Biotechnology Progress最新文献_第3页

In silico optimization of a challenging bispecific antibody chromatography step.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3531

Zsófia Bencze, Tobias Hahn, Henri Kornmann, Pia Graf, Tatjana Trunzer

{"title":"In silico optimization of a challenging bispecific antibody chromatography step.","authors":"Zsófia Bencze, Tobias Hahn, Henri Kornmann, Pia Graf, Tatjana Trunzer","doi":"10.1002/btpr.3531","DOIUrl":"https://doi.org/10.1002/btpr.3531","url":null,"abstract":"Mechanistic modeling of chromatographic steps is an effective tool in biopharma process development that enhances process understanding and accelerates optimization efforts and subsequent risk assessment. A relatively new model for ion exchange chromatography is the colloidal particle adsorption (CPA) formalism, which promises improved separation of material and molecule-specific parameters. This case study demonstrates a straightforward CPA modeling workflow to describe an ion exchange chromatography polishing step of a knobs-into-holes construct bispecific antibody molecule. An adapted Yamamoto method was used to calculate charge and equilibrium parameters at three pH values. The remaining model parameters, binding kinetics, and effective mass transfer coefficients were determined via inverse fitting. The model was created from six experiments in total, tested on model parameter uncertainty, and evaluated on its power to predict changes in the biomolecule's retention behavior when variations in elution salt concentration occur. Finally, a three-step-gradient experiment was optimized, separating the desired bispecific antibody from its low and high molecular weight impurities, achieving a monomer yield of 68% and purity of 96%. Testing the model against a different load composition demonstrated its ability to extrapolate. An in silico one-factor-at-time and two-parameter screening of the optimized method identified the salt concentration to elute weaker binding impurities as a critical process attribute, while deviations in the buffer pH had a minor influence.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3531"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022046","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}

引用次数: 0

A method for facile production of variable lymphocyte receptors using SHuffle Escherichia coli.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3530

Elizabeth A Appelt, James B Thoden, Candis Dancy, Hannah D Bachmeier, Seth A Gehrke, Melanie A Staffenson, Ivan Rayment, Moriah E Katt, Hazel M Holden, Eric V Shusta

{"title":"A method for facile production of variable lymphocyte receptors using SHuffle Escherichia coli.","authors":"Elizabeth A Appelt, James B Thoden, Candis Dancy, Hannah D Bachmeier, Seth A Gehrke, Melanie A Staffenson, Ivan Rayment, Moriah E Katt, Hazel M Holden, Eric V Shusta","doi":"10.1002/btpr.3530","DOIUrl":"10.1002/btpr.3530","url":null,"abstract":"Variable lymphocyte receptors (VLRs) are the antigen receptors of jawless vertebrates such as lamprey. VLRs are of growing biotechnological interest for their ability to bind certain antigenic targets with higher affinity than traditional immunoglobulins. However, VLRs are disulfide-bonded proteins that are often challenging to produce requiring genetic modifications, fusion partners, non-scalable host cell lines or inclusion body formation and refolding. As a potential VLR expression platform option, the SHuffle Escherichia coli strain has been genetically altered to allow cytoplasmic disulfide bond formation by mutations to thioredoxin reductase (trxB) and glutathione reductase (gor) to create an oxidative cytoplasm. Furthermore, the SHuffle strain expresses disulfide bond isomerase DsbC in the cytoplasm to promote correct disulfide bond pairing. Here, we demonstrate that the SHuffle strain can produce high yield VLRs with titers ranging from 2 to 32 mg of VLR per liter of SHuffle culture. Three VLRs (P1C10, RBC36, VLRA.R2.1) were expressed in SHuffle E. coli and the products were compared directly to those generated using the Rosetta E. coli strain. All VLRs were validated for correct sequence, purity, and activity. For all VLRs, SHuffle E. coli produced 2-9 times more soluble VLRs than Rosetta E. coli. Furthermore, the soluble protein fraction was 2-6 times greater in SHuffle E. coli than Rosetta E. coli for all VLRs. Overall, these results suggest that the E. coli SHuffle strain is a convenient and effective expression system for producing large amounts of VLRs.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3530"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022035","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}

引用次数: 0

Coupling high-throughput and modeling approaches to streamline early-stage process development for biologics.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3523

John P Welsh, Scott H Altern, Jessica Y Lyall, Sean Burgess, Michael A Rauscher, Abraham M Lenhoff, Steven M Cramer, Chris Williams

