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After the expiration of its original patents, interest in producing biosimilars has increased, particularly to reduce treatment-related side effects. In this study, we compared two production methods for EcA2, purifying the enzyme from broth and from the soluble fraction of the cell pellet lysate. Using a converging purification workflow, we obtained 66.3 (\u00b12.3) mg\/L of l-asparaginase from broth and 29.2 (\u00b14.6) mg\/L from the cell pellet lysate, with specific activities of 136.3 (\u00b113.3) and 123.5 (\u00b110.3) IU\/mg, respectively. Both versions showed similar three-dimensional structures, thermal stability, and specific activity, with no significant differences in performance. Proteomic analysis revealed that purification from broth resulted in fewer host cell proteins than purification from the cell pellet lysate. Our results further suggest that the purification process from cell lysate is more susceptible to variability than purification from the broth. These findings demonstrate that while both production methods yield comparable enzymes in terms of structure and activity, purifying from broth may offer advantages in terms of lower contamination and better reproducibility.<\/jats:p>","DOI":"10.1021\/acsomega.5c01816","type":"journal-article","created":{"date-parts":[[2025,6,13]],"date-time":"2025-06-13T10:51:58Z","timestamp":1749811918000},"page":"29166-29179","source":"Crossref","is-referenced-by-count":1,"title":["Investigation of\nthe Impact of a Protein Source on\nthe Purification of\n                    <scp>l<\/scp>\n                    \u2011Asparaginase Type II from\n                    <i>Escherichia coli<\/i>"],"prefix":"10.1021","volume":"10","author":[{"given":"Anna Catharinna","family":"da Costa","sequence":"first","affiliation":[{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio de Janeiro, Brazil, RJ 21941-902"]},{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio de Janeiro, Brazil, RJ 21941-902"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Talita Stelling","family":"de Araujo","sequence":"additional","affiliation":[{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio de Janeiro, Brazil, RJ 21941-902"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna Carolina","family":"Lomba Pereira","sequence":"additional","affiliation":[{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio de Janeiro, Brazil, RJ 21941-902"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luis Ariel","family":"Espinosa Rodri\u0301guez","sequence":"additional","affiliation":[{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio de Janeiro, Brazil, RJ 21941-902"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leonardo","family":"Dingo do Lago","sequence":"additional","affiliation":[{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio de Janeiro, Brazil, RJ 21941-902"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Camila","family":"Dias Leite da Silva","sequence":"additional","affiliation":[{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio de Janeiro, Brazil, RJ 21941-902"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rafael Alves","family":"de Andrade","sequence":"additional","affiliation":[{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio de Janeiro, Brazil, RJ 21941-902"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lui\u0301s Mauri\u0301cio","family":"Trambaioli da Rocha e Lima","sequence":"additional","affiliation":[{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio de Janeiro, Brazil, RJ 21941-902"]},{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio de Janeiro, Brazil, RJ 21941-902"]},{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio\nde Janeiro, Brazil, RJ 21941-902"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5507-7142","authenticated-orcid":true,"given":"Fa\u0301bio C. S.","family":"Nogueira","sequence":"additional","affiliation":[{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio