{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,8]],"date-time":"2025-11-08T18:02:22Z","timestamp":1762624942769,"version":"build-2065373602"},"reference-count":42,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2022,11,17]],"date-time":"2022-11-17T00:00:00Z","timestamp":1668643200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"iNOVA4Health","award":["UIDB\/04462\/2020","UIDP\/04462\/2020","LA\/P\/0087\/2020","PTDC\/BTM-ORG\/1383\/2020","2020.01216.CEECIND","PD\/BD\/2020.06003"],"award-info":[{"award-number":["UIDB\/04462\/2020","UIDP\/04462\/2020","LA\/P\/0087\/2020","PTDC\/BTM-ORG\/1383\/2020","2020.01216.CEECIND","PD\/BD\/2020.06003"]}]},{"name":"iNOVA4Health","award":["UIDB\/04462\/2020","UIDP\/04462\/2020","LA\/P\/0087\/2020","PTDC\/BTM-ORG\/1383\/2020","2020.01216.CEECIND","PD\/BD\/2020.06003"],"award-info":[{"award-number":["UIDB\/04462\/2020","UIDP\/04462\/2020","LA\/P\/0087\/2020","PTDC\/BTM-ORG\/1383\/2020","2020.01216.CEECIND","PD\/BD\/2020.06003"]}]},{"name":"Associate Laboratory LS4FUTURE","award":["UIDB\/04462\/2020","UIDP\/04462\/2020","LA\/P\/0087\/2020","PTDC\/BTM-ORG\/1383\/2020","2020.01216.CEECIND","PD\/BD\/2020.06003"],"award-info":[{"award-number":["UIDB\/04462\/2020","UIDP\/04462\/2020","LA\/P\/0087\/2020","PTDC\/BTM-ORG\/1383\/2020","2020.01216.CEECIND","PD\/BD\/2020.06003"]}]},{"name":"FCT","award":["UIDB\/04462\/2020","UIDP\/04462\/2020","LA\/P\/0087\/2020","PTDC\/BTM-ORG\/1383\/2020","2020.01216.CEECIND","PD\/BD\/2020.06003"],"award-info":[{"award-number":["UIDB\/04462\/2020","UIDP\/04462\/2020","LA\/P\/0087\/2020","PTDC\/BTM-ORG\/1383\/2020","2020.01216.CEECIND","PD\/BD\/2020.06003"]}]},{"name":"FCT","award":["UIDB\/04462\/2020","UIDP\/04462\/2020","LA\/P\/0087\/2020","PTDC\/BTM-ORG\/1383\/2020","2020.01216.CEECIND","PD\/BD\/2020.06003"],"award-info":[{"award-number":["UIDB\/04462\/2020","UIDP\/04462\/2020","LA\/P\/0087\/2020","PTDC\/BTM-ORG\/1383\/2020","2020.01216.CEECIND","PD\/BD\/2020.06003"]}]},{"name":"FCT","award":["UIDB\/04462\/2020","UIDP\/04462\/2020","LA\/P\/0087\/2020","PTDC\/BTM-ORG\/1383\/2020","2020.01216.CEECIND","PD\/BD\/2020.06003"],"award-info":[{"award-number":["UIDB\/04462\/2020","UIDP\/04462\/2020","LA\/P\/0087\/2020","PTDC\/BTM-ORG\/1383\/2020","2020.01216.CEECIND","PD\/BD\/2020.06003"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Virus-based biopharmaceutical products are used in clinical applications such as vaccines, gene therapy, and immunotherapy. However, their manufacturing remains a challenge, hampered by the lack of appropriate analytical tools for purification monitoring or characterization of the final product. This paper describes the implementation of a highly sensitive method, capillary electrophoresis (CE)-sodium dodecyl sulfate (SDS) combined with a laser-induced fluorescence (LIF) detector to monitor the impact of various bioprocess steps on the quality of different viral vectors. The fluorescence labelling procedure uses the (3-(2-furoyl) quinoline-2-carboxaldehyde dye, and the CE-SDS LIF method enables the evaluation of in-process besides final product samples. This method outperforms other analytical methods, such as SDS\u2013polyacrylamide gel electrophoresis with Sypro Ruby staining, in terms of sensitivity, resolution, and high-throughput capability. Notably, this CE-SDS LIF method was also successfully implemented to characterize enveloped viruses such as Maraba virus and lentivirus, whose development as biopharmaceuticals is now restricted by the lack of suitable analytical tools. This method was also qualified for quantification of rAAV2 according to the International Council for Harmonisation guidelines. Overall, our work shows that CE-SDS LIF is a precise and sensitive analytical platform for in-process sample analysis and quantification of different virus-based targets, with a great potential for application in biomanufacturing.