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In addition, RBCs are constantly exposed to oxidative stress due to the intracellular reactive oxygen species (ROS) generated during Hb auto\u2010oxidation. Antioxidant enzymes within RBCs, such as superoxide dismutase (SOD), catalase (CAT), and peroxiredoxin (Prx), counteract ROS generation to protect the RBC from oxidative stress. Therefore, this study presents a scaled\u2010up method to extract an enzyme cocktail from lysed human RBCs, enriched with the major RBC enzymes with minimal Hb contamination. Using ethanol\u2010chloroform precipitation and multiple biophysical analyses (SDS\u2010PAGE, SEC\u2010HPLC, MALDI\u2010TOF, and LC\u2010MS\/MS), the RBC enzymes were successfully separated from Hb in the hemolysate. The purified enzyme cocktail exhibited minimal Hb contamination and retained a significant amount of CA, and antioxidative enzymes like SOD and CAT. Therefore, this scalable RBC enzyme purification method provides an efficient approach for isolating RBC enzymes with broad biomedical relevance.<\/jats:p>","DOI":"10.1002\/btpr.70072","type":"journal-article","created":{"date-parts":[[2025,9,19]],"date-time":"2025-09-19T12:45:38Z","timestamp":1758285938000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Scalable production and biophysical characterization of an enzyme cocktail derived from human red blood cells"],"prefix":"10.1002","volume":"42","author":[{"given":"Mohd Asim","family":"Khan","sequence":"first","affiliation":[{"name":"William G. Lowrie Department of Chemical and Biomolecular Engineering The Ohio State University  Columbus Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Griffin J.","family":"Beyer","sequence":"additional","affiliation":[{"name":"William G. 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