de Janeiro, Brazil, RJ 21941-902"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1329-6483","authenticated-orcid":true,"given":"Gilberto Barbosa","family":"Domont","sequence":"additional","affiliation":[{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio de Janeiro, Brazil, RJ 21941-902"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4921-8185","authenticated-orcid":true,"given":"Marcius da Silva","family":"Almeida","sequence":"additional","affiliation":[{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio de Janeiro, Brazil, RJ 21941-902"]},{"name":"Universidade Federal do Rio de Janeiro , , ,","place":["Rio de Janeiro, Brazil, RJ 21941-902"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2025,6,13]]},"reference":[{"issue":"2","key":"2026081812112901600_cit1","first-page":"535","article-title":"L-Asparaginase Vincristine, and Prednisone\nfor Induction of First Remission in Acute Lymphocytic Leukemia","volume":"37","author":"Ortega","year":"1977","journal-title":"Cancer Res."},{"issue":"4","key":"2026081812112901600_cit2","doi-asserted-by":"publisher","first-page":"387","DOI":"10.1002\/mpo.2950030410","article-title":"Optimal Use\nof L-Asparaginase (NSC-109229) in Acute Lymphocytic Leukemia","volume":"3","author":"Jones","year":"1977","journal-title":"Med. Pediatr. Oncol."},{"issue":"11","key":"2026081812112901600_cit3","doi-asserted-by":"publisher","first-page":"657","DOI":"10.1056\/NEJM198609113151101","article-title":"Four-Agent Induction and Intensive\nAsparaginase Therapy for Treatment of Childhood Acute Lymphoblastic\nLeukemia","volume":"315","author":"Clavell","year":"1986","journal-title":"N. Engl. J. Med."},{"issue":"3","key":"2026081812112901600_cit4","doi-asserted-by":"publisher","first-page":"335","DOI":"10.1038\/sj.leu.2401310","article-title":"Intensive High-Dose\nAsparaginase Consolidation Improves\nSurvival for Pediatric Patients with T Cell Acute Lymphoblastic Leukemia\nand Advanced Stage Lymphoblastic Lymphoma: A Pediatric Oncology Group\nStudy","volume":"13","author":"Amylon","year":"1999","journal-title":"Leukemia"},{"issue":"11","key":"2026081812112901600_cit5","doi-asserted-by":"publisher","first-page":"3310","DOI":"10.1182\/blood.V95.11.3310","article-title":"Improved Outcome in Childhood Acute\nLymphoblastic Leukemia despite Reduced Use of Anthracyclines and Cranial\nRadiotherapy: Results of Trial ALL-BFM 90. German-Austrian-Swiss ALL-BFM\nStudy Group","volume":"95","author":"Schrappe","year":"2000","journal-title":"Blood"},{"issue":"3","key":"2026081812112901600_cit6","doi-asserted-by":"publisher","first-page":"356","DOI":"10.1038\/sj.leu.2401704","article-title":"Benefit of\nIntensified Treatment for All Children with Acute Lymphoblastic Leukaemia:\nResults from MRC UKALL XI and MRC ALL97 Randomised Trials","volume":"14","author":"Hann","year":"2000","journal-title":"Leukemia"},{"issue":"1","key":"2026081812112901600_cit7","doi-asserted-by":"publisher","first-page":"237","DOI":"10.1200\/JCO.2002.20.1.237","article-title":"Treatment\nof Childhood Acute Lymphoblastic Leukemia: Results of Dana-Farber\nALL Consortium Protocol 87\u201301","volume":"20","author":"LeClerc","year":"2002","journal-title":"J. Clin.\nOncol."},{"issue":"16","key":"2026081812112901600_cit8","doi-asserted-by":"publisher","first-page":"1677","DOI":"10.1016\/S1470-2045(15)00363-0","article-title":"Intravenous\nPegylated Asparaginase versus Intramuscular Native Escherichia Coli\nL-Asparaginase in Newly Diagnosed Childhood Acute Lymphoblastic Leukaemia\n(DFCI 05\u2013001): A Randomised, Open-Label Phase 3 Trial","volume":"16","author":"Place","year":"2015","journal-title":"Lancet Oncol."