Bioprocess and Biosystems Engineering最新文献

{"title":"Hybrid dynamic flux balance modeling approach for bioprocesses: an E. coli case study.","authors":"Zahra Negahban, Valerie Ward, Anne Richelle, Chris McCready, Hector Budman","doi":"10.1007/s00449-025-03147-z","DOIUrl":"10.1007/s00449-025-03147-z","url":null,"abstract":"<p><p>In this study, we present a hybrid dynamic flux balance analysis (DFBA) model, combined with Partial Least Squares (PLS) regression, to simulate cell culture behavior in response to variations in media composition. DFBA models typically incorporate a stoichiometric matrix representing metabolic reactions, leveraging the pseudo-stationarity assumption to reduce the number of parameters, which in turn minimizes the risk of overfitting. Here, PLS regression is employed to define kinetic rate constraints within the DFBA model, capturing the dynamic and non-linear nature of reaction rates over different culture phases. An optimization approach identifies the minimal number of kinetic constraints required, ensuring model accuracy without excessive complexity. Our hybrid model is validated through simulation case studies using an E. coli system, demonstrating its effectiveness in adjusting to changes in initial media composition. The case studies reveal that the model's accuracy improves with a more detailed stoichiometric matrix, particularly when larger networks or more varied metabolic environments are present. Additionally, the hybrid DFBA-PLS approach provides a robust and scalable modeling framework adaptable to other bioprocesses, offering insights into medium composition effects and highlighting its potential for bioprocess optimization.</p>","PeriodicalId":9024,"journal":{"name":"Bioprocess and Biosystems Engineering","volume":" ","pages":"841-856"},"PeriodicalIF":3.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143708346","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 tandem conversion process for the synthesis of polyitaconic acid from glucose in the Aspergillus terreus culture.","authors":"Ikumi Kinoshita, Tomonari Tanaka, Yuji Aso","doi":"10.1007/s00449-025-03139-z","DOIUrl":"10.1007/s00449-025-03139-z","url":null,"abstract":"<p><p>In this study, a bio-based vinylidene monomer, itaconic acid (IA), produced by a fungus Aspergillus terreus NBRC 6123 was polymerized in the culture. The inhibition of IA polymerization by the culture components was eased by extraction with 4-methyltetrahydropyran (4mTHP). The extraction with 4mTHP for 4 days under aerobic condition resulted in IA production at 67.9 g/L (522 mM) in the organic phase from 180 g/L glucose. Interestingly, 4mTHP did not affect to the glucose consumption and respiration of A. terreus. IA polymerization in 4mTHP for 72 h using 400 mM IA and 1 mM 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) as a radical initiator under anaerobic condition by N₂ gas purging resulted in that the IA conversion, and the weight-averaged molecular weight (Mw) and the molecular weight distribution (MWD) of the synthesized polyitaconic acid (polyIA) were 11.5%, 79.8 × 10³ g/mol, and 1.26, respectively. The respiration by the IA producer itself was used to remove the residual oxygen in the flask by sealing after the IA production. Colorimetric evaluation with a redox indicator, resazurin, revealed that an anaerobic condition which is suitable for the IA polymerization can be accomplished by just incubation for 1 day after the sealing. The synthesis of polyIA from glucose in the same flask with 4mTHP was demonstrated, resulting in that the IA conversion, and Mw and MWD of the synthesized polyIA were 13.8%, 9.1 × 10³ g/mol, and 1.24, respectively, when 47.1 g/L (362 mM) IA was produced by the extraction for 3 days. To our knowledge, this is the first demonstration of radical polymerization of vinylidene monomer in microbial cultures.</p>","PeriodicalId":9024,"journal":{"name":"Bioprocess and Biosystems Engineering","volume":" ","pages":"737-748"},"PeriodicalIF":3.