David Vonester, Kyra Hoffmann, Thomas Palmen, Lena Regestein, Ulrike Richter, Anna-Lena Altenhoff, Maximilian Hoffmann, Yulia Radeva, Jochen Büchs, Jørgen Barsett Magnus
Foam formation in stirred tank fermentation processes is a well-studied phenomenon. However, foaming in shake flask cultivations is rarely considered. Non-baffled shake flasks, in particular, are generally considered to prevent foaming problems. However, under certain process conditions, foaming in non-baffled shake flasks can occur. In this study, phenomena of foam formation in shake flasks, their impact on the maximum oxygen transfer capacity (OTRmax), and experimental reproducibility are investigated. It is shown that foaming events in shake flasks can increase the OTRmax by up to threefold. This enhanced OTRmax alters process conditions and, thereby, affects the reproducibility of experiments. Foaming in shake flasks can be induced by elements such as conventional baffles or sensor spots that are used for online measurement. Moreover, a connection between the out-of-phase phenomenon and foam formation was discovered in non-baffled shake flasks. This is especially important when cultivating microorganisms at elevated viscosities. Hence, foaming in shake flasks should be considered as significantly altering process conditions, compared to non-foaming cultures. Ensuring in-phase cultivation conditions and unhindered liquid flow in shake flasks may help to avoid foaming.
Practical application: This work provides insights into foam formation in non-baffled shake flasks and its resulting implications. Foaming can increase the maximum oxygen transfer capacity and, thus, affect process conditions. The reproducibility can be severely reduced, and a comparison between foaming and non-foaming cultivations is only possible to a limited extent. Foaming can be induced by baffles or internals, such as small sensor spots, used for online monitoring. Additionally, foaming can be caused by the out-of-phase phenomenon. This is of particular importance when cultivating microorganisms at elevated viscosities. This paper is intended to raise awareness of the topic of foam formation in the shake flask and help to correctly interpret this phenomenon.
{"title":"Foam Formation in Shake Flasks and Its Consequences","authors":"David Vonester, Kyra Hoffmann, Thomas Palmen, Lena Regestein, Ulrike Richter, Anna-Lena Altenhoff, Maximilian Hoffmann, Yulia Radeva, Jochen Büchs, Jørgen Barsett Magnus","doi":"10.1002/elsc.70057","DOIUrl":"10.1002/elsc.70057","url":null,"abstract":"<p>Foam formation in stirred tank fermentation processes is a well-studied phenomenon. However, foaming in shake flask cultivations is rarely considered. Non-baffled shake flasks, in particular, are generally considered to prevent foaming problems. However, under certain process conditions, foaming in non-baffled shake flasks can occur. In this study, phenomena of foam formation in shake flasks, their impact on the maximum oxygen transfer capacity (OTR<sub>max</sub>), and experimental reproducibility are investigated. It is shown that foaming events in shake flasks can increase the OTR<sub>max</sub> by up to threefold. This enhanced OTR<sub>max</sub> alters process conditions and, thereby, affects the reproducibility of experiments. Foaming in shake flasks can be induced by elements such as conventional baffles or sensor spots that are used for online measurement. Moreover, a connection between the out-of-phase phenomenon and foam formation was discovered in non-baffled shake flasks. This is especially important when cultivating microorganisms at elevated viscosities. Hence, foaming in shake flasks should be considered as significantly altering process conditions, compared to non-foaming cultures. Ensuring in-phase cultivation conditions and unhindered liquid flow in shake flasks may help to avoid foaming.</p><p><i>Practical application:</i> This work provides insights into foam formation in non-baffled shake flasks and its resulting implications. Foaming can increase the maximum oxygen transfer capacity and, thus, affect process conditions. The reproducibility can be severely reduced, and a comparison between foaming and non-foaming cultivations is only possible to a limited extent. Foaming can be induced by baffles or internals, such as small sensor spots, used for online monitoring. Additionally, foaming can be caused by the out-of-phase phenomenon. This is of particular importance when cultivating microorganisms at elevated viscosities. This paper is intended to raise awareness of the topic of foam formation in the shake flask and help to correctly interpret this phenomenon.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 10","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12541551/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145354080","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}
Fixed-bed bioreactors for anchorage-dependent cells are an obvious choice for development because of their large-scale capabilities, allowing manufacturing with reduced cost and footprint. In this study, a serum-free production process for Japanese encephalitis virus (JEV) in a single-use fixed-bed bioreactor was developed and compared to conventional roller bottle production as a productivity benchmark. After optimization of serum-free cell culture conditions, an initial media screening in roller bottles showed a strong impact of growth and production media on virus yields. Selected optimized medium combinations were assessed in roller bottles and the fixed-bed bioreactor. Both systems proved to be excellent production systems for JEV, but media choice was key to achieve the highest titers. In particular, DMEM with its enriched glucose content beneficially affected viral yields, enabling potential large-scale manufacturing using the fixed-bed reactor with serum-containing or serum-free media.
Practical application: Data presented in this work show feasible ways of serum-free virus production with Vero cells, a common cell substrate in vaccine development. The fixed-bed bioreactor process described here could facilitate manufacturing activities to reduce cost and footprint while simultaneously achieving higher process control compared to conventional manufacturing systems like roller bottles. With a much better upscale potential (up to 500 m2) the fixed-bed bioreactor showed comparable or better yields to roller bottles depending on media used, even with serum-free media. This research article further emphasizes the need to optimize cell culture media or media combinations for each virus individually to achieve the highest titers. As shown, performing a simple media screening experiment to optimize yields early in process development could lead to better productivity, with a high business impact in later development stages.