},{"issue":"20","key":"2026081812112901600_cit9","doi-asserted-by":"publisher","first-page":"1927","DOI":"10.1021\/acs.biochem.0c00116","article-title":"Mechanism of Catalysis by l-Asparaginase","volume":"59","author":"Lubkowski","year":"2020","journal-title":"Biochemistry"},{"issue":"4","key":"2026081812112901600_cit10","first-page":"294","article-title":"Biochemical Characterization\nof U937\nCells Resistant to L-Asparaginase: The Role of Asparagine Synthetase","volume":"3","author":"Kiriyama","year":"1989","journal-title":"Leukemia"},{"issue":"1","key":"2026081812112901600_cit11","doi-asserted-by":"publisher","first-page":"43","DOI":"10.1016\/0006-291X(68)90028-4","article-title":"Asparagine Synthetase in Asparaginase Resistant and\nSusceptible Mouse Lymphomas","volume":"31","author":"Prager","year":"1968","journal-title":"Biochem. Biophys.\nRes. Commun."},{"issue":"6","key":"2026081812112901600_cit12","doi-asserted-by":"publisher","first-page":"565","DOI":"10.1084\/jem.98.6.565","article-title":"Regression\nOf Transplanted Lymphomas Induced In Vivo By Means Of Normal Guinea\nPig Serum","volume":"98","author":"Kidd","year":"1953","journal-title":"J. Exp. Med."},{"issue":"6","key":"2026081812112901600_cit13","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1084\/jem.98.6.583","article-title":"Regression\nOf Transplanted Lymphomas Induced In Vivo By Means Of Normal Guinea\nPig Serum","volume":"98","author":"Kidd","year":"1953","journal-title":"J. Exp. Med."},{"issue":"1","key":"2026081812112901600_cit14","doi-asserted-by":"publisher","first-page":"99","DOI":"10.1084\/jem.118.1.99","article-title":"Evidence\nThat The L-Asparaginase Of Guinea Pig Serum Is Responsible For Its\nAntilymphoma Effects","volume":"118","author":"Broome","year":"1963","journal-title":"J. Exp. Med."},{"issue":"2","key":"2026081812112901600_cit15","doi-asserted-by":"publisher","first-page":"450","DOI":"10.1016\/0003-9861(64)90032-3","article-title":"Tumor Inhibitory\nEffect of L-Asparaginase from Escherichia\nColi","volume":"105","author":"Mashburn","year":"1964","journal-title":"Arch. Biochem. Biophys."},{"issue":"3","key":"2026081812112901600_cit16","doi-asserted-by":"publisher","first-page":"721","DOI":"10.1021\/bi00855a011","article-title":"Two L-Asparaginases\nfrom Escherichia Coli B. Their Separation, Purification, and Antitumor\nActivity","volume":"6","author":"Campbell","year":"1967","journal-title":"Biochemistry"},{"issue":"6","key":"2026081812112901600_cit17","doi-asserted-by":"publisher","first-page":"2386","DOI":"10.1021\/bi00834a020","article-title":"Purification and Properties of Asparaginase\nfrom Escherichia\nColi B","volume":"8","author":"Whelan","year":"1969","journal-title":"Biochemistry"},{"issue":"3","key":"2026081812112901600_cit18","doi-asserted-by":"publisher","first-page":"408","DOI":"10.1002\/cpt1970113408","article-title":"Clinical Pharmacologic\nStudies of L-Asparaginase","volume":"11","author":"Ho","year":"1970","journal-title":"Clin. Pharmacol.\nTher."},{"issue":"11","key":"2026081812112901600_cit19","doi-asserted-by":"publisher","DOI":"10.1002\/pbc.29046","article-title":"Unsatisfactory\nQuality of E. Coli Asparaginase Biogenerics in India:\nImplications for Clinical Outcomes in Acute Lymphoblastic Leukaemia","volume":"68","author":"Sidhu","year":"2021","journal-title":"Pediatr. Blood Cancer"},{"issue":"6","key":"2026081812112901600_cit20","doi-asserted-by":"publisher","first-page":"1081","DOI":"10.1111\/bcp.14216","article-title":"A Comparison of Asparaginase Activity in Generic Formulations of E.Coli Derived L- Asparaginase: In-vitro Study and Retrospective\nAnalysis of Asparaginase Monitoring in Pediatric Patients with Leukemia","volume":"86","author":"Sankaran","year":"2020","journal-title":"Br. J. Clin. Pharmacol."},{"issue":"9","key":"2026081812112901600_cit21","doi-asserted-by":"publisher","first-page":"1149","DOI":"10.1016\/S1470-2045(18)30584-9","article-title":"Monitoring\nAsparaginase Activity in Middle-Income Countries","volume":"19","author":"Cecconello","year":"2018","journal-title":"Lancet Oncol."},{"issue":"2","key":"2026081812112901600_cit22","doi-asserted-by":"publisher","first-page":"97","DOI":"10.1016\/S1040-8428(98)00015-8","article-title":"Use of L-Asparaginase in Childhood\nALL","volume":"28","author":"Mu\u0308ller","year":"1998","journal-title":"Crit.