{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T10:17:19Z","timestamp":1785406639127,"version":"3.56.0"},"reference-count":33,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T00:00:00Z","timestamp":1776902400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000005","name":"United States Department of Defense","doi-asserted-by":"publisher","award":["W911SR22D0009"],"award-info":[{"award-number":["W911SR22D0009"]}],"id":[{"id":"10.13039\/100000005","id-type":"DOI","asserted-by":"publisher"}]},{"award":["W911SR22D0009"],"award-info":[{"award-number":["W911SR22D0009"]}],"id":[{"id":"https:\/\/ror.org\/0447fe631","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000774","name":"Defense Threat Reduction Agency","doi-asserted-by":"publisher","award":["W15QKN-16-9-1002"],"award-info":[{"award-number":["W15QKN-16-9-1002"]}],"id":[{"id":"10.13039\/100000774","id-type":"DOI","asserted-by":"publisher"}]},{"award":["W15QKN-16-9-1002"],"award-info":[{"award-number":["W15QKN-16-9-1002"]}],"id":[{"id":"https:\/\/ror.org\/04tz64554","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>A highly potent antitoxin for botulinum neurotoxin (BoNT) serotypes A and B has been developed that comprises three monoclonal antibodies (mAbs) targeting BoNT\/A and three targeting BoNT\/B. These oligoclonal antibody combinations neutralize toxin by simultaneously binding non-overlapping epitopes, thereby promoting rapid toxin clearance. All six mAbs use the same human Fc and framework and have been individually manufactured using the same expression platform and purification process. To minimize the time and labor required to produce the divalent antitoxin, we tested a co-expression and co-purification strategy for the three mAbs per serotype. The mAbs were expressed in CHO-K1 cells, and the media were optimized for co-expression in 10 L bioreactors. Chromatographic co-purification consisted of Protein A capture, followed by strong anion exchange chromatography in flow-through mode and cation-exchange chromatography in bind-elute mode. Co-expression experiments demonstrated that expression of the three anti-BoNT\/A antibodies remained within approximately \u00b130% of the optimal equimolar ratio, whereas the anti-BoNT\/B antibodies showed greater variability. Downstream purification steps achieved recoveries greater than 95% per chromatographic step, resulting in overall process yields of approximately 63\u201375%. This strategy provided sufficient purity of all six mAbs while largely preserving their relative ratios. These results demonstrate the feasibility of producing oligoclonal antitoxin antibodies using co-expression and shared purification strategies. Such approaches may simplify the manufacturing of antibody cocktails while maintaining product quality and biological activity.<\/jats:p>","DOI":"10.3390\/toxins18050199","type":"journal-article","created":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T17:04:59Z","timestamp":1776963899000},"page":"199","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Co-Expression and Co-Purification Enable Manufacturing of a Six-Monoclonal Antibody Botulinum Antitoxin Cocktail"],"prefix":"10.3390","volume":"18","author":[{"given":"Andrew","family":"Davis","sequence":"first","affiliation":[{"name":"Resilience Government Services, 2061 Challenger Drive, Alameda, CA 94501, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kamaljit","family":"Bajwa","sequence":"additional","affiliation":[{"name":"Resilience Government Services, 2061 Challenger Drive, Alameda, CA 94501, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zachary","family":"Martinez","sequence":"additional","affiliation":[{"name":"Resilience Government Services, 2061 Challenger Drive, Alameda, CA 94501, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9695-4356","authenticated-orcid":false,"given":"Ryan R.","family":"Davis","sequence":"additional","affiliation":[{"name":"Resilience Government Services, 13200 NW Nano Court, Alachua, FL 32615, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"April","family":"Green","sequence":"additional","affiliation":[{"name":"Resilience Government Services, 13200 NW Nano Court, Alachua, FL 32615, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fletcher","family":"Suber","sequence":"additional","affiliation":[{"name":"Resilience Government Services, 13200 NW Nano Court, Alachua, FL 32615, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4130-6332","authenticated-orcid":false,"given":"Shauna","family":"Farr-Jones","sequence":"additional","affiliation":[{"name":"Department of Anesthesia, University of California, 1001 Potrero Ave, San Francisco, CA 94110, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Milan T.","family":"Tomic","sequence":"additional","affiliation":[{"name":"Resilience Government Services, 2061 Challenger Drive, Alameda, CA 