{"title":"Serum-Free Japanese Encephalitis Virus Production in a Single-Use Fixed-Bed Bioreactor","authors":"Marco Kress, Robert Schlegl, Alois Jungbauer","doi":"10.1002/elsc.70052","DOIUrl":"10.1002/elsc.70052","url":null,"abstract":"<p>Fixed-bed bioreactors for anchorage-dependent cells are an obvious choice for development because of their large-scale capabilities, allowing manufacturing with reduced cost and footprint. In this study, a serum-free production process for Japanese encephalitis virus (JEV) in a single-use fixed-bed bioreactor was developed and compared to conventional roller bottle production as a productivity benchmark. After optimization of serum-free cell culture conditions, an initial media screening in roller bottles showed a strong impact of growth and production media on virus yields. Selected optimized medium combinations were assessed in roller bottles and the fixed-bed bioreactor. Both systems proved to be excellent production systems for JEV, but media choice was key to achieve the highest titers. In particular, DMEM with its enriched glucose content beneficially affected viral yields, enabling potential large-scale manufacturing using the fixed-bed reactor with serum-containing or serum-free media.</p><p><i>Practical application:</i> Data presented in this work show feasible ways of serum-free virus production with Vero cells, a common cell substrate in vaccine development. The fixed-bed bioreactor process described here could facilitate manufacturing activities to reduce cost and footprint while simultaneously achieving higher process control compared to conventional manufacturing systems like roller bottles. With a much better upscale potential (up to 500 m<sup>2</sup>) the fixed-bed bioreactor showed comparable or better yields to roller bottles depending on media used, even with serum-free media. This research article further emphasizes the need to optimize cell culture media or media combinations for each virus individually to achieve the highest titers. As shown, performing a simple media screening experiment to optimize yields early in process development could lead to better productivity, with a high business impact in later development stages.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 10","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12541546/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145354054","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}
Kira Müntjes, Lesley Plücker, Magnus Philipp, Paul Richter, Marcel Mann, Michael Feldbrügge, Kerstin Schipper
<div> <section> <p>Single cell oils produced in microorganisms constitute appealing alternatives to plant oils. Oleaginous fungi accumulate triacylglycerols in lipid droplets (LD). Their biosynthesis is typically induced under nitrogen limitation. We exploit the fungal model <i>Ustilago maydis</i> for oil production. The stain 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) can be used to track LD formation during cultivation but this expensive compound is only affordable at small-scale. Therefore, mutant screening for optimization of oil production and composition would benefit from an inexpensive online monitoring system. Accordingly, we aimed at developing an intrinsic reporter that is suitable to track oil formation even in larger cultures. From three tested candidates, the potential delta(24)-sterol C-methyltransferase Erg6 turned out to be the best reporter. Fluorescence microscopy confirmed its localization at the LD membrane. After optimization, Erg6 fused to mKate2, expressed from a promoter derived from glycolipid biosynthesis, showed a good correlation of fluorescence with oil accumulation. Time course experiments in micro-cultivators demonstrated that the fluorescence read-out can be used to track oil formation starting at the onset of nitrogen limitation to approximate the LD amount. In essence, our study introduces a biosensor for oil monitoring that can easily be transferred to other oleaginous yeasts.</p> </section> <section> <h3> Summary</h3> <div> <ul> <li> <p>Microbial oils are promising, environmentally friendly alternatives to plant oils and have the potential of a huge market share once competitive production and isolation processes are accomplished.</p> </li> <li> <p>Online monitoring is key to efficient engineering of single cell oil producing microorganisms and bioprocess optimization in order to achieve competitive products.</p> </li> <li> <p>Here, we present an inexpensive, fluorescence-based reporter that can be used to track the approximate oil accumulation of microbial cultures in vivo. This omits the use of expensive dyes or offline methodology with a high workload.</p> </li> <li> <p>While we established the biosensor in the yeast form of the fungal microorganism <i>U. maydis</i>, the evolutionary conservation of the underlying protein Erg6 will allow for a straightforward transfer of the methodology to other oleaginous yeasts.</p> </li> <
{"title":"In Vivo Online Monitoring of Intracellular Lipid Accumulation in Ustilago maydis","authors":"Kira Müntjes, Lesley Plücker, Magnus Philipp, Paul Richter, Marcel Mann, Michael Feldbrügge, Kerstin Schipper","doi":"10.1002/elsc.70053","DOIUrl":"10.1002/elsc.70053","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <p>Single cell oils produced in microorganisms constitute appealing alternatives to plant oils. Oleaginous fungi accumulate triacylglycerols in lipid droplets (LD). Their biosynthesis is typically induced under nitrogen limitation. We exploit the fungal model <i>Ustilago maydis</i> for oil production. The stain 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) can be used to track LD formation during cultivation but this expensive compound is only affordable at small-scale. Therefore, mutant screening for optimization of oil production and composition would benefit from an inexpensive online monitoring system. Accordingly, we aimed at developing an intrinsic reporter that is suitable to track oil formation even in larger cultures. From three tested candidates, the potential delta(24)-sterol C-methyltransferase Erg6 turned out to be the best reporter. Fluorescence microscopy confirmed its localization at the LD membrane. After optimization, Erg6 fused to mKate2, expressed from a promoter derived from glycolipid biosynthesis, showed a good correlation of fluorescence with oil accumulation. Time course experiments in micro-cultivators demonstrated that the fluorescence read-out can be used to track oil formation starting at the onset of nitrogen limitation to approximate the LD amount. In essence, our study introduces a biosensor for oil monitoring that can easily be transferred to other oleaginous yeasts.