Biotechnology Progress最新文献

{"title":"A systemic approach to identifying sequence frameworks that decrease mAb production in a transient Chinese hamster ovary cell expression system","authors":"Alana C. Szkodny,&nbsp;Kelvin H. Lee","doi":"10.1002/btpr.3466","DOIUrl":"10.1002/btpr.3466","url":null,"abstract":"<p>Monoclonal antibodies (mAbs) are often engineered at the sequence level for improved clinical performance yet are rarely evaluated prior to candidate selection for their “developability” characteristics, namely expression, which can necessitate additional resource investments to improve the manufacturing processes for problematic mAbs. A strong relationship between primary sequence and expression has emerged, with slight differences in amino acid sequence resulting in titers differing by up to an order of magnitude. Previous work on these “difficult-to-express” (DTE) mAbs has shown that these phenotypes are driven by post-translational bottlenecks in antibody folding, assembly, and secretion processes. However, it has been difficult to translate these findings across cell lines and products. This work presents a systematic approach to study the impact of sequence variation on mAb expression at a larger scale and under more industrially relevant conditions. The analysis found 91 mutations that decreased transient expression of an IgG₁κ in Chinese hamster ovary (CHO) cells and revealed that mutations at inaccessible residues, especially those leading to decreases in residue hydrophobicity, are not favorable for high expression. This workflow can be used to better understand sequence determinants of mAb expression to improve candidate selection procedures and reduce process development timelines.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140566587","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":"Online monitoring of the respiration activity in 96-deep-well microtiter plate Chinese hamster ovary cultures streamlines kill curve experiments","authors":"Anne Neuss,&nbsp;Nele von Vegesack,&nbsp;Raoul Liepelt,&nbsp;Jochen Büchs,&nbsp;Jørgen Barsett Magnus","doi":"10.1002/btpr.3468","DOIUrl":"10.1002/btpr.3468","url":null,"abstract":"<p>Cell line generation of mammalian cells is a time-consuming and labor-intensive process, especially because of challenges in clone selection after transfection. Antibiotics are common selection agents for mammalian cells due to their simplicity of use. However, the optimal antibiotic concentration must be determined with a kill curve experiment before clone selection starts. The traditional kill curve experiments are resource-intensive and time-consuming due to necessary sampling and offline analysis effort. This study, thus, explores the potential of online monitoring the oxygen transfer rate (OTR), as a non-invasive and efficient alternative for kill curve experiments. The OTR is monitored using the Transfer-rate Online Measurement (TOM) system and the micro(μ)-scale Transfer-rate Online Measurement (μTOM) device, which was used for mammalian cells first. It could be shown that the OTR curves for both devices align perfectly, affirming consistent cultivation conditions. The μTOM device proves effective in performing kill curve experiments in 96-deep-well plates without the need for sampling and offline analysis. The streamlined approach reduces medium consumption by 95%, offering a cost-effective and time-efficient solution for kill curve experiments. The study validates the generalizability of the method by applying it to two different CHO cell lines (CHO-K1 and sciCHO) with two antibiotics (puromycin and hygromycin B) each. In conclusion, the broad application of OTR online monitoring for CHO cell cultures in 96-deep-well plates is highlighted. The μTOM device proves as a valuable tool for high-throughput experiments, paving the way for diverse applications, such as media and clone screening, cytotoxicity tests, and scale-up experiments.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3468","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140566484","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mesenchymal stem cells derived-exosomes enhanced amniotic membrane extract promotes corneal keratocyte proliferation","authors":"Fatma Zehra Erkoc-Biradli,&nbsp;Berkay Erenay,&nbsp;Alp Ozgun,&nbsp;Hayriye Öztatlı,&nbsp;Ferda Işık,&nbsp;Utku Ateş,&nbsp;Rıfat Rasier,&nbsp;Bora Garipcan","doi":"10.1002/btpr.3465","DOIUrl":"10.1002/btpr.3465","url":null,"abstract":"<p>Amniotic membrane extract (AME) and Wharton's jelly mesenchymal stem cells derived-exosomes (WJ-MSC-Exos) are promising therapeutic solutions explored for their potential in tissue engineering and regenerative medicine, particularly in skin and corneal wound healing applications. AME is an extract form of human amniotic membrane and known to contain a plethora of cytokines and growth factors, making it a highly attractive option for topical applications. Similarly, WJ-MSC-Exos have garnered significant interest for their wound healing properties. Although WJ-MSC-Exos and AME have been used separately for wound healing research, their combined synergistic effects have not been