94501, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"11346","DOI":"10.1073\/pnas.172229899","article-title":"Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody","volume":"99","author":"Nowakowski","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Fan, Y., Garcia-Rodriguez, C., Lou, J., Wen, W., Conrad, F., Zhai, W., Smith, T.J., Smith, L.A., and Marks, J.D. (2017). A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0174187"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Fan, Y., Lou, J., Tam, C.C., Wen, W., Conrad, F., Leal da Silva Alves, P., Cheng, L.W., Garcia-Rodriguez, C., Farr-Jones, S., and Marks, J.D. (2023). A Three-Monoclonal Antibody Combination Potently Neutralizes BoNT\/G Toxin in Mice. Toxins, 15.","DOI":"10.3390\/toxins15050316"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Garcia-Rodriguez, C., Razai, A., Geren, I.N., Lou, J., Conrad, F., Wen, W.-H., Farr-Jones, S., Smith, T.J., Brown, J.L., and Skerry, J.C. (2018). A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype E subtypes. Toxins, 10.","DOI":"10.3390\/toxins10030105"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Garcia-Rodriguez, C., Yan, S., Geren, I.N., Knopp, K.A., Dong, J., Sun, Z., Lou, J., Conrad, F., Wen, W.-H., and Farr-Jones, S. (2021). A Four-Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotypes C and D. Toxins, 13.","DOI":"10.3390\/toxins13090641"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"5047","DOI":"10.1128\/AAC.02830-14","article-title":"Safety and pharmacokinetics of XOMA 3AB, a novel mixture of three monoclonal antibodies against botulinum toxin A","volume":"58","author":"Nayak","year":"2014","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e0232920","DOI":"10.1128\/AAC.02329-20","article-title":"Safety, Tolerability, and Pharmacokinetics of NTM-1632, a Novel Mixture of Three Monoclonal Antibodies against Botulinum Toxin B","volume":"65","author":"Guptill","year":"2021","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Snow, D.M., Cobb, R.R., Martinez, J., Finger-Baker, I., Collins, L., Terpening, S., Syar, E.S., Niemuth, N., Kobs, D., and Barnewall, R. (2021). A Monoclonal Antibody Combination against both Serotypes A and B Botulinum Toxin Prevents Inhalational Botulism in a Guinea Pig Model. Toxins, 13.","DOI":"10.3390\/toxins13010031"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Tomic, M.T., Farr-Jones, S., Syar, E.S., Niemuth, N., Kobs, D., Hackett, M.J., Espinoza, Y., Martinez, Z., Pham, K., and Snow, D.M. (2021). Neutralizing Concentrations of Anti-Botulinum Toxin Antibodies Positively Correlate with Mouse Neutralization Assay Results in a Guinea Pig Model. Toxins, 13.","DOI":"10.3390\/toxins13090671"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1016\/j.tibtech.2010.02.001","article-title":"Recent advances in large-scale production of monoclonal antibodies and related proteins","volume":"28","author":"Shukla","year":"2010","journal-title":"Trends Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e3139","DOI":"10.1002\/btpr.3139","article-title":"Process and operations strategies to enable global access to antibody therapies","volume":"37","author":"Kelley","year":"2021","journal-title":"Biotechnol. Prog."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1016\/j.abb.2012.07.001","article-title":"Recombinant antibody mixtures: Production strategies and cost considerations","volume":"526","author":"Rasmussen","year":"2012","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"753","DOI":"10.1038\/nbt.2621","article-title":"Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies","volume":"31","author":"Spiess","year":"2013","journal-title":"Nat. Biotechnol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s12929-019-0592-z","article-title":"Development of therapeutic antibodies for the treatment of diseases","volume":"27","author":"Lu","year":"2020","journal-title":"J. Biomed. Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"480","DOI":"10.4161\/mabs.2.5.12645","article-title":"Recovery and purification process development for monoclonal antibody production","volume":"2","author":"Liu","year":"2010","journal-title":"MAbs"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"635","DOI":"10.2105\/AJPH.2020.306138","article-title":"The Pandemic and the Supply Chain: Gaps in Pharmaceutical Production and Distribution","volume":"111","author":"Socal","year":"2021","journal-title":"Am. J. Public Health"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1016\/j.nbt.2011.03.018","article-title":"Antibodies in infectious diseases: Polyclonals, monoclonals and niche biotechnology","volume":"28","author":"Berry","year":"2011","journal-title":"New Biotechnol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1014","DOI":"10.1126\/science.abd0831","article-title":"Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies","volume":"369","author":"Baum","year":"2020","journal-title":"Science"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3534","DOI":"10.1038\/s41591-024-03247-5","article-title":"Safety and antiviral effect of a triple combination of HIV-1 broadly neutralizing antibodies: A phase 1\/2a trial","volume":"30","author":"Julg","year":"2024","journal-title":"Nat. Med."