{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,15]],"date-time":"2025-11-15T05:23:29Z","timestamp":1763184209021,"version":"3.45.0"},"reference-count":38,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T00:00:00Z","timestamp":1762992000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012190","name":"Ministry of Science and Higher Education of the Russian Federation","doi-asserted-by":"crossref","award":["075-15-2025-526"],"award-info":[{"award-number":["075-15-2025-526"]}],"id":[{"id":"10.13039\/501100012190","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background\/Objectives: The neonatal Fc receptor (FcRn) is a heterodimeric protein composed of a heavy \u03b1-chain with an MHC class I-like fold and \u03b22-microglobulin. It plays a crucial role in maintaining the homeostasis and pharmacokinetics of immunoglobulin G (IgG) and albumin through pH-dependent recycling. The production of soluble recombinant FcRn is technically challenging due to its heterodimeric structure and the presence of a transmembrane domain. This study aimed to develop a polycistronic construct enabling the co-expression of FcRn subunits from a single transcript and to evaluate the functional activity of the resulting protein in CHO-K1 cells. Methods: Integration vectors (pComV-FcRn-B2M) were designed to encode FcRn and \u03b22-microglobulin linked via self-cleaving 2A peptides (P2A, E2A, F2A, T2A). Stable producer cell lines were generated using the Sleeping Beauty transposon system. The purified proteins were characterized by SDS-PAGE, Western blotting, and size-exclusion chromatography (SEC). Functional activity was assessed by ELISA and bio-layer interferometry (BLI). Results: Electrophoretic and chromatographic analyses confirmed the expected subunit composition and demonstrated that over 95% of the recombinant protein was monomeric. Functional assays revealed pH-dependent IgG binding, with strong interaction at pH 6.0 and negligible binding at pH 7.5. BLI measurements showed high affinity consistent with native FcRn function (KD = 3.15 nM at pH 6.0). Conclusions: The developed polycistronic construct containing a P2A peptide with a GSG linker enabled efficient production of functional FcRn in CHO-K1 cells (yield up to 2.23 mg\/mL). The P2A variant demonstrated the highest efficiency and can serve as a reference system for screening Fc-engineered antibodies with optimized pharmacokinetic properties.<\/jats:p>","DOI":"10.3390\/pharmaceutics17111463","type":"journal-article","created":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T09:10:45Z","timestamp":1763025045000},"page":"1463","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Does a Polycistronic 2A Design Enable Functional FcRn Production for Antibody Pharmacokinetic Studies?"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1091-3586","authenticated-orcid":false,"given":"Valentina S.","family":"Nesmeyanova","sequence":"first","affiliation":[{"name":"State Scientific Center of Virology and Biotechnology \u201cVector\u201d, Rospotrebnadzor, 630559 Koltsovo, Novosibirsk Region, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2171-7444","authenticated-orcid":false,"given":"Nikita D.","family":"Ushkalenko","sequence":"additional","affiliation":[{"name":"State Scientific Center of Virology and Biotechnology \u201cVector\u201d, Rospotrebnadzor, 630559 Koltsovo, Novosibirsk Region, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sergei E.","family":"Olkin","sequence":"additional","affiliation":[{"name":"State Scientific Center of Virology and Biotechnology \u201cVector\u201d, Rospotrebnadzor, 630559 Koltsovo, Novosibirsk Region, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8023-0601","authenticated-orcid":false,"given":"Maksim N.","family":"Kosenko","sequence":"additional","affiliation":[{"name":"State Scientific Center of Virology and Biotechnology \u201cVector\u201d, Rospotrebnadzor, 630559 Koltsovo, Novosibirsk Region, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3465-4577","authenticated-orcid":false,"given":"Elena A.","family":"Rukhlova","sequence":"additional","affiliation":[{"name":"State Scientific Center of Virology and Biotechnology \u201cVector\u201d, Rospotrebnadzor, 630559 Koltsovo, Novosibirsk Region, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9718-7339","authenticated-orcid":false,"given":"Ivan M.","family":"Susloparov","sequence":"additional","affiliation":[{"name":"State Scientific Center of Virology and Biotechnology \u201cVector\u201d, Rospotrebnadzor, 630559 Koltsovo, Novosibirsk Region, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8023-4453","authenticated-orcid":false,"given":"Dmitry N.","family":"Shcherbakov","sequence":"additional","affiliation":[{"name":"State Scientific Center of Virology and Biotechnology \u201cVector\u201d, Rospotrebnadzor, 630559 Koltsovo, Novosibirsk Region, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"729","DOI":"10.1007\/s13311-021-01175-7","article-title":"Neonatal Fc Receptor-Targeted Therapies in Neurology","volume":"19","author":"Nelke","year":"2022","journal-title":"Neurotherapeutics"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2393","DOI":"10.1007\/s13555-024-01253-6","article-title":"Extended Half-life Antibodies: A Narrative Review of a New Approach in the Management of Atopic Dermatitis","volume":"14","author":"Yilmaz","year":"2024","journal-title":"Dermatol. 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