\nRev. Oncol. Hematol."},{"issue":"1","key":"2026081812112901600_cit23","doi-asserted-by":"publisher","first-page":"151","DOI":"10.1099\/00221287-132-1-151","article-title":"Cloning\nand Expression\nof the Erwinia Chrysanthemi Asparaginase Gene in Escherichia Coli\nand Erwinia Carotovora","volume":"132","author":"Gilbert","year":"1986","journal-title":"Microbiology"},{"issue":"2\u20133","key":"2026081812112901600_cit24","doi-asserted-by":"publisher","first-page":"144","DOI":"10.1016\/1046-5928(91)90063-O","article-title":"Construction of\nExpression Systems for Escherichia\nCofi Asparaginase II and Two-Step Purification of the Recombinant\nEnzyme from Periplasmic Extracts","volume":"2","author":"Harms","year":"1991","journal-title":"Protein Expression\nPurif."},{"issue":"1","key":"2026081812112901600_cit25","doi-asserted-by":"publisher","first-page":"29","DOI":"10.1016\/j.pep.2004.07.009","article-title":"Extracellular Expression and Single\nStep Purification of Recombinant Escherichia Coli L-Asparaginase II","volume":"38","author":"Khushoo","year":"2004","journal-title":"Protein Expression Purif."},{"issue":"2","key":"2026081812112901600_cit26","doi-asserted-by":"publisher","first-page":"189","DOI":"10.1007\/s00253-004-1867-0","article-title":"Optimization\nof Extracellular Production of Recombinant\nAsparaginase in Escherichia Coli in Shake-Flask and Bioreactor","volume":"68","author":"Khushoo","year":"2005","journal-title":"Appl. Microbiol. Biotechnol."},{"issue":"11","key":"2026081812112901600_cit27","doi-asserted-by":"publisher","first-page":"4515","DOI":"10.1007\/s00253-021-11359-y","article-title":"L-Asparaginase\nProduction Review: Bioprocess Design and Biochemical Characteristics","volume":"105","author":"Castro","year":"2021","journal-title":"Appl. Microbiol. Biotechnol."},{"issue":"2","key":"2026081812112901600_cit28","doi-asserted-by":"publisher","first-page":"99","DOI":"10.1007\/BF02898715","article-title":"Merits\nof Secretion of Heterologous Proteins from Industrial Microorganisms","volume":"42","author":"Quax","year":"1997","journal-title":"Folia Microbiol."},{"key":"2026081812112901600_cit29","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/S0065-2164(10)73001-X","article-title":"Heterologous protein secretion by bacillus species\nfrom the cradle to the grave","volume":"73","author":"Pohl","year":"2010","journal-title":"Adv. Appl. Microbiol."},{"issue":"1","key":"2026081812112901600_cit30","doi-asserted-by":"publisher","first-page":"196","DOI":"10.1186\/s12934-018-1041-5","article-title":"Developing\nGram-Negative Bacteria for the Secretion of Heterologous Proteins","volume":"17","author":"Burdette","year":"2018","journal-title":"Microb. Cell Fact."},{"issue":"3","key":"2026081812112901600_cit31","doi-asserted-by":"publisher","first-page":"177","DOI":"10.1016\/j.biotechadv.2004.11.003","article-title":"Recombinant Protein Secretion in\nEscherichia Coli","volume":"23","author":"Mergulha\u0303o","year":"2005","journal-title":"Biotechnol. Adv."},{"key":"2026081812112901600_cit32","doi-asserted-by":"publisher","first-page":"797334","DOI":"10.3389\/fbioe.2021.797334","article-title":"Strategies to Enhance\nPeriplasmic Recombinant Protein\nProduction Yields in Escherichia Coli","volume":"9","author":"Karyolaimos","year":"2021","journal-title":"Front.\nBioeng. Biotechnol."},{"issue":"1","key":"2026081812112901600_cit33","doi-asserted-by":"publisher","first-page":"32","DOI":"10.1186\/1471-2091-9-32","article-title":"Modification of Carbonic Anhydrase\nII with Acetaldehyde, the First Metabolite of Ethanol, Leads to Decreased\nEnzyme Activity","volume":"9","author":"Bootorabi","year":"2008","journal-title":"BMC Biochem."},{"key":"2026081812112901600_cit34","doi-asserted-by":"publisher","first-page":"158","DOI":"10.1016\/j.ebiom.2018.03.005","article-title":"Low Bioavailability\nand High Immunogenicity of a New Brand of E. 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