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Summary</h3>\u0000 \u0000 <div>\u0000 <ul>\u0000 \u0000 <li>\u0000 <p>Microbial oils are promising, environmentally friendly alternatives to plant oils and have the potential of a huge market share once competitive production and isolation processes are accomplished.</p>\u0000 </li>\u0000 \u0000 <li>\u0000 <p>Online monitoring is key to efficient engineering of single cell oil producing microorganisms and bioprocess optimization in order to achieve competitive products.</p>\u0000 </li>\u0000 \u0000 <li>\u0000 <p>Here, we present an inexpensive, fluorescence-based reporter that can be used to track the approximate oil accumulation of microbial cultures in vivo. This omits the use of expensive dyes or offline methodology with a high workload.</p>\u0000 </li>\u0000 \u0000 <li>\u0000 <p>While we established the biosensor in the yeast form of the fungal microorganism <i>U. maydis</i>, the evolutionary conservation of the underlying protein Erg6 will allow for a straightforward transfer of the methodology to other oleaginous yeasts.</p>\u0000 </li>\u0000 <","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 10","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12541552/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145354062","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}
Jesper W. Jensen, Pablo I. Nikel, John M. Woodley, Helena Junicke
The broad adoption of the obligate aerobe Pseudomonas putida in industrial-scale production requires a good understanding of the effect of changing oxygen availability due to the dissolved oxygen (DO) gradients apparent at such a scale. To that end, both wild-type P. putida KT2440 and a genome-reduced derivative (strain SEM10) were subjected to different oxygen partial pressures (pO2) in the aeration gas to evaluate the effect of low oxygen availability on growth characteristics in batch mode. Strain SEM10 consistently achieved a 12.7% higher biomass yield on glucose than the wild-type strain during non-DO limited growth, suggesting that genome reduction had no adverse effects on the overall growth properties. Furthermore, when exposed to oxygen depletion in cultivations at low pO2 (0.0525 atm), strain SEM10 kept a similar biomass yield and maximum specific growth rate. In fact, the genome-reduced strain significantly outcompeted the wild-type strain under these conditions. SEM10 achieved 23.3% and 35.5% higher biomass yields on glucose and oxygen, respectively, compared to strain KT2440 at low pO2. These findings indicate that the genome-reduced strain, SEM10, could endure oxygen depletion during growth and even outcompete the wild-type strain under these conditions, highlighting the advantages of using streamlined strains as a platform for industrial bioprocesses.
Summary
An energy-efficient, genome-reduced strain of the Pseudomonas putida KT2440 has the potential to increase yields and rates in biochemical production.
A lower energy consumption for futile processes, such as flagellar, allows allocation of this energy for product synthesis or survival. The latter being of importance when the strain is applied for the production of harsh biochemicals or intermediates.
These attributes are of no use unless the obligate aerobe P. putida can tolerate scarce oxygen supplies that may occur in large-scale cultivations.
Our findings suggest that the genome-reduced strain performs equally well under oxygen-limited and non-limited conditions and even outcompetes the wildtype under oxygen-limited conditions.
{"title":"Genome-Reduced Pseudomonas putida Outcompetes the Wild-Type Strain Upon Oxygen Depletion","authors":"Jesper W. Jensen, Pablo I. Nikel, John M. Woodley, Helena Junicke","doi":"10.1002/elsc.70046","DOIUrl":"https://doi.org/10.1002/elsc.70046","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <p>The broad adoption of the obligate aerobe <i>Pseudomonas putida</i> in industrial-scale production requires a good understanding of the effect of changing oxygen availability due to the dissolved oxygen (DO) gradients apparent at such a scale. To that end, both wild-type <i>P. putida</i> KT2440 and a genome-reduced derivative (strain SEM10) were subjected to different oxygen partial pressures (pO<sub>2</sub>) in the aeration gas to evaluate the effect of low oxygen availability on growth characteristics in batch mode. Strain SEM10 consistently achieved a 12.7% higher biomass yield on glucose than the wild-type strain during non-DO limited growth, suggesting that genome reduction had no adverse effects on the overall growth properties. Furthermore, when exposed to oxygen depletion in cultivations at low pO<sub>2</sub> (0.0525 atm), strain SEM10 kept a similar biomass yield and maximum specific growth rate. In fact, the genome-reduced strain significantly outcompeted the wild-type strain under these conditions. SEM10 achieved 23.3% and 35.5% higher biomass yields on glucose and oxygen, respectively, compared to strain KT2440 at low pO<sub>2</sub>. These findings indicate that the genome-reduced strain, SEM10, could endure oxygen depletion during growth and even outcompete the wild-type strain under these conditions, highlighting the advantages of using streamlined strains as a platform for industrial bioprocesses.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Summary</h3>\u0000 \u0000 <div>\u0000 <ul>\u0000 \u0000 <li>\u0000 <p>An energy-efficient, genome-reduced strain of the <i>Pseudomonas putida</i> KT2440 has the potential to increase yields and rates in biochemical production.</p>\u0000 </li>\u0000 \u0000 <li>\u0000 <p>A lower energy consumption for futile processes, such as flagellar, allows allocation of this energy for product synthesis or survival. The latter being of importance when the strain is applied for the production of harsh biochemicals or intermediates.</p>\u0000 </li>\u0000 \u0000 <li>\u0000 <p>These attributes are of no use unless the obligate aerobe <i>P. putida</i> can tolerate scarce oxygen supplies that may occur in large-scale cultivations.</p>\u0000 </li>\u0000 \u0000 <li>\u0000 <p>Our findings suggest that the genome-reduced strain performs equally well under oxygen-limited and non-limited conditions and even outcompetes the wildtype under oxygen-limited conditions.</p>\u0000 </li>\u0000 \u0000 <li>\u0000 <p>This highlights, for the first time, the potentia","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 10","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70046","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145317509","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}