<\/jats:p>","DOI":"10.3390\/v14112539","type":"journal-article","created":{"date-parts":[[2022,11,17]],"date-time":"2022-11-17T03:26:04Z","timestamp":1668655564000},"page":"2539","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Assessing Multi-Attribute Characterization of Enveloped and Non-Enveloped Viral Particles by Capillary Electrophoresis"],"prefix":"10.3390","volume":"14","author":[{"given":"Rita P.","family":"Fernandes","sequence":"first","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Avenida da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4857-9413","authenticated-orcid":false,"given":"Jos\u00e9 M.","family":"Escandell","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Avenida da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7733-6336","authenticated-orcid":false,"given":"Ana C. L.","family":"Guerreiro","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Avenida da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Filipa","family":"Moura","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Avenida da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Tiago Q.","family":"Faria","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Avenida da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5000-7217","authenticated-orcid":false,"given":"Sofia B.","family":"Carvalho","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Avenida da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2959-102X","authenticated-orcid":false,"given":"Ricardo J. S.","family":"Silva","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Avenida da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7245-6785","authenticated-orcid":false,"given":"Patr\u00edcia","family":"Gomes-Alves","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Avenida da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Cristina","family":"Peixoto","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Avenida da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2022,11,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1080\/14712598.2020.1693541","article-title":"Current challenges in biotherapeutic particles manufacturing","volume":"20","author":"Moleirinho","year":"2020","journal-title":"Expert Opin. Biol. Ther."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"237","DOI":"10.4155\/pbp.14.15","article-title":"Manufacturing of viral vectors: Part II. Downstream processing and safety aspects","volume":"2","author":"Merten","year":"2014","journal-title":"Pharm. Bioprocess."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"117681","DOI":"10.1016\/j.seppur.2020.117681","article-title":"3D-printed ordered bed structures for chromatographic purification of enveloped and non-enveloped viral particles","volume":"254","author":"Moleirinho","year":"2021","journal-title":"Sep. Purif. Technol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1700031","DOI":"10.1002\/biot.201700031","article-title":"Universal label-free in-process quantification of influenza virus-like particles","volume":"12","author":"Carvalho","year":"2017","journal-title":"Biotechnol. J."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1029","DOI":"10.1080\/14760584.2022.2072302","article-title":"Quantification methods for viruses and virus-like particles applied in biopharmaceutical production processes","volume":"21","author":"Lothert","year":"2022","journal-title":"Expert Rev. Vaccines"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"S152","DOI":"10.1016\/j.jcyt.2020.03.315","article-title":"The use of capillary electrophoresis in Gene Therapy","volume":"22","author":"Lock","year":"2020","journal-title":"Cytotherapy"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"338811","DOI":"10.1016\/j.aca.2021.338811","article-title":"Capillary electrophoresis of DNA with high resolution based on copoly(pentaerythritoltetra succinimidylcarboxypentyl\/aminopropyl polyoxyethylene) hydrogel","volume":"1178","author":"Wang","year":"2021","journal-title":"Anal. Chim. Acta"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1016\/j.chroma.2008.03.035","article-title":"Sodium dodecyl sulfate-capillary gel electrophoresis analysis of rotavirus-like particles","volume":"1192","author":"Mellado","year":"2008","journal-title":"J. Chromatogr. A"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"10218","DOI":"10.1021\/ac401930j","article-title":"Capillary electrophoresis\/mass spectrometry of APTS-labeled glycans for the identification of unknown glycan species in capillary electrophoresis\/laser-induced fluorescence systems","volume":"85","author":"Bunz","year":"2013","journal-title":"Anal. Chem."},{"key":"ref_10","first-page":"1133","article-title":"Application of affinity capillary electrophoresis in the study of protein-DNA interactions","volume":"38","author":"Yu","year":"2020","journal-title":"Se pu = Chin. J. Chromatogr."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2257","DOI":"10.1002\/elps.200405954","article-title":"On-line capillary electrophoresis-mass spectrometry for the analysis of biomolecules","volume":"25","author":"Cifuentes","year":"2004","journal-title":"Electrophoresis"},{"key":"ref_12","first-page":"1013","article-title":"Analysis of metabolomics and proteomics based on capillary electrophoresis-mass spectrometry","volume":"38","author":"Wang","year":"2020","journal-title":"Se pu = Chin. J. Chromatogr."