</i> BL21-Gold(DE3) to obtain a catalase-deficient strain. A short UPO, <i>Dca</i>UPO, and two CYP peroxygenases, <i>Ssca</i>CYP_E284A and CYP102A1_21B3, were used to compare the strains for peroxygenase expression and subsequent sulfoxidation, epoxidation, and benzylic hydroxylation activity. While 10 mM H₂O₂ was depleted within 10 min after addition to WCs and CFEs of the wild-type strain, at least 60% remained after 24 h in WCs and CFEs of the catalase-deficient strain. CYP peroxygenase reactions, with generally lower turnover frequencies, benefited the most from the use of the catalase-deficient strain. Comparison of purified peroxygenases in buffer versus CFEs of the catalase-deficient strain revealed that the peroxygenases in CFEs generally performed as well as the purified proteins. We also used WCs from catalase-deficient <i>E. coli</i> to screen three CYP peroxygenases, wild-type SscaCYP, <i>Ssca</i>CYP_E284A, and <i>Ssca</i>CYP_E284I for activity against 10 substrates comparing H₂O₂ consumption with substrate consumption and product formation. Finally, the enzyme-substrate pair with highest activity, <i>Ssca</i>CYP_E284I, and <i>trans</i>-β-methylstyrene, were used in a preparative scale reaction with catalase-deficient WCs. Use of WCs or CFEs from catalase-deficient <i>E. coli</i> instead of purified enzymes can greatly benefit the high-throughput screening of enzyme or substrate libraries for peroxygenase activity, while they can also be used for preparative scale reactions.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"32 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143495269","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":"Multiple Use of Regenerated Depth Filters in Antibody Purification Processes","authors":"Bernhard Spensberger, Ferdinand Stueckler, Marc Pompiati, Christoph Feistl, Thorsten Lemm, Roberto Falkenstein","doi":"10.1002/bit.28957","DOIUrl":"https://doi.org/10.1002/bit.28957","url":null,"abstract":"During the manufacturing of therapeutic antibodies, disposable depth filters are used after affinity chromatography to remove haze and process-related impurities such as host cell proteins (HCP) and DNA known as critical quality attributes. The present study reports on the regeneration of depth filters allowing their reuse for at least 10 times while retaining sufficiently high clarification capacity. Three filter types were evaluated including standard cellulose-based and fully synthetic matrix materials using acidic or alkaline solutions in alternating cycles of loading and regeneration. Both alkaline and acidic solutions were effective, however, overall acidic regeneration of the filter material appeared superior for multiple use. This was especially evident for the silica-containing XOSP filter, where HCP and DNA were almost completely removed and remained low over 10 applications. Simultaneously preserved product quality indicated a high resistance of the filter matrix toward regeneration. These unexpected findings offer improved flexibility for available filter capacity in downstream processing along with ecologic advantages over the single use applications. Regarding the carbon footprint of the filtration process, calculated potential savings by a factor of four can be achieved, mainly accounting for reduced plastic waste. Therefore, depth filter reuse supports sustainability and carbon dioxide reduction during production processes.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"65 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143485955","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}