studied extensively. In this study, we evaluated the effects of both AME and WJ-MSC-Exos, individually and together, on the proliferation of corneal keratocytes as well as their ability to promote in vitro cell migration, wound healing, and their impact on cellular morphology. Our findings indicated that the presence of both exosomes (3 × 10⁵ Exo/mL) and AME (50 μg/mL) synergistically enhance the proliferation of corneal keratocytes. Combined use of these solutions (3 × 10⁵ Exo/mL + 50 μg/mL) increased cell proliferation compared to only 50 μg/mL AME treatment on day 3 (**** <i>p</i> &lt; 0.0001). This mixture treatment (3 × 10⁵ Exo/mL + 50 μg/mL) increased wound closure rate compared to isolated WJ-MSC-Exo treatment (3 × 10⁵ Exo/mL) (*<i>p</i> &lt; 0.05). Overall, corneal keratocytes treated with AME and WJ-MSC-Exo (3 × 10⁵ Exo/mL + 50 μg/mL) mixture resulted in enhanced proliferation and wound healing tendency. Utilization of combined use of AME and WJ-MSC-Exo can pave the way for a promising foundation for corneal repair research.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140566579","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 a pan-tau multivalent nanobody that binds tau aggregation motifs and recognizes pathological tau aggregates","authors":"Nikki McArthur,&nbsp;Bokyung Kang,&nbsp;Felix G. Rivera Moctezuma,&nbsp;Akber T. Shaikh,&nbsp;Kathryn Loeffler,&nbsp;Nemil N. Bhatt,&nbsp;Madison Kidd,&nbsp;Jennifer M. Zupancic,&nbsp;Alec A. Desai,&nbsp;Naima Djeddar,&nbsp;Anton Bryksin,&nbsp;Peter M. Tessier,&nbsp;Rakez Kayed,&nbsp;Levi B. Wood,&nbsp;Ravi S. Kane","doi":"10.1002/btpr.3463","DOIUrl":"10.1002/btpr.3463","url":null,"abstract":"<p>Alzheimer's disease and other tauopathies are characterized by the misfolding and aggregation of the tau protein into oligomeric and fibrillar structures. Antibodies against tau play an increasingly important role in studying these neurodegenerative diseases and the generation of tools to diagnose and treat them. The development of antibodies that recognize tau protein aggregates, however, is hindered by complex immunization and antibody selection strategies and limitations to antigen presentation. Here, we have taken a facile approach to identify single-domain antibodies, or nanobodies, that bind to many forms of tau by screening a synthetic yeast surface display nanobody library against monomeric tau and creating multivalent versions of our lead nanobody, MT3.1, to increase its avidity for tau aggregates. We demonstrate that MT3.1 binds to tau monomer, oligomers, and fibrils, as well as pathogenic tau from a tauopathy mouse model, despite being identified through screens against monomeric tau. Through epitope mapping, we discovered binding epitopes of MT3.1 contain the key motif VQIXXK which drives tau aggregation. We show that our bivalent and tetravalent versions of MT3.1 have greatly improved binding ability to tau oligomers and fibrils compared to monovalent MT3.1. Our results demonstrate the utility of our nanobody screening and multivalent design approach in developing nanobodies that bind amyloidogenic protein aggregates. This approach can be extended to the generation of multivalent nanobodies that target other amyloid proteins and has the potential to advance the research and treatment of neurodegenerative diseases.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3463","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140566721","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimizing glutamine concentration enhances ex vivo expansion of natural killer cells through improved redox status","authors":"Danni Ying,&nbsp;Guofeng Zhang,&nbsp;Huimin Huang,&nbsp;Wen-song Tan,&nbsp;Haibo Cai","doi":"10.1002/btpr.3464","DOIUrl":"10.1002/btpr.3464","url":null,"abstract":"<p>Amino acids are vital components of the serum-free medium that influence the expansion and function of NK cells. This study aimed to clarify the relationship between amino acid metabolism and expansion and cytotoxicity of NK cells. Based on analyzing the mino acid metabolism of NK-92 cells and Design of Experiments (DOE), we optimized the combinations and concentrations of amino acids in NK-92 cells culture medium. The results demonstrated that NK-92 cells showed a pronounced demand for glutamine, serine, leucine, and arginine, in which glutamine played a central role. Significantly, at a glutamine concentration of 13 mM, NK-92 cells expansion reached 161.9 folds, which was significantly higher than 55.5 folds at 2.5 mM. Additionally, under higher glutamine concentrations, NK-92 cells expressed elevated levels of cytotoxic molecules, the level of cytotoxic molecules expressed by NK-92 cells was increased and the cytotoxic rate was 68.42%, significantly higher than that of 58.08% under low concentration. In view of the close relationship between glutamine metabolism and intracellular redox state, we investigated the redox status within the cells. This study demonstrated that intracellular ROS levels in higher glutamine concentrations were significantly lower than those under lower concentration cultures with decreased intracellular GSH/GSSG ratio, NADPH/NADP⁺ ratio, and apoptosis rate. These findings indicate that NK-92 cells exhibit improved redox status when cultured at higher glutamine concentrations. Overall, our research provides valuable insights into the development of serum-free culture medium for ex vivo expansion of NK-92 cells.