A mechanism of action-reflective, dual cell-based bioassay for determining the bioactivity of sclerostin-neutralizing antibodies

<!DOCTYPE html> <html lang="en" class="pb-page" data-request-id="b41a9edf-8521-4e1a-a152-06939d4af269" ><head data-pb-dropzone="head"><meta name="pbContext" content=";requestedJournal:journal:slasd;website:website:slasd-site;ctype:string:Journal Content;article:article:pii\:S2472555224000492;journal:journal:slasd;issue:issue:pii\:S2472555224X00088;pageGroup:string:Article View;wgroup:string:Migrated Websites;page:string:Show Full Text;subPage:string:Full Text;product:product:elsevier\:product\:ha"/>  <meta name="robots" content="noarchive,nocache" /> <meta name="viewport" content="width=device-width,initial-scale=1,maximum-scale=1, user-scalable=1"/> <meta property="og:title" content="A mechanism of action-reflective, dual cell-based bioassay for determining the bioactivity of sclerostin-neutralizing antibodies" /> <meta property="og:type" content="Article" /> <meta property="og:url" content="https://slas-discovery.org/article/S2472-5552(24)00049-2/abstract" /> <meta property="og:site_name" content="SLAS Discovery" /> <meta property="og:image" content="https://slas-discovery.org/cms/asset/db3adad0-afb1-4595-8b10-f37263c39220/ga1.jpg" /> <meta property="og:description" content="Osteoporosis is a skeletal condition characterized by the reduction in density (mass/volume) of normally mineralized bone. On a cellular level, osteoporosis arises from osteoclastic bone resorption that is not compensated by osteoblastic bone formation [1]. One of the pathways that plays a crucial role during skeletal development and bone homeostasis is the Wnt signaling pathway [2]. Wnt ligands are a family of secreted glycoproteins which undergo post-translational modification with palmitoylation." /> <meta name="twitter:title" content="A mechanism of action-reflective, dual cell-based bioassay for determining the bioactivity of sclerostin-neutralizing antibodies" /> <meta name="twitter:image" content="https://slas-discovery.org/cms/asset/db3adad0-afb1-4595-8b10-f37263c39220/ga1.jpg" /> <meta name="twitter:description" content="Osteoporosis is a skeletal condition characterized by the reduction in density (mass/volume) of normally mineralized bone. On a cellular level, osteoporosis arises from osteoclastic bone resorption that is not compensated by osteoblastic bone formation [1]. One of the pathways that plays a crucial role during skeletal development and bone homeostasis is the Wnt signaling pathway [2]. Wnt ligands are a family of secreted glycoproteins which undergo post-translational modification with palmitoylation." /> <meta name="twitter:card" content="summary_large_image"> <link rel="canonical" href="https://slas-discovery.org/article/S2472-5552(24)00049-2/fulltext"> <title>A mechanism of action-reflective, dual cell-based bioassay for determining the bioactivity of sclerostin-neutralizing antibodies - SLAS Discovery</title><meta name="citation_journal_title" content="SLAS Discovery"/> <meta name="citation_journal_abbrev" content="SLAS Discovery"/> <meta name="citation_publisher" content="Elsevier"/> <meta name="citation_title" content="A mechanism of action-reflective, dual cell-based bioassay for determining the bioactivity of sclerostin-neutralizing antibodies"/> <meta name="citation_date" content="2024/10/01"/> <meta name="citation_online_date" content="2024/10/07"/> <meta name="citation_volume" content="29"/> <meta name="citation_issue" content="7"/> <meta name="citation_pmid" content="39389544"/> <meta name="citation_abstract_html_url" content="http://slas-discovery.org/article/S2472555224000492/abstract"/> <meta name="citation_fulltext_html_url" content="http://slas-discovery.org/article/S2472555224000492/fulltext"/> <meta name="citation_pdf_url" content="http://slas-discovery.org/article/S2472555224000492/pdf"/> <meta name="citation_issn" content="2472-5552"/> <meta name="citation_issn" content="2472-5560"/> <meta name="citation_language" content="English"/> <meta name="citation_doi" content="10.1016/j.slasd.2024.100187"/> <meta name="citation_pmid" content="39389544"/> <meta name="citation_author" content="Suzhen Wei"></meta><meta name="citation_author_institution" content="Zhuhai United Biopharma Co., Ltd, 399 Airport West Road, Zhuhai, Guangdong, China"></meta><meta name="citation_author" content="Qiang Wu"></meta><meta name="citation_author_institution" content="Zhuhai United Laboratories Co., Ltd, 2428 Anji Road, Zhuhai, Guangdong, China"></meta><meta