{"title":"Coupling high-throughput and modeling approaches to streamline early-stage process development for biologics.","authors":"John P Welsh, Scott H Altern, Jessica Y Lyall, Sean Burgess, Michael A Rauscher, Abraham M Lenhoff, Steven M Cramer, Chris Williams","doi":"10.1002/btpr.3523","DOIUrl":"https://doi.org/10.1002/btpr.3523","url":null,"abstract":"Platforms have long been implemented for downstream process development of monoclonal antibodies (mAbs) to streamline development and reduce timelines. These platforms are also increasingly being used for other complex biologics modalities. While development has traditionally been conducted at the lab bench scale in a sequential manner, automated miniaturized and parallelized approaches like RoboColumns and resin plates have also been implemented for chromatographic screening. Additionally, mechanistic modeling for chromatographic separations has also seen increased use for development applications. In this manuscript, we propose a workflow with elements of both high-throughput screening and modeling that provides a streamlined roadmap for early process development. The workflow utilizes automated resin plate screens to both narrow screening conditions and calibrate binding isotherm parameters. Mechanistic models are then used to characterize a robust range of conditions suitable for an early manufacturing process. Miniaturized RoboColumns then confirm the process space, thus completing the development without the use of any traditional lab-scale columns. Case studies demonstrate the utility of this workflow for both cation-exchange (CEX) and multimodal cation-exchange (MMCEX) processes. Process parameter sensitivities across process ranges for the models are compared with typical design-of-experiment (DOE) statistical models. The models are able to predict the mAb product as well as aggregate impurities. This workflow provides a practical method to enable increased process understanding while also reducing timeline and material requirements for development.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3523"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022038","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}

引用次数: 0

Insights into the morphology-productivity relationship of filamentous fungi through small-scale cultivation and automated microscopy of Thermothelomyces thermophilus.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3528

Katja Rohr, Bertram Geinitz, Johannes Seiffarth, Aydin Anbarani, Sören Bernauer, Matthias Moch, Julia Tenhaef, Wolfgang Wiechert, Katharina Nöh, Marco Oldiges

{"title":"Insights into the morphology-productivity relationship of filamentous fungi through small-scale cultivation and automated microscopy of Thermothelomyces thermophilus.","authors":"Katja Rohr, Bertram Geinitz, Johannes Seiffarth, Aydin Anbarani, Sören Bernauer, Matthias Moch, Julia Tenhaef, Wolfgang Wiechert, Katharina Nöh, Marco Oldiges","doi":"10.1002/btpr.3528","DOIUrl":"https://doi.org/10.1002/btpr.3528","url":null,"abstract":"Filamentous fungi are a cornerstone in the biotechnological production of enzymes, proteins, and organic acids. However, challenges in understanding and controlling the relationship between morphology and productivity can limit their application. This study addresses these challenges using Thermothelomyces thermophilus, a promising thermophilic fungus known for the production of thermostable enzymes. We investigated the effects of environmental conditions on fungal morphology and enzyme production using a combination of microbioreactor cultivation, automated liquid handling, and automated microscopy. Specifically, batch and fed batch cultivations were performed at different pH levels and glucose feeding rates to study their effects on secretory phytase production, fungal growth, and morphology. Results from batch cultivations revealed a two-fold higher phytase activity at pH 5.5 compared to pH 6.5, with notably smaller fungal fragments at the end of cultivation. Conversely, fed batch cultivations at a feeding rate of 1 g (l h)-1 glucose showed a 1.6-fold higher enzyme activity at pH 5.5, accompanied by much larger fungal aggregates throughout the feeding phase. These findings suggest that large aggregates are associated with high productivity; however, their breakdown further enhances enzyme release, increasing activity in the supernatant. This study not only provides insights on the morphology-productivity relationship of T. thermophilus, but also demonstrates the efficacy of integrating microbioreactors with automated microscopy. This methodology represents a significant advance in the field of fungal biotechnology, paving the way for more efficient industrial bioprocesses.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3528"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022049","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}

引用次数: 0

Process intensification of the baculovirus expression vector system using a perfusion process with a low multiplicity of infection at high cell concentrations.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3527

Jort J Altenburg, Brenda E Juarez-Garza, Jelle van Keimpema, Linda van Oosten, Gorben P Pijlman, Monique M van Oers, René H Wijffels, Dirk E Martens

