{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T10:14:30Z","timestamp":1788430470890,"version":"build-2803163510"},"reference-count":28,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:00:00Z","timestamp":1788393600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000865","name":"Gates Foundation","doi-asserted-by":"publisher","award":["INV-035430"],"award-info":[{"award-number":["INV-035430"]}],"id":[{"id":"10.13039\/100000865","id-type":"DOI","asserted-by":"publisher"}]},{"award":["INV-035430"],"award-info":[{"award-number":["INV-035430"]}],"id":[{"id":"https:\/\/ror.org\/0456r8d26","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background\/Objectives: The rapid emergence of SARS-CoV-2 variants that evade neutralizing antibodies underscores the need for new antiviral biologics that integrate precise molecular design with scalable, cost-effective manufacturing. Computationally designed miniproteins targeting the spike protein\u2019s receptor-binding domain (RBD) offer a promising alternative to monoclonal antibodies because of their small size, high thermal stability, and compatibility with microbial expression systems. Here, we detail the full development and cGMP manufacturing of IPD-52520, a novel antiviral miniprotein produced using an optimized E. coli platform. Methods: Two candidate miniproteins, a homotrimeric construct (Trimer, IPD-52520, 17 kDa) and a tandem fusion construct (Daisy, IPD-52521, 25 kDa), were evaluated in parallel through systematic optimization of strain selection, media formulation, fed-batch fermentation, inclusion-body solubilization, refolding, and chromatographic purification. The leading candidate was scaled from 5 L to 50 L under cGMP conditions, and biophysical and stability analyses were conducted to support nonclinical and Phase 1 clinical development. Results: The Trimer was selected as the lead molecule based on superior preclinical efficacy, favorable pharmacokinetics, and higher volumetric yields. The optimized process yields about 2 g\/L of purified protein at over 90% purity. Scale-up from 5 L to 50 L demonstrated excellent batch consistency across six independent runs. Biophysical studies confirmed a well-folded, predominantly alpha-helical trimer (Tm = 73.4 \u00b0C; polydispersity = 1.005) with an intact primary structure and strong target binding (KD &lt; 1 pM). Stability studies show the drug remains stable at 2\u20138 \u00b0C for at least 12 months, with ongoing long-term studies. Conclusions: These findings demonstrate that computationally designed antiviral miniproteins can be translated into scalable biologics and establish a platform for rapid therapeutic development against current and future pandemics.<\/jats:p>","DOI":"10.3390\/pharmaceutics18091110","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T09:20:43Z","timestamp":1788427243000},"page":"1110","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Scalable Production of a De Novo SARS-CoV-2 Antiviral Miniprotein in Escherichia coli"],"prefix":"10.3390","volume":"18","author":[{"given":"Jinhwan","family":"Shin","sequence":"first","affiliation":[{"name":"SK bioscience Co., Ltd., 38, Yeongudanji-ro, Yeonsu-gu, Incheon 13493, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eu-min","family":"Kim","sequence":"additional","affiliation":[{"name":"SK bioscience Co., Ltd., 38, Yeongudanji-ro, Yeonsu-gu, Incheon 13493, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun-hong","family":"Jang","sequence":"additional","affiliation":[{"name":"SK bioscience Co., Ltd., 38, Yeongudanji-ro, Yeonsu-gu, Incheon 13493, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-1382-1328","authenticated-orcid":false,"given":"Seok-won","family":"Jee","sequence":"additional","affiliation":[{"name":"SK bioscience Co., Ltd., 38, Yeongudanji-ro, Yeonsu-gu, Incheon 13493, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sang-hoon","family":"Kim","sequence":"additional","affiliation":[{"name":"SK bioscience Co., Ltd., 38, Yeongudanji-ro, Yeonsu-gu, Incheon 13493, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7590-6056","authenticated-orcid":false,"given":"Seonggwan","family":"Yu","sequence":"additional","affiliation":[{"name":"SK bioscience Co., Ltd., 38, Yeongudanji-ro, Yeonsu-gu, Incheon 13493, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7732-9646","authenticated-orcid":false,"given":"Minguen","family":"Yoon","sequence":"additional","affiliation":[{"name":"SK bioscience Co., Ltd., 38, Yeongudanji-ro, Yeonsu-gu, Incheon 13493, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Craig","sequence":"additional","affiliation":[{"name":"International AIDs Vaccine Initiative (IAVI), 125 Broad Street, 9th Floor, New York, NY 10004, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryan","family":"Swoyer","sequence":"additional","affiliation":[{"name":"International AIDs Vaccine Initiative (IAVI), 125 Broad Street, 9th Floor, New York, NY 10004, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-7100-0720","authenticated-orcid":false,"given":"Sandip","family":"Patel","sequence":"additional","affiliation":[{"name":"International AIDs Vaccine Initiative (IAVI), 125 Broad Street, 9th Floor, New York, NY 10004, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Praveen","family":"Alamuri","sequence":"additional","affiliation":[{"name":"International AIDs Vaccine Initiative (IAVI), 125 Broad Street, 9th Floor, New York, NY 10004, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Albert","family":"Price","sequence":"additional","affiliation":[{"name":"International AIDs Vaccine Initiative (IAVI), 125 Broad Street, 9th Floor, New York, NY 10004, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2630-7487","authenticated-orcid":false,"given":"Rashmi","family":"Ravichandran","sequence":"additional","affiliation":[{"name":"Institute for Protein Design (IPD), University of Washington, 330 Yale Ave North, Seattle, WA 98109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lauren","family":"Carter","sequence":"additional","affiliation":[{"name":"Institute for Protein Design (IPD), University of Washington, 330 Yale Ave North, Seattle, WA 98109, USA"},{"name":"Gates Medical Research Institute, One Kendall Square, Building 600, Suite 6-301, Cambridge, MA 02139, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-7973-0332","authenticated-orcid":false,"given":"Sammaiah","family":"Pallerla","sequence":"additional","affiliation":[{"name":"International AIDs Vaccine Initiative (IAVI), 125 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