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In this study a high-cell-density cultivation (HCDC) fed-batch process for an anti-human CD45 NB in\n                      <jats:italic>Escherichia coli<\/jats:italic>\n                      was established and evaluated how process parameters, host background, and subcellular targeting (cytoplasmic vs. periplasmic) affect yield and product quality including correct disulfide bond formation.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Using a two-stage design-of-experiments (DoE) strategy in 2\u00a0L stirred-tank bioreactors, OD\n                      <jats:sub>600nm<\/jats:sub>\n                      at induction and post-induction temperature were identified as the main process variables controlling the post-purification nanobody concentration, whereas inducer concentration had no relevant effect within the tested range. Late induction at OD\n                      <jats:sub>600nm<\/jats:sub>\n                      125\u2013150 combined with production temperatures around 21\u201322.5\u00a0\u00b0C defined a robust operating window and yielded more than 2.5\u00a0g\u00a0L\n                      <jats:sup>\u22121<\/jats:sup>\n                      purified nanobody after Strep-tag affinity chromatography. Benchmarking of five host strain\u2013plasmid combinations under standardized HCDC conditions further increased NB yield indicating that production performance was directly governed by the combined host background and expression architecture. While\n                      <jats:italic>E.\u00a0coli<\/jats:italic>\n                      NEBExpress\u00ae with periplasmic SP\n                      <jats:sub>PelB<\/jats:sub>\n                      targeting reached around 2.4\u00a0g\u00a0L\n                      <jats:sup>\u22121<\/jats:sup>\n                      anti-hCD45 NB and the highest volumetric productivity (0.122\u00a0g\u00a0L\n                      <jats:sup>\u22121<\/jats:sup>\n                      \u00a0h\n                      <jats:sup>\u22121<\/jats:sup>\n                      ),\n                      <jats:italic>E. coli<\/jats:italic>\n                      SHuffle\u00ae\u00a0T7\u00a0Express produced 2.2\u00a0g\u00a0L\n                      <jats:sup>\u22121<\/jats:sup>\n                      anti-hCD45 NB intracellularly. Nanobody-quality analysis revealed a pronounced difference between production routes. NB preparations obtained from periplasmic targeting constructs formed thermally uniform species with single nano differential scanning fluorimetry (DSF) unfolding transitions at 73.27\u201374.05\u00a0\u00b0C, whereas cytoplasmic derived preparations showed biphasic unfolding profiles with lower first transitions at 51.62\u201355.31\u00a0\u00b0C and second transitions at 68.30\u201371.53\u00a0\u00b0C indicating a higher thermal stability and also folding uniformity for secreted anti-hCD45 NB. Finally, flow cytometry confirmed functional binding of all tested anti-hCD45 NB to CD45-positive Jurkat cells.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      High-cell-density fed-batch cultivations of\n                      <jats:italic>E.\u00a0coli<\/jats:italic>\n                      can reach\u2009&gt;\u20092.5\u00a0g\u00a0L\n                      <jats:sup>\u22121<\/jats:sup>\n                      production of functional anti-CD45 nanobodies when induction timing, production temperature, host strain genetics, and subcellular location of the final product are aligned. The highest yield, most uniform, and thermally stable anti-hCD45 NB preparation was obtained with signal-peptide-mediated periplasmic targeting.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12934-026-03093-9","type":"journal-article","created":{"date-parts":[[2026,8,29]],"date-time":"2026-08-29T13:54:58Z","timestamp":1788011698000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["High-cell-density Escherichia coli\u2013based nanobody production: benchmarking of subcellular targeting, strain background and process development strategies"],"prefix":"10.1186","volume":"25","author":[{"given":"Jan-Angelus","family":"Meyer","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marie","family":"Beneke","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sadija","family":"Altm\u00fcller","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Reeb","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jannis","family":"K\u00fcnzl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathalie","family":"Opilo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Theo","family":"Weise","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christos","family":"Gouloudis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebekka","family":"Biedendieck","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rainer","family":"Krull","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,8,29]]},"reference":[{"key":"3093_CR1","doi-asserted-by":"publisher","first-page":"446","DOI":"10.1038\/363446a0","volume":"363","author":"C Hamers-Casterman","year":"1993","unstructured":"Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hammers C, Songa EB. 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No studies involving human participants, human data or animal experiments were performed.","order":1,"name":"Ethics","label":"Ethics approval and consent to participate","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"Not applicable.","order":2,"name":"Ethics","label":"Consent for publication","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"Christos Gouloudis is affiliated with IBA Lifesciences GmbH. IBA Lifesciences GmbH provided the proprietary anti-hCD45 nanobody construct, expression plasmids, and Strep-tag-related materials used in this study. The remaining authors declare no competing interests.","order":3,"name":"Ethics","label":"Competing interests","group":{"name":"EthicsHeading","label":"Declarations"}}],"article-number":"182"}}