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140334607","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":"Digital application for drug product potency target evaluation in biopharmaceutical manufacturing","authors":"Darrick Shen,&nbsp;Shyam Panjwani,&nbsp;Konstantinos Spetsieris","doi":"10.1002/btpr.3461","DOIUrl":"10.1002/btpr.3461","url":null,"abstract":"<p>Biopharmaceutical manufacturing entails a series of highly regulated steps. The manufacturing of safe and efficacious drug product (DP) requires testing of critical quality attributes (CQAs) against specification limits. DP potency concentration, which measures the dosage strength of a particular DP, is a CQA of great interest. In order to minimize the DP potency out-of-specification (OOS) risk, sterile fill finish (SFF) process adjustments may be needed. Varying the potency targets can be one such process adjustment. To facilitate such evaluation, data acquisition and statistical calculations are required. Regularly conducting the OOS risk assessment manually using commercial statistical software can be tedious, error-prone, and impractical, especially when several alternate potency targets are under consideration. In this work, the development of a novel framework for OOS risk assessment and deployment of cloud-based statistical software application to facilitate the risk assessment are presented. This application is intended to streamline the assessment of alternate potency targets for DP in biologics manufacturing. The major aspects of this potency targeting application development are presented in detail. Specifically, data sources, pipeline, application architecture, back-end and front-end development as well as application verification are discussed. Finally, several use cases are presented to highlight the application's utility in biologics manufacturing.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3461","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140334606","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CD133 ligand-enhanced etoposide-liposome complex for targeted killing of lung cancer cells","authors":"Shiwei Nie,&nbsp;Junzheng Zhou,&nbsp;Xiaodong Zheng,&nbsp;Xudong Wei,&nbsp;Jinrui Zhang,&nbsp;Xiaojuan Shen,&nbsp;Weimin Zhang","doi":"10.1002/btpr.3460","DOIUrl":"10.1002/btpr.3460","url":null,"abstract":"<p>Lung cancer has a high incidence rate and a low cure rate, hence the urgent need for effective treatment methods. Current lung cancer drugs have several drawbacks, including low specificity, poor targeting, drug resistance, and irreversible damage to normal tissues. Therefore, there is a need to develop a safe and effective new drug that can target and kill tumor cells. In this study, we combined nanotechnology and biotechnology to develop a CD133 ligand-modified etoposide-liposome complex (Lipo@ETP-CD133) for targeted therapy of lung cancer. The CD133 ligand targeted lung cancer stem cells, causing the composite material to aggregate at the tumor site, where high levels of ETP liposomes could exert a strong tumor-killing effect. Our research results demonstrated that this nano-drug had efficient targeting and tumor-killing effects, indicating its potential for clinical application.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140334605","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":"GMP implementation of a hybrid continuous manufacturing process for a recombinant non-mAb protein—A case study","authors":"Venkatesh Natarajan,&nbsp;Neil Soice,&nbsp;Johanna Mullen,&nbsp;David Bull","doi":"10.1002/btpr.3459","DOIUrl":"10.1002/btpr.3459","url":null,"abstract":"<p>Advances in manufacturing technology coupled with the increased potency of new biotherapeutic modalities have created an external environment where continuous manufacturing (CM) can address a growing need. Amgen has successfully implemented a hybrid CM process for a commercial lifecycle program. In this process, the bioreactor, harvest, capture column, and viral inactivation/depth filtration unit operations were integrated together in an automated, continuous module, while the remaining downstream unit operations took place in stand-alone batch mode. CM operations are particularly suited for so-called “high mix, low volume” manufacturing plants, where a variety of molecules are manufactured in relatively low volumes. The selected molecule fit this mold and was manufactured in a low-capital micro-footprint suite attached to an existing therapeutic production facility. Use of a hybrid process within an already operating facility required less capital and minimized complexity. To enable this hybrid CM process, an established fed-batch process was converted to a perfusion process with continuous harvest. Development efforts included both process changes and the generation of a novel cell line adapted to long-term perfusion. Chromatography resins were