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Larbouret, C., Gros, L., P\u00e8legrin, A., and Chard\u00e8s, T. (2021). Improving Biologics\u2019 Effectiveness in Clinical Oncology: From the Combination of Two Monoclonal Antibodies to Oligoclonal Antibody Mixtures. Cancers, 13.","DOI":"10.3390\/cancers13184620"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1056\/NEJMoa1113216","article-title":"Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer","volume":"366","author":"Baselga","year":"2012","journal-title":"N. Engl. J. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1535","DOI":"10.1056\/NEJMoa1910836","article-title":"Five-Year Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma","volume":"381","author":"Larkin","year":"2019","journal-title":"N. Engl. J. Med."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2293","DOI":"10.1056\/NEJMoa1910993","article-title":"A Randomized, Controlled Trial of Ebola Virus Disease Therapeutics","volume":"381","author":"Mulangu","year":"2019","journal-title":"N. Engl. J. Med."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e231","DOI":"10.1016\/S1473-3099(20)30282-6","article-title":"Recent successes in therapeutics for Ebola virus disease: No time for complacency","volume":"20","author":"Iversen","year":"2020","journal-title":"Lancet Infect. Dis."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Rasetti-Escargueil, C., Avril, A., Miethe, S., Mazuet, C., Derman, Y., Selby, K., Thullier, P., Pelat, T., Urbain, R., and Fontayne, A. (2017). The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies. Toxins, 9.","DOI":"10.3390\/toxins9100309"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e202400348","DOI":"10.1002\/biot.202400348","article-title":"Single-Batch Expression of an Experimental Recombinant Snakebite Antivenom Based on an Oligoclonal Mixture of Human Monoclonal Antibodies","volume":"19","author":"Adams","year":"2024","journal-title":"Biotechnol. J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e0173221","DOI":"10.1128\/aac.01732-21","article-title":"First-in-Human Clinical Trial to Assess the Safety, Tolerability and Pharmacokinetics of Single Doses of NTM-1633, a Novel Mixture of Monoclonal Antibodies against Botulinum Toxin E","volume":"66","author":"Raja","year":"2022","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"e01270-19","DOI":"10.1128\/AAC.01270-19","article-title":"Safety and Pharmacokinetics of a Four Monoclonal Antibody Combination Against Botulinum C and D Neurotoxins","volume":"63","author":"Snow","year":"2019","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Li, M., Lee, D., Obi, C.R., Freeberg, J.K., Farr-Jones, S., and Tomic, M.T. (2018). An ambient temperature-stable antitoxin of nine co-formulated antibodies for botulism caused by serotypes A, B and E. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0197011"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.jbiotec.2021.06.004","article-title":"Improving mAb capture productivity on batch and continuous downstream processing using nanofiber PrismA adsorbents","volume":"336","author":"Davis","year":"2021","journal-title":"J. Biotechnol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2149","DOI":"10.1016\/j.xphs.2022.05.014","article-title":"It is Never Too Late for a Cocktail-Development and Analytical Characterization of Fixed-dose Antibody Combinations","volume":"111","author":"Krieg","year":"2022","journal-title":"J. Pharm. Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/j.ab.2012.08.005","article-title":"Engineered domain-based assays to identify individual antibodies in oligoclonal combinations targeting the same protein","volume":"430","author":"Meng","year":"2012","journal-title":"Anal. Biochem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1016\/j.ab.2011.09.030","article-title":"Domain-based assays of individual antibody concentrations in an oligoclonal combination targeting a single protein","volume":"421","author":"Meng","year":"2012","journal-title":"Anal. Biochem."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/18\/5\/199\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T17:11:11Z","timestamp":1776964271000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/18\/5\/199"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,23]]},"references-count":33,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["toxins18050199"],"URL":"https:\/\/doi.org\/10.3390\/toxins18050199","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,23]]}}}