The enhanced surface cylindrical flask (ESCF) consists of an eight-striped inner arrangement holding 16 standard microscopic slides placed inside a cylindrical vessel. The specially designed spatula-accessible slides can be withdrawn from the vessel during cultivation without disturbing biofilm formation through an innovative window-flap accessibility mechanism. The vessel and its accessories were three-dimensional (3D) printed by applying a fused deposition modeling technique utilizing biodegradable polylactic acid. Biofilms of clinically relevant bacteria namely Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli were successfully grown in the ESCF and observed through confocal laser scanning microscopy. Advantages of the device include an enhanced surface area for biofilm formation, ease of insertion and removal of microscopic slides, convenient fitting into standard rotary shaker platforms, creation of anoxic/microaerophilic environment inside the vessel as well as the feasibility of pH, dissolved gases, and metabolite measurements in the liquid surrounding the biofilm. The ESCF will find widespread application in medical, industrial, and environmental disciplines.
{"title":"Production of an Innovative, Surface Area-Enhanced and Biodegradable Biofilm-Generating Device by 3D Printing","authors":"Atulona Datta, Rituparna Saha, Sovan Sahoo, Arup Ratan Roy, Shayontani Basu, Girish Mahajan, Subhash Chandra Panja, Joydeep Mukherjee","doi":"10.1002/elsc.202400046","DOIUrl":"https://doi.org/10.1002/elsc.202400046","url":null,"abstract":"<p>The enhanced surface cylindrical flask (ESCF) consists of an eight-striped inner arrangement holding 16 standard microscopic slides placed inside a cylindrical vessel. The specially designed spatula-accessible slides can be withdrawn from the vessel during cultivation without disturbing biofilm formation through an innovative window-flap accessibility mechanism. The vessel and its accessories were three-dimensional (3D) printed by applying a fused deposition modeling technique utilizing biodegradable polylactic acid. Biofilms of clinically relevant bacteria namely <i>Klebsiella pneumoniae</i>, <i>Pseudomonas aeruginosa</i>, <i>Staphylococcus aureus</i>, and <i>Escherichia coli</i> were successfully grown in the ESCF and observed through confocal laser scanning microscopy. Advantages of the device include an enhanced surface area for biofilm formation, ease of insertion and removal of microscopic slides, convenient fitting into standard rotary shaker platforms, creation of anoxic/microaerophilic environment inside the vessel as well as the feasibility of pH, dissolved gases, and metabolite measurements in the liquid surrounding the biofilm. The ESCF will find widespread application in medical, industrial, and environmental disciplines.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 2","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.202400046","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143481481","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Wei Long, Lina Apitius, Patrick Lenz, Felix Jakob, Anna Joёlle Ruff , Ulrich Schwaneberg
Antimicrobial peptides (AMPs) are host defense peptides that act against a broad spectrum of microorganisms. AMPs are of high interest as medicinal products, antimicrobial coatings, and for controlling biofilm formation. Applications and research of many AMPs are still hampered by insufficient titers and lack of production platforms that can tolerate high titers of AMPs. Corynebacterium glutamicum is an excellent microbial host for protein secretion and has been barely explored as a host for AMP production. Here, we report the successful production and secretion of two AMPs (amounts of up to 130 mg/L for liquid chromatography peak I [LCI] and 54 mg/L for Psoriasin) by C. glutamicum with low amounts of secreted byproducts.
