Protocol to Assess In Vitro the Biological Activity of Insulin Glulisine, Insulin Detemir and Insulin Degludec and Potential Biosimilars

Protocol to Assess In Vitro the Biological Activity of Insulin Glulisine, Insulin Detemir and Insulin Degludec and Potential Biosimilars | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article Protocol to Assess In Vitro the Biological Activity of Insulin Glulisine, Insulin Detemir and Insulin Degludec and Potential Biosimilars Md Shahinozzaman, Yeonjin Yang, Sujata Bupp, Carole Sourbier This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6620166/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Insulin therapy is essential for helping diabetic patients manage their blood glucose levels. Over the years, various insulin analogs with distinct pharmacokinetic and pharmacodynamic profiles have been developed, offering a range of therapeutic options. Rapid-acting insulins, such as insulin lispro, insulin aspart and insulin glulisine, and long-acting or basal insulins such as insulin detemir and insulin degludec, are commonly used in clinical practice. Each analog features unique formulations and structural modifications compared to endogenous human insulin. As a result, methods used to assess their biological activity in vitro must be specifically optimized for each analog. Previously, we published protocols for evaluating the biological activity of insulin glargine, insulin aspart and insulin lispro using an in vitro cell-based assay. In this report, we present step-by-step protocols for assessing the biological activity of insulin glulisine, insulin detemir and insulin degludec using adaptations to the same in-cell western assay. Drug Discovery, Design, & Development bioassay bioidentity potency long-acting insulin rapid-acting insulin Full Text Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6620166","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":453765045,"identity":"e96982d6-a0ca-4f83-bb1a-a56ca9201685","order_by":0,"name":"Md Shahinozzaman","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Md","middleName":"","lastName":"Shahinozzaman","suffix":""},{"id":453765046,"identity":"87115342-409e-4844-a88f-fc3506d9e698","order_by":1,"name":"Yeonjin Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yeonjin","middleName":"","lastName":"Yang","suffix":""},{"id":453765047,"identity":"989c263c-d38e-453a-850e-93bf6043fab8","order_by":2,"name":"Sujata Bupp","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sujata","middleName":"","lastName":"Bupp","suffix":""},{"id":453765048,"identity":"f2de20af-f2e7-430c-b96a-8b9cdd743589","order_by":3,"name":"Carole Sourbier","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIie2PsWrDMBRFnxHYi4JWTdEvyGToUvDY33gmYC01tHTJEIK7OIuga/wX7datBkMnd89UFAqeOmTsFConBFJQE7oFqrPoCt7RfQLweM4VsztCQIBhVCCQoDih4IEyovWfFEuqTyls/rYyOIGE8bwzZvKudDQ25PZ5BuJKS5fCWzWS2EJaLdSFDXe5pp0kVdtA3LZORUIW8rQElMttwPyFoySDsoZ4kaFTYZ2d3ECyUzaoKFdrq8x+V3g/WUDwuFUKRMqv+xYCgo9r51+WHZH4ytNKd5ENGGv6edMMyoZK2jgV9pAFZj29TFiUhauvKQoaqacPu9hQzO8Ll7Iv+3ntX6fS2XEUcazD4/F4/hPf8n9blNK8P3oAAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Carole","middleName":"","lastName":"Sourbier","suffix":""}],"badges":[],"createdAt":"2025-05-08 11:26:54","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6620166/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6620166/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82344145,"identity":"cece027f-eb38-4b0c-88e5-d69add25cdd7","added_by":"auto","created_at":"2025-05-09 09:43:52","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":532666,"visible":true,"origin":"","legend":"","description":"","filename":"MdShahinozzamanInsulinMethod2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6620166/v1_covered_9a8e020b-2c30-4065-aa7c-f0bb09d9b465.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eProtocol to Assess In Vitro the Biological Activity of Insulin Glulisine, Insulin Detemir and Insulin Degludec and Potential Biosimilars\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"United States Food and Drug Administration","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"bioassay, bioidentity, potency, long-acting insulin, rapid-acting insulin","lastPublishedDoi":"10.21203/rs.3.rs-6620166/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6620166/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInsulin therapy is essential for helping diabetic patients manage their blood glucose levels. 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