{"title":"Process intensification of the baculovirus expression vector system using a perfusion process with a low multiplicity of infection at high cell concentrations.","authors":"Jort J Altenburg, Brenda E Juarez-Garza, Jelle van Keimpema, Linda van Oosten, Gorben P Pijlman, Monique M van Oers, René H Wijffels, Dirk E Martens","doi":"10.1002/btpr.3527","DOIUrl":"https://doi.org/10.1002/btpr.3527","url":null,"abstract":"The emergence of new viruses and the spread of existing pathogens necessitate efficient vaccine production methods. The baculovirus expression vector system (BEVS) is an efficient and scalable system for subunit and virus-like particle vaccine production and gene therapy vectors. However, current production processes are often limited to low cell concentrations (1-4 × 106 cells/mL) in fed-batch mode. To improve the volumetric productivity of the BEVS, a medium exchange strategy was investigated. Screening experiments were performed to test baculovirus (expressing green fluorescent protein; GFP) infection and productivity of insect cell cultures infected at high cell concentration (1-2 × 107 cells/mL), showing that infection at high cell concentrations was possible with medium exchange. Next, duplicate perfusion runs with baculovirus infection were performed using a cell concentration upon infection (CCI) of 1.2 × 107 cells/mL and a multiplicity of infection (MOI) of 0.01, reaching a maximum viable cell concentration of 2.8 × 107 cells/mL and a maximum GFP production of 263 mg/L. The volumetric productivity of these perfusion runs was 4.8 times higher than for reference batch processes with a CCI of 3 × 106 cells/mL and an MOI of 1. These results demonstrate that process intensification can be achieved for the BEVS by implementing perfusion, resulting in a higher volumetric productivity.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3527"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022060","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}

引用次数: 0

Stipulations of cell and gene therapy and the ties to biomanufacturing.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3521

Justin Allisha, Juthika Das, Thomas Dunnigan, Susan T Sharfstein, Payel Datta

{"title":"Stipulations of cell and gene therapy and the ties to biomanufacturing.","authors":"Justin Allisha, Juthika Das, Thomas Dunnigan, Susan T Sharfstein, Payel Datta","doi":"10.1002/btpr.3521","DOIUrl":"https://doi.org/10.1002/btpr.3521","url":null,"abstract":"Cell and gene therapy (CGT) products are emerging and innovative biopharmaceuticals that hold promise for treating diseases that are otherwise beyond the scope of conventional medicines. The evolution of CGT from a research idea to a promising therapeutic product is due to the complementary advancements across various scientific disciplines. First, the innovations and advancements in gene editing and delivery technology have provided fundamental tools to manipulate genes and cells for therapeutic pursuits. Second, advancements in applied and translational research, including how clinical trials are designed, performed, evaluated, and analyzed, have transformed the technology into a potential therapeutic product. Third, advancements in scaling up the production of CGT products have been critical in delivering the product for preclinical studies, clinical trials, and approved treatments. In parallel, regulatory requirements have continuously evolved, with lessons learned from translational studies and biomanufacturing. These combined efforts have transformed CGT products from a promising concept into a reality with the potential to treat a wide range of diseases. However, continued R&D and regulatory oversight are crucial to further improve the safety, efficacy, and accessibility of CGT products.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3521"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143021625","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}

引用次数: 0

A design space for the filtration of challenging monoclonal antibodies using Planova™ S20N, a new regenerated cellulose virus removal filter.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3533

Asami Inoue, Satoshi Murakami, Akika Futamura, Naoto Watanabe, Yumiko Masuda

引用次数: 0

Highly iterated palindrome 1 sequence improves Synechococcus sp. PCC 7002 transformation efficiencies in a homology- and methylation-dependent manner.

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2025-01-23 DOI: 10.1002/btpr.3518

Cody Kamoku, David R Nielsen

{"title":"Highly iterated palindrome 1 sequence improves Synechococcus sp. PCC 7002 transformation efficiencies in a homology- and methylation-dependent manner.","authors":"Cody Kamoku, David R Nielsen","doi":"10.1002/btpr.3518","DOIUrl":"https://doi.org/10.1002/btpr.3518","url":null,"abstract":"The ability to precisely engineer cyanobacterial metabolism first requires the ability to efficiently deliver engineered DNA constructs. Here, we investigate how natural transformation efficiencies in Synechococcus sp. PCC 7002 can be greatly improved by leveraging the native and abundant cyanobacterial Highly Iterated Palindrome 1 (HIP1) sequence. While including at least one homologous HIP1 site within the homology arms of an integrating plasmid increased integration efficiency by up to 7-fold, methylation of those sites by HIP1 methyltransferase (encoded by slr0214 from Synechococcus sp. PCC 6803) boosted this to greater than a 100-fold improvement overall. Non-homologous HIP1 sites also improved transformation efficiencies of both integrating and replicating episomal plasmids (by up to 60- and 9-fold, respectively), but only if methylated. The collective data further reveal that HIP1 does not function as part of a native restriction enzyme system in PCC 7002, but rather may improve transformation efficiency via an alternative mechanism(s), occurring prior to and/or during homologous recombination. Future studies are needed, however, to more clearly elucidate the specific role of HIP1 during natural transformation of cyanobacteria.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3518"},"PeriodicalIF":2.5,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022044","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}