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143603711","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":"Degradation and transformation of tylvalosin by newly selected Providencia vermicola strain CT1: removal efficiency, pathways, mechanisms, and actual applications.","authors":"Ruina Chai, Meng Meng, Qi Li, Hansong Zhao, Yinglin Zhao, Jianxiang Zhong, Yunying Liu, Wenyuan Zhao, Xiaoxia Wang, Jianguo Cheng","doi":"10.1007/s00449-025-03140-6","DOIUrl":"10.1007/s00449-025-03140-6","url":null,"abstract":"<p><p>Tylvalosin (TAT) is a widely used veterinary antibiotic whose residual contaminants promote antibiotic resistance and pose potential risks to human health and ecosystems. This study successfully isolated and identified a TAT-degrading bacterial strain, Providencia vermicola strain CT1, through 16S rRNA analysis and biochemical tests. Under optimized conditions (30 °C, pH = 6, initial TAT concentration of 300 mg/L, and bacterial culture volume of 50 mL), strain CT1 achieved a TAT degradation percentage of 97.1%. The degradation process followed a first-order kinetic model and was primarily driven by extracellular metabolites. GC-MS analysis identified that strain CT1 degrades TAT to produce small molecules such as 3-methylbutanoic acid, ethyl acetate, and 3-iminopentan-2-one. Strain CT1 effectively degraded TAT in actual wastewater samples, achieving 95% degradation within 60 h, and significantly reduced the COD, BOD₅, and NH₄⁺-N concentrations. These findings provide theoretical guidelines for removing TAT and other macrolide antibiotic contaminants from the environment.</p>","PeriodicalId":9024,"journal":{"name":"Bioprocess and Biosystems Engineering","volume":" ","pages":"749-760"},"PeriodicalIF":3.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143613056","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":"Enhancement of natamycin production by combining ARTP mutagenesis with temperature control strategy development in Streptomyces gilvosporeus.","authors":"Jian Xue, Wen Xiao, Yuxiu Xu, Liang Wang, Jianhua Zhang, Hongjian Zhang, Xusheng Chen","doi":"10.1007/s00449-025-03145-1","DOIUrl":"10.1007/s00449-025-03145-1","url":null,"abstract":"<p><p>Natamycin, a natural antifungal compound produced by Streptomyces, possesses antibacterial activity against yeast and mold. However, its low yield hinders widespread application in the food and pharmaceutical industries. This study aims to enhance natamycin production of Streptomyces gilvosporeus through engineering strain and optimization bioprocess. A high-yield strain exhibiting robust genetic stability was bred, yielding a 19.8% increase in shake flask fermentation and a 26.3% increase in fed-batch fermentation compared to the starting strain. The influence of temperature on high-yield strains was examined separately through batch fermentation and fed-batch fermentation. Subsequently, based on comprehensive analysis of fermentation kinetic parameters, a two-stage temperature control strategy was proposed. Specifically, the temperature was maintained at 30 ℃ for the first 18 h to shorten the lag phase, followed by a reduction to 26 ℃ and maintaining this temperature until the end of fermentation. Under this strategy, the natamycin production reached 14.4 g·L^-1, representing a 25.2% increase compared to constant temperature fermentation at 28 ℃. This study provided an efficient production strategy for natamycin.</p>","PeriodicalId":9024,"journal":{"name":"Bioprocess and Biosystems Engineering","volume":" ","pages":"817-827"},"PeriodicalIF":3.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143655939","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":"Carbon paper anodes decorated with TiO₂ nanowires and Au nanoparticles for facilitating bacterial extracellular electron transfer.","authors":"Zhixing Qiao, Xiaoyu Dong, Tong Yang, Lichen Hu, Tao Yin","doi":"10.1007/s00449-025-03141-5","DOIUrl":"10.1007/s00449-025-03141-5","url":null,"abstract":"<p><p>Au nanoparticles-composite TiO₂ nanowires (NWs) modified carbon paper (CP) anode was synthesized via the hydrothermal method. Field emission scanning electron microscopy (FESEM) images demonstrate that the modified nanocomposite electrode features a rough and bumpy surface structure. The electrochemical activities of TiO₂-Au/CP and the control electrodes (TiO₂-NWs/CP, Au/CP, CP) for microbial fuel cell (MFC) are investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). When using TiO₂-Au/CP as a bioanode, the maximum power output density of Shewanella loihica PV-4 inoculated MFC increases by 49.7%, 26.5% and 190.6% compared with that when using TiO₂-NWs/CP, Au/CP and bare CP as bioanodes, respectively. CV analysis indicates that TiO₂-Au mediates direct and indirect electron transfer between the electrode and the bacteria, as evidenced by the appearance of redox peaks with mid-point potentials E_m of - 0.305 