Mariia Abyzova, Lasse Schoppe, Marline Kirsch, Martin Muuß, Sina Zargarchi, Jordi Morales-Dalmau, Tuba Esatbeyoglu, Ulrich Krings, Antonina Lavrentieva
The research field of cellular agriculture has developed rapidly in recent years. Despite many successes, there is an urgent need for innovative methods to culture adherent cells. Edible scaffolds offer a promising solution for anchorage-dependent cells from agriculturally relevant species. In this study, we present a novel approach using plant-based scaffolds for the production of cultivated fat. Our findings indicate that coating of electrospun-derived plant-based scaffolds with poly-L-lysine significantly enhances cell adhesion and proliferation, offering a more cost-effective alternative to coating with extracellular matrix (ECM) components. Furthermore, we investigated the influence of various adipogenic media formulations on the fatty acid composition of the cultivated fat. Notably, the incorporation of intralipid significantly changed the lipid profile, leading to an increased proportion of stearic acid with a simultaneous reduction in the proportions of oleic, linoleic, and alpha-linolenic acid. This modulation allows for the customization of lipid profiles to satisfy diverse user requirements. However, our analysis showed that both types of matrices and the basal media formulations exerted only moderate to negligible effects on the overall fatty acid composition of the cultivated fat.
Practical application: In this study, we evaluated the impact of cold plasma and coating treatments on plant-based scaffold materials to improve porcine mesenchymal stem cell adhesion and growth. Additionally, the influence of different basal media formulations and the addition of intralipid on the fatty acid composition of the cultivated fat accumulated in differentiated adipocytes were examined. Our results provide valuable insights into how these variables can be adjusted to influence the fatty acid profile of differentiated cells, to meet the requirements of customers with variable nutritional and functional needs. Discovered findings can be used for further development of sustainable alternatives within the food technology sector.
{"title":"Comparative Study of Porcine Mesenchymal Stem Cells Behavior and Lipid Metabolism on Plant-Based Scaffolds and Two-Dimensional Systems for Cultivated Fat","authors":"Mariia Abyzova, Lasse Schoppe, Marline Kirsch, Martin Muuß, Sina Zargarchi, Jordi Morales-Dalmau, Tuba Esatbeyoglu, Ulrich Krings, Antonina Lavrentieva","doi":"10.1002/elsc.70050","DOIUrl":"10.1002/elsc.70050","url":null,"abstract":"<p>The research field of cellular agriculture has developed rapidly in recent years. Despite many successes, there is an urgent need for innovative methods to culture adherent cells. Edible scaffolds offer a promising solution for anchorage-dependent cells from agriculturally relevant species. In this study, we present a novel approach using plant-based scaffolds for the production of cultivated fat. Our findings indicate that coating of electrospun-derived plant-based scaffolds with poly-L-lysine significantly enhances cell adhesion and proliferation, offering a more cost-effective alternative to coating with extracellular matrix (ECM) components. Furthermore, we investigated the influence of various adipogenic media formulations on the fatty acid composition of the cultivated fat. Notably, the incorporation of intralipid significantly changed the lipid profile, leading to an increased proportion of stearic acid with a simultaneous reduction in the proportions of oleic, linoleic, and alpha-linolenic acid. This modulation allows for the customization of lipid profiles to satisfy diverse user requirements. However, our analysis showed that both types of matrices and the basal media formulations exerted only moderate to negligible effects on the overall fatty acid composition of the cultivated fat.</p><p><b><i>Practical application</i></b>: In this study, we evaluated the impact of cold plasma and coating treatments on plant-based scaffold materials to improve porcine mesenchymal stem cell adhesion and growth. Additionally, the influence of different basal media formulations and the addition of intralipid on the fatty acid composition of the cultivated fat accumulated in differentiated adipocytes were examined. Our results provide valuable insights into how these variables can be adjusted to influence the fatty acid profile of differentiated cells, to meet the requirements of customers with variable nutritional and functional needs. Discovered findings can be used for further development of sustainable alternatives within the food technology sector.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 10","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12518025/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145298795","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}
Ulrich Thiele, Tobias Kaloghlian, Jonas Wohlgemuth, Gerald Brenner-Weiß, André Tschöpe, Matthias Franzreb, Katharina Bleher
Glycans, a diverse group of complex oligosaccharides, are critical to human physiology and hold significant potential in medical applications and as food additives. However, their synthesis by glycosyltransferases produces intricate mixtures comprising saccharides, nucleosides, and reaction buffer components, posing substantial challenges for downstream processing and purification. This study aims to establish a continuous, single-pass nanofiltration process for purifying oligosaccharide-nucleoside mixtures using a novel dual-membrane module. We investigated the influence of a static mixer, along with varying flow rates for both the diafiltration and feed streams, on the recovery rate and purity of the final product. Measurements using ESI-MS assessed product recovery and purity, while buffer removal was monitored through online conductivity measurement. The results demonstrate that incorporating a static mixer nearly doubled the saccharide recovery rate, achieving product purities exceeding 99% and 95%, along with high product recovery rates. Additionally, the reaction buffer system was found to significantly impact the overall process performance. These findings suggest that our novel dual-membrane module can be effectively utilized for the purification of enzymatically synthesized glycan products.
