Biotechnology and Bioengineering最新文献

{"title":"AtLEC2-Mediated Enhancement of Endoplasmic Reticulum-Targeted Foreign Protein Synthesis in Nicotiana benthamiana Leaves: Insights From Transcriptomic Analysis.","authors":"Carolina G Ocampo, Florencia Vignolles, Marina A Pombo, Maria Laura Colombo, Hernan G Rosli, Silvana Petruccelli","doi":"10.1002/bit.28893","DOIUrl":"https://doi.org/10.1002/bit.28893","url":null,"abstract":"<p><p>Many proteins used in industrial and pharmaceutical applications are typically synthesized within the secretory pathway. While yeast and mammalian cells have been engineered to enhance the production of endomembrane-targeted proteins, similar strategies in plant cells remain underexplored. This study investigates the potential of arabidopsis leafy cotyledon 2 (AtLEC2), a key regulator of seed development, to enhance the production of proteins targeted to the endoplasmic reticulum (ER) in Nicotiana benthamiana leaves. Through transient expression experiments, we demonstrate that AtLEC2 selectively increases the production of ER-targeted GUS without affecting its cytosolic variant. Moreover, leaves agroinfiltrated with AtLEC2 show a significant increase in ER-GFP accumulation compared to controls lacking AtLEC2. Transcriptomic analysis reveals that AtLEC2 promotes ribosome and chloroplast biogenesis, along with the upregulation of genes involved in photosynthesis, translation, and membrane synthesis. Notably, seed-specific poly(A) binding proteins involved in RNA stability and translation initiation, as well as 3-hydroxy-3-methylglutaryl coenzyme A reductase-linked to ER hypertrophy-are highly upregulated. This study establishes a novel connection between AtLEC2 and the enhancement of ER-targeted foreign protein synthesis, paving the way for innovative strategies in plant cellular engineering.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142726180","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":"Deciphering the Catalytic Proficiency and Mechanism of the N-Acetylglucosamine Deacetylase From Pantoea dispersa.","authors":"Wentao Yang, Xiao Chen, Li Pang, Hong Tian, Liang Yang, Bo Xia","doi":"10.1002/bit.28894","DOIUrl":"https://doi.org/10.1002/bit.28894","url":null,"abstract":"<p><p>Glucosamine (GlcN) is a widely utilized amino monosaccharide. It is traditionally synthesized from N-acetylglucosamine (GlcNAc) via chemical processes that pose environmental threats. In pursuit of a greener alternative, our investigation explored biocatalysis with a Pantoea dispersa derived deacetylase (Pd-nagA), showcasing its efficacy as a catalyst in GlcN production. As a result, this work provides a comprehensive characterization of Pd-nagA, scrutinizes its enzymatic behavior, and delves into the deacetylation mechanism in detail. Heterologous expression methods were utilized for the production and isolation of Pd-nagA, followed by a kinetic evaluation highlighting its enzymatic activity. The complex interactions between the enzyme and its substrate were investigated by integrating classical molecular dynamics, quantum mechanics/molecular mechanics simulations, funnel metadynamics, and on-the-fly probability enhanced sampling techniques, thereby elucidating the precise deacetylation pathway. Rigorous computational analysis results demonstrated that Pd-nagA exhibited promising specificity and efficiency for GlcNAc with a high turnover rate. The catalytic residues central to the reaction were identified, and the underlying quantum reaction mechanism was detailed. Our findings suggest an approach to GlcN production using eco-friendly biocatalysis, positioning Pd-nagA at the forefront of industrial application not only because of its remarkable catalytic capabilities but also due to its potential for enzyme optimization.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142726183","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":"Development of a Microphysiological System to Model Human Cancer Metastasis From the Colon to the Liver","authors":"Paula G. Miller, Emina Huang, Robert Fisher, Michael L. Shuler","doi":"10.1002/bit.28890","DOIUrl":"https://doi.org/10.1002/bit.28890","url":null,"abstract":"We describe a novel device to mimic the metastasis of cancer cells from the colon into the liver in a human model. The colon mimic is connected to the liver model by a gravity-driven recirculating unidirectional flow of a blood surrogate and can mimic the five steps of the metastatic cascade: invasion in the colon, intravasation into the bloodstream, systemic transportation, extravasation into the liver, and colonization in the liver. The colon mimic uses established normal colon epithelial organoid cells (NL) and human umbilical vein endothelial cells (HUVEC) plated