V and -0.465 V, respectively. The generation of bioelectricity reveals the formation of a biofilm on the electrode surface. Furthermore, compared with the control electrodes, the MFC assembled with a TiO₂-Au anode exhibits a smaller semicircle in the high-frequency region, representing a lower charge transfer resistance (R_ct). The improvement in MFC performance can be attributed to the fact that the combination of TiO₂ and Au enhances the conductivity and electrochemical activity of the electrode, along with its good biocompatibility and large specific surface area, which are favorable for bacterial colonization. Thus, TiO₂-Au/CP serves as an ideal anode material featuring simple synthesis. Additionally, its surface modifier, TiO₂-Au can be extended for the modification of other base electrodes, enabling the acquisition of high-quality anodes for MFCs.</p>","PeriodicalId":9024,"journal":{"name":"Bioprocess and Biosystems Engineering","volume":" ","pages":"761-769"},"PeriodicalIF":3.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143596175","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":"Highly efficient production of prodigiosin from corn stover hydrolysate in Serratia marcescens mutant RZ 21-6C generated by atmospheric and room‑temperature plasma mutagenesis.","authors":"Zi-Ang Zhang, Meng Tang, Jing Yang, Qi-Yin Yang, Chuan-Chao Dai, Fei Chen","doi":"10.1007/s00449-025-03144-2","DOIUrl":"10.1007/s00449-025-03144-2","url":null,"abstract":"<p><p>Prodigiosin, a natural pigment mainly produced by microorganisms, has a wide range of applications in medicine, agriculture, and environmental protection. To improve the production efficiency of prodigiosin and develop a low-cost biomass carbon source to reduce the fermentation cost, we mutated Serratia marcescens strain isolated previously in our laboratory through atmospheric and room-temperature plasma (ARTP) mutation breeding and obtained a mutant strain RZ 21-6C with a high pigment production and high genetic stability. The fermentation performance analysis of different carbon sources showed that the mutant strain not only significantly improved the conversion of conventional carbon source - sucrose, but also synthesized prodigiosin from xylose and glucose. In particular, the utilization efficiency of xylose was very high. Based on the above characteristics, low-cost biomass carbon source corn stover hydrolysate with xylose as the main component was developed for the production of prodigiosin. The highest concentration of prodigiosin in fed-batch fermentation reached 16.17 g.L^-1, with a production efficiency of 0.12 g.L^-1.h^-1, and a total sugar conversion rate of 20.21%. The transmission electron microscopy (TEM) observation of strains and of cell membrane components and permeability showed significant changes in the physiological state of the mutant strain to facilitate pigment efflux and substrate pumping. Finally, combined with the physiological data and proteomic results, the underlying mechanism of efficient prodigiosin production by RZ 21-6C was explained from the perspective of phenotypic changes, prodigiosin synthesis, membrane transport, glycogen utilization, and primary metabolism. In this study, a S. marcescens RZ 21-6C strain with excellent characteristics was obtained by modern physical mutagenesis for the biosynthesis of prodigiosin using the hydrolysate of corn stover, an agricultural waste, as a fermentation substrate, which provides an important technological support for the renewable biorefinery of prodigiosin bio-based products.</p>","PeriodicalId":9024,"journal":{"name":"Bioprocess and Biosystems Engineering","volume":" ","pages":"799-816"},"PeriodicalIF":3.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143662155","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":"Development of oyster protein-enhanced scaffolds with seven-band grouper muscle satellite cells for cultured seafood production.","authors":"Selvakumari Ulagesan, Sathish Krishnan, Taek-Jeong Nam, Youn-Hee Choi","doi":"10.1007/s00449-025-03148-y","DOIUrl":"10.1007/s00449-025-03148-y","url":null,"abstract":"<p><p>Cell-based aquaculture holds significant promise for revolutionizing the seafood industry by addressing the limitations of traditional fishing and aquaculture practices. In this study muscle cells from Seven-band grouper (SBG) Epinephelus septemfasciatus were isolated using an enzymatic digestion method. Initial cell attachment