name="citation_author" content="Chunlai Cao"></meta><meta name="citation_author_institution" content="Zhuhai United Biopharma Co., Ltd, 399 Airport West Road, Zhuhai, Guangdong, China"></meta><meta name="citation_author" content="Zhuoni Yang"></meta><meta name="citation_author_institution" content="Zhuhai United Biopharma Co., Ltd, 399 Airport West Road, Zhuhai, Guangdong, China"></meta><meta name="citation_author" content="Jianrui Shi"></meta><meta name="citation_author_institution" content="Zhuhai United Biopharma Co., Ltd, 399 Airport West Road, Zhuhai, Guangdong, China"></meta><meta name="citation_author" content="Jingqun Huang"></meta><meta name="citation_author_institution" content="Zhuhai United Biopharma Co., Ltd, 399 Airport West Road, Zhuhai, Guangdong, China"></meta><meta name="citation_author" content="Hua He"></meta><meta name="citation_author_institution" content="Zhuhai United Biopharma Co., Ltd, 399 Airport West Road, Zhuhai, Guangdong, China"></meta><meta name="citation_author" content="Yongjie Lai"></meta><meta name="citation_author_email" content="laiyongjie121@163.com"></meta><meta name="citation_author_institution" content="Department of Microbiology and Immunology, Zunyi Medical University (Zhuhai Campus), 368 Golden Coast Avenue, Zhuhai, Guangdong, China"></meta><meta name="citation_author" content="Jing Li"></meta><meta name="citation_author_email" content="lijvica@163.com"></meta><meta name="citation_author_orcid" content="0000-0002-7418-8101"></meta><meta name="citation_author_institution" content="Zhuhai United Biopharma Co., Ltd, 399 Airport West Road, Zhuhai, Guangdong, China"></meta><meta name="citation_author_institution" content="Zhuhai United Laboratories Co., Ltd, 2428 Anji Road, Zhuhai, Guangdong, China"></meta><meta name="citation_keywords" content="Bioassay; Bioactivity; Sclerostin; Neutralizing; Antibody"></meta><meta name="citation_reference" content="citation_title=Osteoporosis: pathophysiology and therapeutic options; citation_author=U. 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Statistical analysis of results of biological assays and tests general."></meta><meta name="citation_reference" content="citation_title=Department of Health and Human Services, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM): Bioanalytical Method Validation; citation_author=Food and Drug Administration; citation_firstpage=1; citation_lastpage=35; citation_publication_date=2018; citation_publisher=Guidance for Industry. Biopharmaceutics, Ed.; Food Drug and Administration; "></meta><meta name="citation_reference" content="citation_title=Validation of analytical procedures: text and methodology Q2(R1); citation_author=International conference for harmonization; citation_firstpage=4; citation_publication_date=2005; "></meta><meta name="citation_reference" content="citation_title=Wnt signalling pathway parameters for mammalian cells; citation_author=C.W. Tan; citation_author=B.S. Gardiner; citation_author=Y. Hirokawa; citation_author=M.J. Layton; citation_author=D.W. Smith; citation_author=A.W. Burgess; citation_journal_title=PLoS One; citation_volume=7; citation_doi=10.1371/journal.pone.0031882; citation_firstpage=e31882; citation_publication_date=2012; "></meta><meta name="citation_reference" content="citation_title=Analysis of Wnt signaling β-catenin spatial dynamics in HEK293T cells; citation_author=C.W. Tan; citation_author=B.S. Gardiner; citation_author=Y. Hirokawa; citation_author=D.W. Smith; citation_author=A.W. Burgess; citation_journal_title=BMC Syst Biol; citation_volume=8; citation_doi=10.1186/1752-0509-8-44; citation_firstpage=44; citation_publication_date=2014; "></meta><meta name="citation_abstract" content="<h2>Abstract</h2><p>Osteoporosis is a major threat to the elderly worldwide. The Wnt signaling pathway plays a critical role in bone development and homeostasis. Sclerostin, a Wnt ligand inhibitor, competes with Wnt ligands for low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) on osteoblasts, thereby suppressing bone formation. Sclerostin-neutralizing monoclonal antibodies (mAbs) have emerged as a potential bone-forming therapy for osteoporosis. A cell-based bioassay which determines the relative activity of a product, related to its mechanism of action, is of great importance from drug discovery to quality control and batch release. Currently used cell-based bioassays for sclerostin-neutralizing mAbs usually use Wnt1 or Wnt3a to stimulate the Wnt pathway; sclerostin is a direct inhibitor of Wnt1 but not Wnt3a. Wnt1 is a highly hydrophobic protein that binds to the producing cell membrane and acts in a juxtacrine manner to stimulate the Wnt pathway in neighboring cells. Bioassays for drugs that induce Wnt1 signaling should be performed in a juxtacrine manner. 