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1002\/elps.202100182","article-title":"Applications of capillary electrophoresis for biopharmaceutical product characterization","volume":"43","author":"Kumar","year":"2022","journal-title":"Electrophoresis"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1016\/j.copbio.2021.12.009","article-title":"Leveraging rAAV bioprocess understanding and next generation bioanalytics development","volume":"74","author":"Escandell","year":"2022","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"116407","DOI":"10.1016\/j.trac.2021.116407","article-title":"Capillary electrophoresis and the biopharmaceutical industry: Therapeutic protein analysis and characterization","volume":"144","author":"Kaur","year":"2021","journal-title":"TrAC-Trends Anal. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"6583","DOI":"10.1021\/ac060828p","article-title":"Optimization and validation of a quantitative capillary electrophoresis sodium dodecyl sulfate method for quality control and stability monitoring of monoclonal antibodies","volume":"78","author":"Tomlinson","year":"2006","journal-title":"Anal. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"815","DOI":"10.1002\/elps.201100528","article-title":"Quantitative impurity analysis of monoclonal antibody size heterogeneity by CE-LIF: Example of development and validation through a quality-by-design framework","volume":"33","author":"Michels","year":"2012","journal-title":"Electrophoresis"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"628","DOI":"10.1089\/hum.2020.233","article-title":"Capillary Electrophoresis-Sodium Dodecyl Sulfate with Laser-Induced Fluorescence Detection as a Highly Sensitive and Quality Control-Friendly Method for Monitoring Adeno-Associated Virus Capsid Protein Purity","volume":"32","author":"Zhang","year":"2021","journal-title":"Hum. Gene Ther."},{"key":"ref_19","unstructured":"EMA (1995). ICH Topic Q 2 (R1) Validation of Analytical Procedures: Text and Methodology."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1089\/hgtb.2013.046","article-title":"Generation and characterization of adeno-associated virus producer cell lines for research and preclinical vector production","volume":"24","author":"Martin","year":"2013","journal-title":"Hum. Gene Ther. Methods"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1016\/j.jviromet.2013.10.038","article-title":"Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery","volume":"196","author":"Chahal","year":"2014","journal-title":"J. Virol. Methods"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"5271","DOI":"10.1038\/s41598-018-23593-y","article-title":"LentiPro26: Novel stable cell lines for constitutive lentiviral vector production","volume":"8","author":"Rodrigues","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"118598","DOI":"10.1016\/j.seppur.2021.118598","article-title":"Enhancing the purification of Lentiviral vectors for clinical applications","volume":"274","author":"Moreira","year":"2021","journal-title":"Sep. Purif. Technol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/j.omtm.2017.12.004","article-title":"Universal Method for the Purification of Recombinant AAV Vectors of Differing Serotypes","volume":"9","author":"Nass","year":"2018","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_25","unstructured":"Li, T., Santos, M., and Guttman, A. (2022, October 19). Ultrahigh Sensitivity Analysis of Adeno-associated Virus (AAV) Capsid Proteins by Sodium Dodecyl Sulphate Capillary Gel Electrophoresis. Chromatography Today, Available online: https:\/\/www.chromatographytoday.com\/article\/bioanalytical\/40\/sciex\/ultrahigh-sensitivity-analysis-of-adeno-associated-virus-aavbr-capsid-proteins-by-sodium-dodecyl-sulphate-capillary-gel-electrophoresis\/2852."},{"key":"ref_26","unstructured":"Li, T., Malik, M., Yowanto, H., and Mollah, S. (2022, October 19). Purity Analysis of Adeno-Associated Virus (AAV) Capsid Proteins using CE-SDS Method. Available online: https:\/\/lcms.cz\/labrulez-bucket-strapi-h3hsga3\/AAV_purity_73f90a1968\/AAV-purity.pdf."