and growth were monitored, showing robust proliferation when cultured in L-15 medium supplemented with 10% fetal bovine serum (FBS) and basic fibroblast growth factor (bFGF). We assessed the biocompatibility and cytotoxicity of two protein sources, oyster protein (OP) and soy protein (SP), for their effects on grouper muscle cell viability and growth. OP demonstrated strong biocompatibility, effectively supporting cell viability and significantly promoting muscle cell proliferation. At a concentration of 10,000 µg/mL, OP increased muscle cell proliferation by up to 90% after 48 h of incubation. SP, however, exhibited dose-dependent cytotoxic effects, with reduced cell viability observed at higher concentrations (10,000 µg/mL) over 48 h. This comparative analysis indicates that OP maintains cellular health and enhances cell growth, while SP may limit cell viability at elevated concentrations. Following these findings, we prepared a scaffold using Alginate-κ-Carrageenan (Alg-κ-Car) combined with oyster protein (Alg-κ-Car-OP), which showed enhanced gelation and printability properties. 3D bioprinting of grouper muscle satellite cells (GMSC) within Alg-κ-Car-OP scaffolds resulted in higher cell viability than Alg-κ-Car scaffolds alone. Taste sensory analysis using an electronic tongue revealed distinct taste profiles, with Alg-κ-Car-OP-GMSC scaffolds exhibiting the highest umami score. Flavor analysis using flash gas chromatography and an electronic nose differentiated between scaffold types and protein samples, highlighting potential flavor markers. These findings underscore the potential of cell-based aquaculture, especially with OP-incorporated scaffolds, to meet the demand for sustainable and nutritious seafood alternatives. Further research is warranted to optimize production processes and explore commercial applications.</p>","PeriodicalId":9024,"journal":{"name":"Bioprocess and Biosystems Engineering","volume":" ","pages":"857-875"},"PeriodicalIF":3.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143690965","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":"Bioprocess biomarker identification and diagnosis for industrial mAb production based on metabolic profiling and multivariate data analysis.","authors":"Yingting Shi, Yuxiang Wan, Jiayu Yang, Yuting Lu, Xinyuan Xie, Jianyang Pan, Haibin Wang, Haibin Qu","doi":"10.1007/s00449-025-03142-4","DOIUrl":"10.1007/s00449-025-03142-4","url":null,"abstract":"<p><p>Monoclonal antibody (mAb) production is a complex bioprocess influenced by various cellular and metabolic factors. Understanding these interactions is critical for optimizing manufacturing and improving yields. In this study, we proposed a diagnostic and identification strategy using quantitative proton nuclear magnetic resonance (¹H qNMR) technology-based pharmaceutical process-omics to analyze bioprocess variability and unveil significant metabolites affecting cell growth and yield during industrial mAb manufacturing. First, batch level model (BLM) and orthogonal partial least squares-discriminant analysis (OPLS-DA) identified glucose and lactate as primary contributors to culture run variability. Maintaining an optimal glucose set point was crucial for high-yield runs. Second, a partial least squares (PLS) regression model was established, which revealed viable cell density (VCD), along with glutamine, maltose, tyrosine, citrate, methionine, and lactate, as critical variables impacting mAb yield. Finally, hierarchical clustering analysis (HCA) highlighted one-carbon metabolism metabolites, such as choline, pyroglutamate, and formate, as closely associated with VCD. These findings provide a foundation for future bioprocess optimization through cell line engineering and media formulation adjustments, ultimately enhancing mAb production efficiency.</p>","PeriodicalId":9024,"journal":{"name":"Bioprocess and Biosystems Engineering","volume":" ","pages":"771-783"},"PeriodicalIF":3.