{"title":"Fractionation of Oligosaccharide Nucleoside Mixtures by Single Pass Nano-Diafiltration","authors":"Ulrich Thiele, Tobias Kaloghlian, Jonas Wohlgemuth, Gerald Brenner-Weiß, André Tschöpe, Matthias Franzreb, Katharina Bleher","doi":"10.1002/elsc.70055","DOIUrl":"10.1002/elsc.70055","url":null,"abstract":"<p>Glycans, a diverse group of complex oligosaccharides, are critical to human physiology and hold significant potential in medical applications and as food additives. However, their synthesis by glycosyltransferases produces intricate mixtures comprising saccharides, nucleosides, and reaction buffer components, posing substantial challenges for downstream processing and purification. This study aims to establish a continuous, single-pass nanofiltration process for purifying oligosaccharide-nucleoside mixtures using a novel dual-membrane module. We investigated the influence of a static mixer, along with varying flow rates for both the diafiltration and feed streams, on the recovery rate and purity of the final product. Measurements using ESI-MS assessed product recovery and purity, while buffer removal was monitored through online conductivity measurement. The results demonstrate that incorporating a static mixer nearly doubled the saccharide recovery rate, achieving product purities exceeding 99% and 95%, along with high product recovery rates. Additionally, the reaction buffer system was found to significantly impact the overall process performance. These findings suggest that our novel dual-membrane module can be effectively utilized for the purification of enzymatically synthesized glycan products.</p><p><b>Summary</b>\u0000 \u0000 </p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 10","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12518163/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145298958","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}
Sara Cardoso, Sinan Oender, Jana Engelhardt, Alexander Tappe
The growing demand for large quantities of high-purity lentiviral vectors (LVs) has driven the development of scalable, cost-effective purification strategies. Membrane chromatography is particularly well-suited for purifying large biological entities like LVs due to its low mass transfer resistance and minimal back pressure. Among these technologies, anion exchange (AEX) chromatography serves as a key unit operation during the intermediate purification stage of industrial-scale bioprocesses. Sartobind Convec D, a weak AEX membrane with reduced ligand density, lack of hydrogel grafting, and adjusted pore size distribution, was specifically designed for LVs capture. While AEX is commonly employed immediately after clarification to capture LVs, incorporating a tangential flow filtration (TFF) step beforehand can offer several advantages, including product concentration, buffer exchange, and removal of low-molecular-weight impurities. Hydrosart High-Performance TFF membranes, composed of regenerated cellulose, are characterized by high flux rates and are marketed as well-suited for viral vector purification. This study investigated the integration of these two technologies into intermediate purification workflows for LVs. Specifically, the impact of introducing a TFF step prior to AEX chromatography (TFF-AEX workflow) was compared to a process utilizing only AEX chromatography following harvest and clarification (AEX-only workflow). In the AEX-only workflow, Sartobind Convec D membranes were used directly after clarification to capture LVs. These membrane adsorbers can accommodate high flow rates, making them suitable for early downstream processing. In the TFF-AEX workflow, clarified LVs harvests were first concentrated and diafiltrated using Hydrosart TFF membranes, allowing for product enrichment, buffer conductivity adjustment, and removal of smaller contaminants prior to AEX loading. As a result, the TFF-AEX workflow demonstrated improved dynamic binding capacity and enhanced overall impurity removal compared to the AEX-only approach.
