{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T14:33:55Z","timestamp":1786977235636,"version":"build-2736575974"},"reference-count":49,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2026,4,5]],"date-time":"2026-04-05T00:00:00Z","timestamp":1775347200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"},{"start":{"date-parts":[[2026,4,5]],"date-time":"2026-04-05T00:00:00Z","timestamp":1775347200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Biotech &amp; Bioengineering"],"published-print":{"date-parts":[[2026,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>Cell\u2010free gene expression systems offer cell\u2010like functionalities outside the confines of the cell, garnering increasing interest for applications from biomanufacturing to sensing. As applications expand, the need to implement economically scaled processes to produce cellular lysates grows. The protocols to produce these cellular lysates are complex, and the impact of altering many of the process variables remains understudied. Here, we set out to evaluate the effect of extended incubations at several points in the extract preparation process with the goal of identifying breakpoints that would enable flexibility in process implementation. As a model, we prepared lysates from 50\u2009L cultures instead of typical 1\u2009L volumes. We produced 72 lysates, 36 that were incubated overnight before and after culture centrifugation, and 36 that were incubated with and without a run\u2010off reaction, each across different temperatures. We found that incubations before and after culture centrifugation substantially increased variability between culture replicates but did not reduce cell\u2010free protein synthesis activity, contrary to conventional wisdom that materials should be kept cold as much as possible throughout the process. We also observed that omitting the run\u2010off reaction reduced yields but resulted in lysates that were robust to incubation up to room temperature overnight. When a run\u2010off reaction was included, activity dropped both as a function of duration and temperature, and the overall variability increased. Our work offers potential options for flexibility in implementing lysate production processes and motivates further investigation into how key processing steps relate to cell\u2010free expression activity.<\/jats:p>","DOI":"10.1002\/bit.70199","type":"journal-article","created":{"date-parts":[[2026,4,5]],"date-time":"2026-04-05T16:13:38Z","timestamp":1775405618000},"page":"1615-1623","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Impact of Process Interruptions in the Production of Lysates for Cell\u2010Free Expression Systems"],"prefix":"10.1002","volume":"123","author":[{"given":"Katherine A.","family":"Rhea","sequence":"first","affiliation":[{"name":"US Army Combat Capabilities Development Command Chemical Biological Center, Aberdeen Proving Grounds Aberdeen Maryland USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ethan 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