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143596174","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":"Aegle marmelos-mediated zinc oxide nanoparticles for mosquito control, cancer therapy, and antibacterial applications.","authors":"Nandhini Vasu, Thangamathi Perumal, Prabu Kumar Seetharaman, Ananth Sivapunniyam, Gnanasoundari Anthoni, Karthik Raja Ramalingam, Vahitha Rajamohan, Sharmiladevi Thangaiyan","doi":"10.1007/s00449-025-03162-0","DOIUrl":"https://doi.org/10.1007/s00449-025-03162-0","url":null,"abstract":"<p><p>The study investigated the synthesis and characterization of zinc oxide nanoparticles (Am-ZnO-NPs) using Aegle marmelos leaves and their mosquitocidal, antioxidant, and anticancer efficacy. Am-ZnO-NPs were synthesized via combustion method under optimized conditions, including 5% plant extract, temperature of 70 °C, a contact time of 60 min, and pH of 10. The resulting nanoparticles were characterized using advanced analytical techniques. The Am-CuO-NPs were hexagonal wurtzite in shape and had an average particle size of 37.44 nm. The Am-CuO-NPs are highly stable with a surface charge of - 26.77 mV. Gas chromatography-mass spectrometry (GC-MS) analysis identified several compounds with insecticidal, antioxidant, and anticancer properties, including hexadecenoic acid, ethyl ester (25.61%), 9,12-octadecadienoic acid, ethyl ester (12.64%), and 9,12,15-octadecatrienoic acid, ethyl ester (6.31%). The aqueous extract of A. marmelos (100 µg/mL) demonstrated the highest larvicidal (57.33%) and pupicidal (42.67%) activities compared to other solvent extracts. In contrast, Am-ZnO-NPs exhibited relatively higher larvicidal activity (86.67%) and pupicidal (56.00%) at 10 µg/mL. The nanoparticles also inhibited the activities of acetylcholinesterase (AChE) and carboxylesterases and reduced the total protein (TP) levels in Ae. aegypti larvae. Further, both the aqueous leaf extract and Am-ZnO-NPs were effective in scavenging DPPH free radicals, achieving 76% and 94% inhibition, respectively, at a concentration of 125 µg/mL. Am-ZnO-NPs also exhibited cytotoxicity, induced cell cycle arrest, enhanced cell adhesion, and promoted apoptosis in L-132 lung cancer cell lines. Further, the Am-ZnO-NPs display potent antibacterial activity against clinical pathogens. These results highlight the role of phytochemicals in A. marmelos leaves for their effective biological activities.</p>","PeriodicalId":9024,"journal":{"name":"Bioprocess and Biosystems Engineering","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143964246","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":"Improved productivity of recombinant adeno-associated virus (rAAV) via triple transfection of HEK293 cells using perfusion cultivation.","authors":"Jianqi Nie, Zhaojing Xu, Yang Sun, He Ren, Zichuan Song, Yan Zhang, Zhonghu Bai","doi":"10.1007/s00449-025-03167-9","DOIUrl":"https://doi.org/10.1007/s00449-025-03167-9","url":null,"abstract":"<p><p>In recent years, recombinant adeno-associated virus (rAAV) vectors are the promising viral transfer tools for gene therapy and clinical trials, thanks to their favorable safety profile and long-term transgene expression. The increasing demand for rAAVs for gene therapy led to a rise in the amount of these vectors required for pre-clinical trials, clinical trials, and approved therapeutic applications. A majority of suspension HEK293 cell-based rAAV production protocols reported rely on a triple transfection at cell density below 2 × 10⁶ cells/mL. However, the low yield of such biomanufacturing challenges bioprocess engineers to develop more efficient strategies capable of increasing volumetric productivity. In this study, we developed a perfusion bioprocess to enable rAAV production efficiently at high cell density. We first optimized three key process parameters (the total DNA amount, ratio of polyethyleneimine (PEI) to DNA, and proportion of the three plasmids) of rAAV production at cell density of 2 × 10⁶ cells/mL by the design of experiment method, from which the robust setpoint (total DNA amount of 1.37 μg/mL, ratio of PEI to DNA of 1.52 μL/μg, the proportion of plasmids pHelper 24%, pRC 46%, pGOI 30%) was explored. We then developed a rAAV production process at a cell density of ~ 8 × 10⁶ cells/mL, with increasing DNA amount on a cell basis and optimizing transfection complex preparation. This approach was confirmed in a 5 L benchtop bioreactor connected with a perfusion system, resulting in a viral genomic titer of 7.28 × 10¹¹ vg/mL and a cell-specific viral genomic titer of 4.97 × 10⁴ vg/cell. This study demonstrates that the perfusion process coupled with optimized transfection complex preparation has the potential to improve manufacturing productivity.</p>","PeriodicalId":9024,"journal":{"name":"Bioprocess and Biosystems Engineering","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143962006","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}