{"title":"Downstream Workflows for Intermediate Purification of Lentiviral Vectors Using Tangential Flow Filtration and AEX Membrane Chromatography","authors":"Sara Cardoso, Sinan Oender, Jana Engelhardt, Alexander Tappe","doi":"10.1002/elsc.70056","DOIUrl":"10.1002/elsc.70056","url":null,"abstract":"<p>The growing demand for large quantities of high-purity lentiviral vectors (LVs) has driven the development of scalable, cost-effective purification strategies. Membrane chromatography is particularly well-suited for purifying large biological entities like LVs due to its low mass transfer resistance and minimal back pressure. Among these technologies, anion exchange (AEX) chromatography serves as a key unit operation during the intermediate purification stage of industrial-scale bioprocesses. Sartobind Convec D, a weak AEX membrane with reduced ligand density, lack of hydrogel grafting, and adjusted pore size distribution, was specifically designed for LVs capture. While AEX is commonly employed immediately after clarification to capture LVs, incorporating a tangential flow filtration (TFF) step beforehand can offer several advantages, including product concentration, buffer exchange, and removal of low-molecular-weight impurities. Hydrosart High-Performance TFF membranes, composed of regenerated cellulose, are characterized by high flux rates and are marketed as well-suited for viral vector purification. This study investigated the integration of these two technologies into intermediate purification workflows for LVs. Specifically, the impact of introducing a TFF step prior to AEX chromatography (TFF-AEX workflow) was compared to a process utilizing only AEX chromatography following harvest and clarification (AEX-only workflow). In the AEX-only workflow, Sartobind Convec D membranes were used directly after clarification to capture LVs. These membrane adsorbers can accommodate high flow rates, making them suitable for early downstream processing. In the TFF-AEX workflow, clarified LVs harvests were first concentrated and diafiltrated using Hydrosart TFF membranes, allowing for product enrichment, buffer conductivity adjustment, and removal of smaller contaminants prior to AEX loading. As a result, the TFF-AEX workflow demonstrated improved dynamic binding capacity and enhanced overall impurity removal compared to the AEX-only approach.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 10","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12518160/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145298853","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}
Antonio C. F. dos Santos, Riya Debbarma, Kayla Hinton, Mazin Hakim, Ronghua (Andy) Bei, Luis Solorio, Eduardo Ximenes, Shiven Kapur, Vince Corvari, Michael Ladisch
In vitro measurement of protein diffusion within matrices that simulate the subcutaneous (SQ) environment is of interest, given that protein-based therapeutics formulated for SQ injection comprise the largest class of biologics. To mimic the in vivo transport of a biologic from the SQ injection site through the extracellular matrix (ECM), in vitro diffusion assays typically utilize hyaluronic acid (HA) matrices, as it is the principal component of ECM. However, broad utility has been hampered by inherent lot-to-lot variability in commercially sourced HA, wherein key properties that impact protein diffusion (for example, molecular weight distribution and viscosity) differ across lots, even when nominal molecular weights are identical, making it challenging to compare results across matrices prepared from different HA lots. To address this gap, we report a facile approach wherein binary HA blends generated from individual HA matrices derived from distinct HA lots are functionally equivalent with respect to protein diffusion, that is, the diffusion of a representative set of proteins matches that in a previously reported single HA lot-derived matrix that served as a representative reference. Taken altogether, our protocols enable preparing blended HA matrices with consistent diffusion properties, enabling the use of in vitro assays that leverage this capability.
Practical application: The measurement of in vitro diffusion of IgG-type proteins enables calculation of diffusion coefficients that could help to guide the formulation of protein-based therapeutics, administered by subcutaneous (SQ) injection, and used for treating a range of diseases, including cancer. The side-by-side comparison of these proteins over a period of time provides confirmation of consistency of properties when in vitro hyaluronic acid matrices, within which injected protein diffusion is measured, are also consistent. However, their broad utility has been hindered by the inherent variability of commercial sources of HA used to make-up matrices that simulate the SQ environment in a predictable manner. Our research addresses this gap by defining an approach (validated with rheological and diffusion measurements) that facilitates the preparation of blended matrices from different lots of HA. The resulting matrix properties enable reliable measurement of protein diffusion from one lot to the next.
{"title":"Hyaluronic Acid Matrices for In Situ Measurement of Protein Diffusion Coefficients","authors":"Antonio C. F. dos Santos, Riya Debbarma, Kayla Hinton, Mazin Hakim, Ronghua (Andy) Bei, Luis Solorio, Eduardo Ximenes, Shiven Kapur, Vince Corvari, Michael Ladisch","doi":"10.1002/elsc.70048","DOIUrl":"https://doi.org/10.1002/elsc.70048","url":null,"abstract":"<p>In vitro measurement of protein diffusion within matrices that simulate the subcutaneous (SQ) environment is of interest, given that protein-based therapeutics formulated for SQ injection comprise the largest class of biologics. To mimic the in vivo transport of a biologic from the SQ injection site through the extracellular matrix (ECM), in vitro diffusion assays typically utilize hyaluronic acid (HA) matrices, as it is the principal component of ECM. However, broad utility has been hampered by inherent lot-to-lot variability in commercially sourced HA, wherein key properties that impact protein diffusion (for example, molecular weight distribution and viscosity) differ across lots, even when nominal molecular weights are identical, making it challenging to compare results across matrices prepared from different HA lots. To address this gap, we report a facile approach wherein binary HA blends generated from individual HA matrices derived from distinct HA lots are functionally equivalent with respect to protein diffusion, that is, the diffusion of a representative set of proteins matches that in a previously reported single HA lot-derived matrix that served as a representative reference. Taken altogether, our protocols enable preparing blended HA matrices with consistent diffusion properties, enabling the use of in vitro assays that leverage this capability.</p><p><i>Practical application:</i> The measurement of in vitro diffusion of IgG-type proteins enables calculation of diffusion coefficients that could help to guide the formulation of protein-based therapeutics, administered by subcutaneous (SQ) injection, and used for treating a range of diseases, including cancer. The side-by-side comparison of these proteins over a period of time provides confirmation of consistency of properties when in vitro hyaluronic acid matrices, within which injected protein diffusion is measured, are also consistent. However, their broad utility has been hindered by the inherent variability of commercial sources of HA used to make-up matrices that simulate the SQ environment in a predictable manner. Our research addresses this gap by defining an approach (validated with rheological and diffusion measurements) that facilitates the preparation of blended matrices from different lots of HA. The resulting matrix properties enable reliable measurement of protein diffusion from one lot to the next.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 10","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70048","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145237101","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}
Karim E. Shalaby, Issam Hmila, S. M. Nasir Uddin, Nasser H. Zawia, Omar M. A. El-Agnaf, Mustapha Aouida
The clinical translation of CRISPR genome-editing therapies is often hindered by inefficient delivery of the CRISPR-Cas RNA-protein complex into target cells. The most widely used CRISPR-Cas9 system poses a significant challenge for efficient delivery into cells due to its large size (∼1.4 kDa). Recently reported compact Cas proteins, such as Cas12f (552 Da), Cas12k (639 Da), and Cas12m (596 Da) represent attractive alternatives as cargoes for delivery. In this brief research report, we employ efficient delivery vectors to evaluate the efficiency of cellular uptake of a compact Cas protein (Cas12f) compared to the widely used larger Cas9 in human cells. Our findings demonstrate that compact Cas proteins may facilitate more efficient cellular penetration and delivery, making them a promising alternative for the development of CRISPR-based therapies.