on opposite sides of a membrane. To better mimic the colon structure the NL side of the membrane is exposed to air to establish an air-liquid interface. The liver mimic consists of human liver sinusoidal endothelial cells (HHSEC) and epithelial hepatic cells (HepG2 C3A) plated in Matrigel on opposite sides of a membrane. Labeled colorectal cancer cells/clusters (CA) from organoids are introduced into an established normal colon epithelial cell (NL) layer from the same patient before assembly of the system or alternatively NL organoids and fluorescently labeled CA organoids from the same patient were prepared as a ratio of 10:1 NL:CA and established together before assembly of the system. Cell viability is greater than 85% in this system. We demonstrate that over 5 days of operation that the five steps of the metastatic cascade are replicated. This novel device allows an in vitro estimate of metastatic capability (as measured by using percentages of the labeled areas per device through ImageJ) in response to selected variables. In this study, the metastatic capability depends on the source of cancer cells (e.g., the patient), the clumping of cancer cells, glucose concentration, and oxygen levels (hypoxia). For the first time, this new in vitro system mimics all five steps of the metastatic cascade in a single device and provides a new device to probe and observe the process of metastasis in a human-based model in only 5 days. The rapid observation is due to the use of a high concentration of cancer cells in the colon (e.g. 10%) and the absence of the immune system. Our device makes it possible to probe aspects of each step of metastasis and interactions between steps.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"16 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142713357","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":"Sequence and Configuration of a Novel Bispecific Antibody Format Impacts Its Production Using Chinese Hamster Ovary (CHO) Cells","authors":"Hirra Hussain, Angelica M. S. Ozanne, Tulshi Patel, Davide Vito, Mark Ellis, Matthew Hinchliffe, David P. Humphreys, Paul E. Stephens, Bernie Sweeney, James White, Alan J. Dickson, C. Mark Smales","doi":"10.1002/bit.28879","DOIUrl":"https://doi.org/10.1002/bit.28879","url":null,"abstract":"There are a number of new format antibody-inspired molecules with multiple antigen binding capabilities in development and clinical evaluation. Here, we describe the impact of the sequence and configuration of a unique bispecific antibody format (termed BYbe) using a panel of four BYbe's and the three IgG1s from which they were derived on their production in a Chinese hamster ovary (CHO) cell expression system. Following transfection and selection, one bispecific antibody format yielded fewer mini-pools in comparison to the other bispecific cell pools. When the top 12 expressing stable mini-pools of all BYbe configurations and sequences were evaluated, both the dsscFv sequence and antibody chain configuration or placement directly impacted productivity. The cell-specific productivity (qP, pg/cell/day) was lower in all BYbe cell pools compared to the IgG1 cell lines. However, when the actual molecules/cell/day produced were considered, three of the four bispecific cell pools outproduced the parental IgG1 cell pools. While gene copy number did not correlate to productivity, mRNA analysis showed that for specific BYbe formats there was a strong correlation with productivity. In summary, we describe how bispecific antibody format configuration impacts the cell line construction process and yield of product from CHO cells.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"24 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142713104","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":"Expression of Viral DNA Polymerase in Synthetic Recombinant Adeno‐Associated Virus Producer Cell Line Enhances Full Particle Productivity","authors":"Yu‐Chieh Lin, Han‐Jung Kuo, Min Lu, Carissa Rungkittikhun, Wei‐Shou Hu","doi":"10.1002/bit.28885","DOIUrl":"https://doi.org/10.1002/bit.28885","url":null,"abstract":"Recombinant adeno‐associated virus (rAAV) is a widely used viral vector in gene therapy. To meet the growing clinical demand, a scalable production technology which can efficiently produce high‐quality products is required. We have developed a synthetic biology strategy to generate HEK293‐based cell lines which have integrated essential AAV and adenoviral helper genes and are capable of producing rAAV upon induction. One such cell line, GX6B, produced up to 10<jats:sup>6</jats:sup> capsids per cell, but only a much lower level of rAAV genomes. The low AAV genome titer limited its rAAV productivity and increased empty viral particle content. To boost AAV genome amplification, the coding sequence of the DNA polymerase complex (UL30/UL42) from helper Herpes Simplex Virus type 1 (HSV‐1) was placed under an inducible promoter control and integrated into GX6B genome at a relatively low level. The resulting clones produced significantly higher titer of viral genomes, while their capsid level was unaffected. As a result, the encapsidated rAAV2 titer and the full particle content were significantly increased. We further demonstrated that this strategy of expressing HSV‐1 DNA polymerase to increase full particle productivity could be implemented in a synthetic cell line producing another serotype rAAV8.