{"title":"Secretory Production of Heterologous Antimicrobial Peptides in Corynebacterium glutamicum","authors":"Wei Long, Lina Apitius, Patrick Lenz, Felix Jakob, Anna Joёlle Ruff , Ulrich Schwaneberg","doi":"10.1002/elsc.70008","DOIUrl":"https://doi.org/10.1002/elsc.70008","url":null,"abstract":"<p>Antimicrobial peptides (AMPs) are host defense peptides that act against a broad spectrum of microorganisms. AMPs are of high interest as medicinal products, antimicrobial coatings, and for controlling biofilm formation. Applications and research of many AMPs are still hampered by insufficient titers and lack of production platforms that can tolerate high titers of AMPs. <i>Corynebacterium glutamicum</i> is an excellent microbial host for protein secretion and has been barely explored as a host for AMP production. Here, we report the successful production and secretion of two AMPs (amounts of up to 130 mg/L for liquid chromatography peak I [LCI] and 54 mg/L for Psoriasin) by <i>C. glutamicum</i> with low amounts of secreted byproducts.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 2","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70008","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143438751","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Rafael Machleid, Suneetha Nunna, Ajith George, Jonas Austerjost, Magda Tomala, Izabella Surowiec
Recombinant adeno-associated virus (rAAV) vector production is a complex process in which the robust cultivation of human embryonic kidney cells (HEK293) plays a critical role in generating high-quality viral vectors. Tracking the viable cell concentration (VCC) during upstream production is essential for process monitoring and for implementing actions that ensure optimal process management. The advent of inline capacitance probes has introduced a crucial process analytical technology (PAT) tool for real-time VCC measurement. Here, we present the development and application of a method for real-time monitoring of VCC in HEK293-based rAAV vector production. In a first step, BioPAT Viamass probes were used to record capacitance data of individual 10 L rAAV-8 batches within a frequency range of 50 kHz–20 MHz. Based on the capacitance data, a linear single-frequency model and an orthogonal partial least square (OPLS) multifrequency model for VCC prediction were developed. Subsequently, these models were deployed inline, and predictions were exposed into BioPAT MFCS bioprocess control software, enabling real-time VCC monitoring in subsequent rAAV-8 production batches. In addition, the continuous VCC signal was used as input for an exponential cell growth model that was deployed inline to provide accurate real-time forecasting of the transfection time point. To the best of our knowledge, this is the first example of inline deployment of VCC and Time-Till-Transfection predictive models to the bioprocess control system for real-time monitoring and forecasting of these parameters in HEK-cell-based transient rAAV vector production.
{"title":"Real-Time VCC Monitoring and Forecasting in HEK-Cell-Based rAAV Vector Production Using Capacitance Spectroscopy","authors":"Rafael Machleid, Suneetha Nunna, Ajith George, Jonas Austerjost, Magda Tomala, Izabella Surowiec","doi":"10.1002/elsc.70004","DOIUrl":"https://doi.org/10.1002/elsc.70004","url":null,"abstract":"<p>Recombinant adeno-associated virus (rAAV) vector production is a complex process in which the robust cultivation of human embryonic kidney cells (HEK293) plays a critical role in generating high-quality viral vectors. Tracking the viable cell concentration (VCC) during upstream production is essential for process monitoring and for implementing actions that ensure optimal process management. The advent of inline capacitance probes has introduced a crucial process analytical technology (PAT) tool for real-time VCC measurement. Here, we present the development and application of a method for real-time monitoring of VCC in HEK293-based rAAV vector production. In a first step, BioPAT Viamass probes were used to record capacitance data of individual 10 L rAAV-8 batches within a frequency range of 50 kHz–20 MHz. Based on the capacitance data, a linear single-frequency model and an orthogonal partial least square (OPLS) multifrequency model for VCC prediction were developed. Subsequently, these models were deployed inline, and predictions were exposed into BioPAT MFCS bioprocess control software, enabling real-time VCC monitoring in subsequent rAAV-8 production batches. In addition, the continuous VCC signal was used as input for an exponential cell growth model that was deployed inline to provide accurate real-time forecasting of the transfection time point. To the best of our knowledge, this is the first example of inline deployment of VCC and Time-Till-Transfection predictive models to the bioprocess control system for real-time monitoring and forecasting of these parameters in HEK-cell-based transient rAAV vector production.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 2","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70004","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143362476","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}