Practical Application:
Our study demonstrates that compact Cas proteins significantly enhance cellular uptake compared to larger Cas proteins. This improved uptake efficiency suggests that compact Cas proteins could be more effective for clinical application, where size constraints and delivery efficiency are critical challenges. Combined with the optimization and refinement of the editing efficiencies of compact Cas systems, our study provokes further exploration of compact Cas proteins in various therapeutic contexts to advance the development of more efficient CRISPR-based therapies.
{"title":"Enhanced Cellular Uptake of Compact Cas Proteins: A Comparative Study of Cas12f and Cas9 in Human Cells","authors":"Karim E. Shalaby, Issam Hmila, S. M. Nasir Uddin, Nasser H. Zawia, Omar M. A. El-Agnaf, Mustapha Aouida","doi":"10.1002/elsc.70042","DOIUrl":"https://doi.org/10.1002/elsc.70042","url":null,"abstract":"<p>The clinical translation of CRISPR genome-editing therapies is often hindered by inefficient delivery of the CRISPR-Cas RNA-protein complex into target cells. The most widely used CRISPR-Cas9 system poses a significant challenge for efficient delivery into cells due to its large size (∼1.4 kDa). Recently reported compact Cas proteins, such as Cas12f (552 Da), Cas12k (639 Da), and Cas12m (596 Da) represent attractive alternatives as cargoes for delivery. In this brief research report, we employ efficient delivery vectors to evaluate the efficiency of cellular uptake of a compact Cas protein (Cas12f) compared to the widely used larger Cas9 in human cells. Our findings demonstrate that compact Cas proteins may facilitate more efficient cellular penetration and delivery, making them a promising alternative for the development of CRISPR-based therapies.</p><p>Practical Application:</p><p>Our study demonstrates that compact Cas proteins significantly enhance cellular uptake compared to larger Cas proteins. This improved uptake efficiency suggests that compact Cas proteins could be more effective for clinical application, where size constraints and delivery efficiency are critical challenges. Combined with the optimization and refinement of the editing efficiencies of compact Cas systems, our study provokes further exploration of compact Cas proteins in various therapeutic contexts to advance the development of more efficient CRISPR-based therapies.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 9","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70042","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145146452","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}
Bilyamin Abdulmumin, Ismaila Mudi, Abdulalim Ibrahim, Abdulwasiu Abdurrahman, Helen Onyeaka
<p>Silica-alginate capsule (G-0) has recently been used in fermentation processes to encapsulate microbial cells for several benefits, including facilitating continuous flow processes and simplifying cell recovery and reuse. However, these conventional silica-coated alginate capsules suffer from poor diffusion channels, which are critical for efficiently transporting substrates and products. This study aimed to develop a novel method for producing silica-coated alginate capsules with improved diffusion channels (G-3). The Ca-alginate capsule was fabricated via a simple dripping method, where a solution of calcium chloride (CaCl<sub>2</sub>) and carboxymethylcellulose (CMC) was dripped into an alginate solution. For the traditional silica coating (G-0), the alginate capsule was mixed with a silica source (hydrolyzed 3-aminopropyl triethoxysilane) under specific conditions. In the modified method, glucose was introduced as a pore-forming agent (PFA), with varying amounts (0.75, 1.5, and 3 g) resulting in capsules labeled G-0.75, G-1.5, and G-3, respectively. The diffusion coefficient for G-3 was found to be the highest, for example, at 313.15 K, it was calculated as <span></span><math> <semantics> <mrow> <mspace></mspace> <mrow> <mo>(</mo> <mrow> <mn>7.77</mn> <mo>±</mo> <mn>0.57</mn> </mrow> <mo>)</mo> </mrow> <mo>×</mo> <msup> <mn>10</mn> <mrow> <mo>−</mo> <mn>3</mn> </mrow> </msup> <mspace></mspace> <mi>m</mi> <msup> <mi>m</mi> <mn>2</mn> </msup> <mo>/</mo> <mi>min</mi> </mrow> <annotation>$ ( {7.77 pm 0.57} ) times {{10}^{ - 3}} {mathrm{m}}{{{mathrm{m}}}^2}/{mathrm{min}}$</annotation> </semantics></math> compared to <span></span><math> <semantics> <mrow> <mrow> <mo>(</mo> <mrow> <mn>3.04</mn> <mo>±</mo> <mn>0.09</mn> </mrow> <mo>)</mo> </mrow> <mo>×</mo> <msup> <mn>10</mn> <mrow> <mo>−</mo> <mn>3</mn> </mrow> </msup>
海藻酸硅胶囊（G-0）最近被用于发酵过程中封装微生物细胞的几个好处，包括促进连续流动过程和简化细胞回收和再利用。然而，这些传统的硅涂层海藻酸盐胶囊的扩散通道很差，这对于有效运输底物和产物至关重要。本研究旨在开发一种具有改进扩散通道（G-3）的硅包被海藻酸盐胶囊的新方法。将氯化钙（CaCl2）和羧甲基纤维素（CMC）溶液滴入海藻酸盐溶液中，通过简单的滴法制备海藻酸钙胶囊。对于传统的二氧化硅涂层（G-0），海藻酸盐胶囊在特定条件下与二氧化硅源（水解3-氨基丙基三乙氧基硅烷）混合。在改进的方法中，葡萄糖作为成孔剂（PFA）引入，不同量（0.75,1.5和3g）的胶囊分别标记为g -0.75, g -1.5和g -3。G-3的扩散系数最高，例如在313.15 K时；计算结果为（7.77±0.57）× 10−3M M 2 / min $ ({7.77 pm 0.57}) 倍{{10}^{- 3}} {mathrm{M}}}}^2}/{mathrm{min}}$相比（3.04±0.57）0.09) × 10−3 m m 2 / min $({3.04 pm0.09}) * {{10} ^ {- 3}} { mathrm {m}} {{{ mathrm {m}}} ^ 2} / { mathrm{分钟 }}$ g 0。这一发现强调了PFA在增强膜孔隙率和扩散率方面的有效性，这对于微生物细胞固定化是有希望的，其中传质是一个重要的问题。
{"title":"Enhancing the Diffusion Channels of Silica-Alginate Capsules for Microbial Encapsulation","authors":"Bilyamin Abdulmumin, Ismaila Mudi, Abdulalim Ibrahim, Abdulwasiu Abdurrahman, Helen Onyeaka","doi":"10.1002/elsc.70002","DOIUrl":"https://doi.org/10.1002/elsc.70002","url":null,"abstract":"<p>Silica-alginate capsule (G-0) has recently been used in fermentation processes to encapsulate microbial cells for several benefits, including facilitating continuous flow processes and simplifying cell recovery and reuse. However, these conventional silica-coated alginate capsules suffer from poor diffusion channels, which are critical for efficiently transporting substrates and products. This study aimed to develop a novel method for producing silica-coated alginate capsules with improved diffusion channels (G-3). The Ca-alginate capsule was fabricated via a simple dripping method, where a solution of calcium chloride (CaCl<sub>2</sub>) and carboxymethylcellulose (CMC) was dripped into an alginate solution. For the traditional silica coating (G-0), the alginate capsule was mixed with a silica source (hydrolyzed 3-aminopropyl triethoxysilane) under specific conditions. In the modified method, glucose was introduced as a pore-forming agent (PFA), with varying amounts (0.75, 1.5, and 3 g) resulting in capsules labeled G-0.75, G-1.5, and G-3, respectively. The diffusion coefficient for G-3 was found to be the highest, for example, at 313.15 K, it was calculated as <span></span><math>\u0000 <semantics>\u0000 <mrow>\u0000 <mspace></mspace>\u0000 <mrow>\u0000 <mo>(</mo>\u0000 <mrow>\u0000 <mn>7.77</mn>\u0000 <mo>±</mo>\u0000 <mn>0.57</mn>\u0000 </mrow>\u0000 <mo>)</mo>\u0000 </mrow>\u0000 <mo>×</mo>\u0000 <msup>\u0000 <mn>10</mn>\u0000 <mrow>\u0000 <mo>−</mo>\u0000 <mn>3</mn>\u0000 </mrow>\u0000 </msup>\u0000 <mspace></mspace>\u0000 <mi>m</mi>\u0000 <msup>\u0000 <mi>m</mi>\u0000 <mn>2</mn>\u0000 </msup>\u0000 <mo>/</mo>\u0000 <mi>min</mi>\u0000 </mrow>\u0000 <annotation>$ ( {7.77 pm 0.57} ) times {{10}^{ - 3}} {mathrm{m}}{{{mathrm{m}}}^2}/{mathrm{min}}$</annotation>\u0000 </semantics></math> compared to <span></span><math>\u0000 <semantics>\u0000 <mrow>\u0000 <mrow>\u0000 <mo>(</mo>\u0000 <mrow>\u0000 <mn>3.04</mn>\u0000 <mo>±</mo>\u0000 <mn>0.09</mn>\u0000 </mrow>\u0000 <mo>)</mo>\u0000 </mrow>\u0000 <mo>×</mo>\u0000 <msup>\u0000 <mn>10</mn>\u0000 <mrow>\u0000 <mo>−</mo>\u0000 <mn>3</mn>\u0000 </mrow>\u0000 </msup>\u0000 ","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 9","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145146330","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}
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