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"255 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2024-11-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142690709","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":"Advances in Artificially Designed Antibacterial Active Antimicrobial Peptides.","authors":"Ying Guo, Muhammad Haris Raza Farhan, Fei Gan, Xiaohan Yang, Yuxin Li, Lingli Huang, Xu Wang, Guyue Cheng","doi":"10.1002/bit.28886","DOIUrl":"https://doi.org/10.1002/bit.28886","url":null,"abstract":"<p><p>Antibacterial resistance has emerged as a significant global concern, necessitating the urgent development of new antibacterial drugs. Antimicrobial peptides (AMPs) are naturally occurring peptides found in various organisms. Coupled with a wide range of antibacterial activity, AMPs are less likely to develop drug resistance and can act as potential agents for treating bacterial infections. However, their characteristics, such as low activity, instability, and toxicity, hinder their clinical application. Consequently, researchers are inclined towards artificial design and optimization based on natural AMPs. This review discusses the research advancements in the field of artificial designing and optimization of various AMPs. Moreover, it highlights various strategies for designing such peptides, aiming to provide valuable insights for developing novel AMPs.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142685988","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":"Streamlined Clarification and Capture Process for Monoclonal Antibodies Using Fluidized Bed Centrifugation and Multi-Column Chromatography With Membrane Adsorbers.","authors":"Fabian Schmitz, Martin Saballus, Thomas Kruse, Mirjana Minceva, Markus Kampmann","doi":"10.1002/bit.28884","DOIUrl":"10.1002/bit.28884","url":null,"abstract":"<p><p>Harmonizing unit operations in the downstream process of monoclonal antibodies (mAbs) has a high potential to overcome throughput limitations and reduce manufacturing costs. This study proposes a streamlined clarification and capture (S-CC) process concept for the continuous processing of cell broth harvested from a connected bioreactor. The process was realized with a fluidized bed centrifuge connected to depth and sterile filters, a surge tank, and a multi-column chromatography (MCC) unit. The MCC unit was operated in the rapid cycling simulated moving bed (RC-BioSMB) mode with five convective diffusive membrane adsorbers (MAs). A control strategy and the surge tank were used to adjust the loading flow rate of the MCC unit. The mAb was recovered with a total process yield of 90%, with high removal of the process-related impurities HCP (2.1 LRV) and DNA (2.9 LRV). Moreover, the S-CC process productivity of 4.2 g h^- ¹ was up to 5.3 times higher than for comparable, hypothetical batch MA processes. In addition, the buffer consumption of the capture step could be reduced from 2.0 L g^- ¹ in batch mode to 1.2 L g^- ¹ in the RC-BioSMB mode. These results demonstrate the high potential of streamlined interconnected unit operations to improve the overall mAb downstream process performance.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2024-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142667065","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":"Regeneration of Spent Culture Media for Sustainable and Continuous mAb Production via Ion Concentration Polarization.","authors":"Eric Wynne, Junghyo Yoon, Dohyun Park, Mingyang Cui, Caitlin Morris, Jaeweon Lee, Zhao Wang, Seongkyu Yoon, Jongyoon Han","doi":"10.1002/bit.28888","DOIUrl":"10.1002/bit.28888","url":null,"abstract":"<p><p>In modern bioprocessing, cell culture media is one of the most significant cost drivers, yet the nutrients and other critical factors in the media are often not fully utilized. With the renewed emphasis on reducing the cost of bioprocessing, there is much interest in reducing the overall use of cell culture media. In this work, we introduce a mesoscale microfluidic separation device based on the