引用次数: 0

Sensitivity of bulk electrical impedance spectroscopy (bio-capacitance) probes to cell and culture properties: Study on CHO cell cultures. 体电阻抗谱（生物电容）探针对细胞和培养特性的敏感性：CHO细胞培养的研究。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2024-12-26 DOI: 10.1002/btpr.3519

Elham Salimi, Sara Absalan, Julien Robitaille, Johnny Montes, Michael Butler, Douglas Thomson, Greg Bridges

{"title":"Sensitivity of bulk electrical impedance spectroscopy (bio-capacitance) probes to cell and culture properties: Study on CHO cell cultures.","authors":"Elham Salimi, Sara Absalan, Julien Robitaille, Johnny Montes, Michael Butler, Douglas Thomson, Greg Bridges","doi":"10.1002/btpr.3519","DOIUrl":"https://doi.org/10.1002/btpr.3519","url":null,"abstract":"Bulk electrical impedance spectroscopy (bio-capacitance) probes, hold significant promise for real-time cell monitoring in bioprocesses. Focusing on Chinese hamster ovary (CHO) cells, we present a sensitivity analysis framework to assess the impact of cell and culture properties on the complex permittivity spectrum, εmix, and its associated parameters, permittivity increment, Δε, critical frequency, fc, and Cole-Cole parameter, α, measured by bio-capacitance probes. Our sensitivity analysis showed that Δε is highly sensitive to cell size and concentration, making it suitable for estimating biovolume during the exponential growth phase, whereas fc provides information about cumulative changes in cell size, membrane permittivity, and cytoplasm conductivity during the transition to death phase. The analysis indicated that specific information about cell membrane permittivity or internal conductivity cannot be extracted from εmix spectrum. Based on the sensitivity analysis, we proposed two alternative parameters for monitoring cells in bioprocesses: Δε1 MHz and Δε1 MHz/Δε0.3 MHz, using measurements at 300 kHz, 1 MHz, and 10 MHz. Δε1 MHz is suitable for estimating viable cell density during the exponential growth phase due to its lower sensitivity to cell size. Δε1 MHz/Δε0.3 MHz can replace fc due to similar sensitivities to cell size and dielectric properties. These frequencies are within most bio-capacitance probes' optimal operation range, eliminating the need for low-frequency electrode polarization and high-frequency stray capacitances corrections. Experimental measurements on CHO cells confirmed the results of sensitivity analysis.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3519"},"PeriodicalIF":2.5,"publicationDate":"2024-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142891842","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}

引用次数: 0

Online monitored characterization of Phocaeicola vulgatus for organic acid production using anaerobic microtiter plate cultivations. 利用厌氧微孔板培养在线监测 Phocaeicola vulgatus 生产有机酸的特征。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2024-12-20 DOI: 10.1002/btpr.3526

Laura Keitel, Benjamin Schick, Gino Pohen, Stanislav Yordanov, Jochen Büchs

{"title":"Online monitored characterization of Phocaeicola vulgatus for organic acid production using anaerobic microtiter plate cultivations.","authors":"Laura Keitel, Benjamin Schick, Gino Pohen, Stanislav Yordanov, Jochen Büchs","doi":"10.1002/btpr.3526","DOIUrl":"https://doi.org/10.1002/btpr.3526","url":null,"abstract":"Phocaeicola vulgatus (formerly Bacteroides vulgatus), an anaerobic gut bacterium, produces several organic acids. Research on P. vulgatus is still in its infancy. However, a detailed understanding of P. vulgatus growth and metabolism is essential for its assessment as an organic acid producer. Media variations, including different initial glucose and NH4Cl concentrations and osmolalities, are significant means to yield higher organic acid titers. Furthermore, examining different nitrogen and carbon sources is important to evaluate the potential of P. vulgatus for growth on renewable resources. Cultivations were performed in an in-house built device for anaerobic online-monitoring of fluorescence and scattered light in microtiter plates. Results revealed that the highest organic acid concentrations were reached while using galactose, glucose, or xylose as a carbon source, high osmolalities, and 0.25 g L-1 NH4Cl. In addition, the organic acid composition changed with changing carbon and nitrogen sources. P. vulgatus was successfully further characterized, thereby contributing to a faster characterization of other anaerobic strains and paving the way for anaerobic organic acid production.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":" ","pages":"e3526"},"PeriodicalIF":2.5,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142862945","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}

引用次数: 0