updated, and purification processes adapted to handle variable inputs due to the fluctuations in harvest titer from the lengthy production process. A novel automated single-use (SU) viral inactivation (VI) skid was introduced, which entailed the development of a robust pH verification and alarm system, along with procedures for product isolation to allow discard of specific cycles. The CM process demonstrated consistent performance, meaning it met predefined performance criteria (including product quality attributes, or PQAs) when operated within established process parameters and manufactured according to applicable procedures. Using a 75% reduction in scale, it resulted in a five-fold reduction in process media and buffer usage, a fifteen-fold increase in mass per thaw, and an overall process productivity increase of 45-fold (as measured by grams drug substance per liter per day.) The hybrid CM process also enabled increased material demand to be met with no change in cost of goods manufactured or plant capacity, due to the repurposing of existing facility space and the flexible duration of the hybrid CM harvest. Overall, the success of the hybrid CM platform represents an exciting opportunity to reduce costs and increase process efficiency in industry.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140326241","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":"Raman spectroscopy and one-dimensional convolutional neural network modeling as a real-time monitoring tool for in vitro transaminase-catalyzed synthesis of a pharmaceutically relevant amine precursor","authors":"Julie Østerby Madsen,&nbsp;Sebastian Olivier Nymann Topalian,&nbsp;Mikkel Fog Jacobsen,&nbsp;Tommy Skovby,&nbsp;Krist V. Gernaey,&nbsp;Allan S. Myerson,&nbsp;John Woodley","doi":"10.1002/btpr.3444","DOIUrl":"10.1002/btpr.3444","url":null,"abstract":"<p>Raman spectroscopy has been used to measure the concentration of a pharmaceutically relevant model amine intermediate for positive allosteric modulators of nicotinic acetylcholine receptor in a ω-transaminase-catalyzed conversion. A model based on a one-dimensional convolutional neural network was developed to translate raw data augmented Raman spectra directly into substrate concentrations, with which the conversion from ketone to amine by ω-transaminase could be determined over time. The model showed very good predictive capabilities, with <i>R</i>² values higher than 0.99 for the spectra included in the modeling and 0.964 for an independent dataset. However, the model could not extrapolate outside the concentrations specified by the model. The presented work shows the potential of Raman spectroscopy as a real-time monitoring tool for biocatalytic reactions.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3444","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140304635","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modeling scalability of impurity precipitation in downstream biomanufacturing","authors":"Jing Guo,&nbsp;Steven J. Traylor,&nbsp;Mohamed Agoub,&nbsp;Weixin Jin,&nbsp;Helen Hua,&nbsp;R. Bertrum Diemer,&nbsp;Xuankuo Xu,&nbsp;Sanchayita Ghose,&nbsp;Zheng Jian Li,&nbsp;Abraham M. Lenhoff","doi":"10.1002/btpr.3454","DOIUrl":"10.1002/btpr.3454","url":null,"abstract":"<p>Precipitation during the viral inactivation, neutralization and depth filtration step of a monoclonal antibody (mAb) purification process can provide quantifiable and potentially significant impurity reduction. However, robust commercial implementation of this unit operation is limited due to the lack of a representative scale-down model to characterize the removal of impurities. The objective of this work is to compare isoelectric impurity precipitation behavior for a monoclonal antibody product across scales, from benchtop to pilot manufacturing. Scaling parameters such as agitation and vessel geometry were investigated, with the precipitate amount and particle size distribution (PSD) characterized via turbidity and flow imaging microscopy. Qualitative analysis of the data shows that maintaining a consistent energy dissipation rate (EDR) could be used for approximate scaling of vessel geometry and agitator speeds in the absence of more detailed simulation. For a more rigorous approach, however, agitation was simulated via computational fluid dynamics (CFD) and these results were applied alongside a population balance model to simulate the trajectory of the size distribution of precipitate. CFD results were analyzed within a framework of a two-compartment mixing model comprising regions of high- and low-energy agitation, with material exchange between the two. Rate terms accounting for particle formation, growth and breakage within each region were defined, accounting for dependence on turbulence. This bifurcated model was successful in capturing the variability in particle sizes over time across scales. Such an approach enhances the mechanistic understanding of impurity precipitation and provides additional tools for model-assisted prediction for process scaling.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140304634","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}