ion concentration polarization (ICP) process to regenerate the spent media for reuse by removing critical waste products from the cell culture that are known to inhibit the growth of the cells. We demonstrated that up to 75% of spent culture media can be regenerated and reused without affecting the cell viability. A detailed analysis of the materials consumed during antibody production indicated that one could improve the water process mass intensity by up to 33% by regenerating and recycling the media. Given that ICP separation systems have already been scaled up to support large-volume processing, it would be feasible to deploy this technology for manufacturing scale bioreactors (e.g., 50 L perfusion culture of CHO cells), reducing the overall operation cost and water use.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2024-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142667064","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":"Adaptation of Aglycosylated Monoclonal Antibodies for Improved Production in Komagataella phaffii.","authors":"Yuchen Yang, Neil C Dalvie, Joseph R Brady, Christopher A Naranjo, Timothy Lorgeree, Sergio A Rodriguez-Aponte, Ryan S Johnston, Mary K Tracey, Carmen M Elenberger, Eric Lee, Mark Tié, Kerry R Love, J Christopher Love","doi":"10.1002/bit.28878","DOIUrl":"10.1002/bit.28878","url":null,"abstract":"<p><p>Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals manufactured by well-established processes using Chinese Hamster Ovary (CHO) cells. Next-generation biomanufacturing using alternative hosts like Komagataella phaffii could improve the accessibility of these medicines, address broad societal goals for sustainability, and offer financial advantages for accelerated development of new products. Antibodies produced by K. phaffii, however, may manifest unique molecular quality attributes, like host-dependent, product-related variants, that could raise potential concerns for clinical use. We demonstrate here conservative modifications to the amino acid sequence of aglycosylated antibodies based on the human IgG1 isotype that minimize product-related variations when secreted by K. phaffii. A combination of 2-3 changes of amino acids reduced variations across six different aglycosylated versions of commercial mAbs. Expression of a modified sequence of NIST mAb in both K. phaffii and CHO cells showed comparable biophysical properties and molecular variations. These results suggest a path toward the production of high-quality mAbs that could be expressed interchangeably by either yeast or mammalian cells. Improving molecular designs of proteins to enable a range of manufacturing strategies for well-characterized biopharmaceuticals could accelerate global accessibility and innovations.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2024-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142614860","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":"Perfusion Process Intensification for Lentivirus Production Using a Novel Scale-Down Model","authors":"Maximilian Klimpel, Beatrice Pflüger-Müller, Marta Arrizabalaga Cascallana, Sarah Schwingal, Nikki Indresh Lal, Thomas Noll, Vicky Pirzas, Holger Laux","doi":"10.1002/bit.28880","DOIUrl":"https://doi.org/10.1002/bit.28880","url":null,"abstract":"Process intensification has become an important strategy to lower production costs and increase manufacturing capacities for biopharmaceutical products. In particular for the production of viral vectors like lentiviruses (LVs), the transition from (fed-)batch to perfusion processes is a key strategy to meet the increasing demands for cell and gene therapy applications. However, perfusion processes are associated with higher medium consumption. Therefore, it is necessary to develop appropriate small-scale models to reduce development costs. In this work, we present the use of the acoustic wave separation technology in combination with the Ambr 250 high throughput bioreactor system for intensified perfusion process development using stable LV producer cells. The intensified perfusion process developed in the Ambr 250 model, performed at a harvest rate of 3 vessel volumes per day (VVD) and high cell densities, resulted in a 1.4-fold higher cell-specific functional virus yield and 2.8-fold higher volumetric virus yield compared to the control process at a harvest rate of 1 VVD. The findings were verified at bench scale after optimizing the bioreactor set-up, resulting in a 1.4-fold higher cell-specific functional virus yield and 3.1-fold higher volumetric virus yield.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"39 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142602027","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}