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1136\/jitc-2019-000188","article-title":"Original research: Oncolytic adenovirus shapes the ovarian tumor microenvironment for potent tumor-infiltrating lymphocyte tumor reactivity","volume":"8","author":"Santos","year":"2020","journal-title":"J. Immunother. Cancer"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1642","DOI":"10.1038\/s41467-021-21935-5","article-title":"Adeno-associated virus capsid assembly is divergent and stochastic","volume":"12","author":"Bennett","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"3285","DOI":"10.1021\/acs.analchem.6b02933","article-title":"Sample Stacking Provides Three Orders of Magnitude Sensitivity Enhancement in SDS Capillary Gel Electrophoresis of Adeno-Associated Virus Capsid Proteins","volume":"89","author":"Zhang","year":"2017","journal-title":"Anal. Chem."},{"key":"ref_30","first-page":"1403","article-title":"Characterization of Adeno-Associated Virus Capsid Proteins with Two Types of VP3-Related Components by Capillary Gel Electrophoresis and Mass Spectrometry","volume":"32","author":"Oyama","year":"2021","journal-title":"Hum. Gene Ther."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1038\/s41434-018-0034-7","article-title":"Optimization of viral protein ratios for production of rAAV serotype 5 in the baculovirus system","volume":"25","author":"Bosma","year":"2018","journal-title":"Gene Ther."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2609","DOI":"10.1016\/j.xphs.2021.03.024","article-title":"Manufacturing challenges and rational formulation development for AAV viral vectors","volume":"110","author":"Srivastava","year":"2021","journal-title":"J. Pharm. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e213","DOI":"10.1002\/hsr2.213","article-title":"Physicochemical susceptibility of SARS-CoV-2 to disinfection and physical approach of prophylaxis","volume":"3","author":"Saadatpour","year":"2020","journal-title":"Health Sci. Rep."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1038\/s41434-019-0072-9","article-title":"Stability of the adeno-associated virus 8 reference standard material","volume":"26","author":"Broucque","year":"2019","journal-title":"Gene Ther."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.omtm.2019.08.001","article-title":"Improved GaLV-TR Glycoproteins to Pseudotype Lentiviral Vectors: Impact of Viral Protease Activity in the Production of LV Pseudotypes","volume":"15","author":"Mestre","year":"2019","journal-title":"Mol. Ther.-Methods Clin. Dev."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1186\/1477-5956-10-32","article-title":"Proteomic analysis of purified Newcastle disease virus particles","volume":"10","author":"Ren","year":"2012","journal-title":"Proteome Sci."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"147","DOI":"10.3390\/v2010147","article-title":"Comparative studies on retroviral proteases: Substrate specificity","volume":"2","year":"2010","journal-title":"Viruses"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Do Minh, A., Star, A.T., Stupak, J., Fulton, K.M., Haqqani, A.S., G\u00e9linas, J.F., Li, J., Twine, S.M., and Kamen, A.A. (2021). Characterization of extracellular vesicles secreted in lentiviral producing hek293sf cell cultures. Viruses, 13.","DOI":"10.20944\/preprints202103.0696.v1"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Labisch, J.J., Philip Wiese, G., Barnes, K., Bollmann, F., and Pflanz, K. (2021). Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach. PLoS ONE, 16.","DOI":"10.1371\/journal.pone.0254739"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"740","DOI":"10.1016\/j.omtm.2021.02.010","article-title":"Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies","volume":"20","author":"Gimpel","year":"2021","journal-title":"Mol. Ther.-Methods Clin. Dev."},{"key":"ref_41","first-page":"447","article-title":"Technical Requirements for Registration of Pharmaceuticals for Human Use: The ICH Process","volume":"1994","author":"Harron","year":"2013","journal-title":"Textb. Pharm. Med."},{"key":"ref_42","unstructured":"Booth, B.P., and Simon, W.C. (2004). Analytical method validation. New Drug Development, CRC Press."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/11\/2539\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:19:57Z","timestamp":1760145597000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/11\/2539"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,11,17]]},"references-count":42,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2022,11]]}},"alternative-id":["v14112539"],"URL":"https:\/\/doi.org\/10.3390\/v14112539","relation":{},"ISSN":["1999-4915"],"issn-type":[{"type":"electronic","value":"1999-4915"}],"subject":[],"published":{"date-parts":[[2022,11,17]]}}}