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js--hidden"><span class="error-hint__text">Your password is a required field.</span></div><div class="actions"><a href="https://slas-discovery.org/action/requestResetPassword" class="link show-request-reset-password">Forgot password?</a></div></div><div class="remember"><input type="checkbox" name="savePassword" id="6ee85045-2441-4b5c-8a4b-4c078d548375-remember" value="1" checked="checked"/><label for="6ee85045-2441-4b5c-8a4b-4c078d548375-remember">Remember me</label></div><div class="submit"><input type="submit" name="submit" value="Log in" class="button submit primary"/></div></form></div><div class="create-account"><div>Don’t have an account?</div><a href="/action/registration?redirectUri=https%3A%2F%2Fslas-discovery.org%2Farticle%2FS2472-5552%2824%2900049-2%2Ffulltext" class="show-registration">Create a Free Account</a></div></div></div></div></div></div> <div role="dialog" aria-modal="true" aria-label="Reset password" class="popup top-drawer request-reset-password-drawer hidden"><div class="content"><a href="#" aria-label="Close reset password popup" title="Close reset password popup" role="button" class="close"><i class="icon-close_thin"></i></a><div class="request-reset"><div class="form"><p class="sub">If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password</p><div tabindex="-1" class="message error"></div><form action="/action/requestResetPassword" method="post" class="request-reset-password-form"><input type="hidden" name="platform"/><input type="hidden" name="requestResetPassword" value="true"/><input type="hidden" name="codeSite" value="slasd-site"/><div class="input-group"><div class="label"><label for="reset-password-email">Email*</label></div><input id="reset-password-email" type="text" name="email" value="" size="15" class="email"/></div><div class="submit"><input type="submit" name="submit" value="Submit" disabled="disabled" class="button primary submit"/></div></form></div><div class="success-template hidden"><p>If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password</p></div></div><div class="request-reset-cancel"><a href="#" aria-label="Close reset password popup" title="Close reset password popup" class="cancel">Cancel</a></div></div></div> <div class="ux3-ads"> <div class="pb-ad">    <div id="gpt-Variables" data-adfile="JBS"> <input type="hidden" id="gptSite" value="slasd"> <input type="hidden" id="gptLoggedIn" value="no"> <input type="hidden" id="gptAccessType" value="ae:ANON_GUEST|ae:ANON_GUEST"> </div> <div id="gpt-Interscroller" class="gpt-InScrollBox" style="display:none;"> <div class="gpt-InScrollLabel">ADVERTISEMENT</div> <div class="gpt-InScrollContainer"> <div class="gpt-InScrollWrapper"> <div class="gpt-InScrollInner"> <div class="gpt-adinterscroller" data-adsize="interscroller"></div> </div> </div> </div> <div class="gpt-InScrollLabel">SCROLL TO CONTINUE WITH CONTENT</div> </div> <div id="gpt-DebugDiv" class="gpt-DebugDiv" style="display:none;"> <div style="width: 100%; display: table;"> <div style="display: table-row"> <div style="width: 200px; display: table-cell;"> <button class="gpt-DebugBtn" onclick="gpt_ShowConsole()">Open GPT Console</button><br /> <button id="gpt_GrapeShotDebugBtn" class="gpt-DebugBtn" onclick="gpt_GrapeShotDebug();">Open Oracle Keywords</button><br /> <br /> <button class="gpt-DebugBtn" onclick="gpt_LoadDebugValues()">Refresh Values</button> </div> <div style="display: table-cell;"> <table class="gpt-DebugTable"> <thead> <tr> <th>Property</th> <th>Value</th> </tr> </thead> <tbody> <tr><td class="gpt-DebugTitle">Status</td><td><div class="gptStatus_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">Version</td><td><div class="gptVersion_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">Ad File</td><td><div class="gptAdFile_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">Disable Ads Flag</td><td><div class="gptDisableAds_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">Environment</td><td><div class="gptTestENV_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">Moat Init</td><td><div class="gptMoatInit_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">Moat Ready</td><td><div class="gptMoatReady_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">Contextual Ready</td><td><div class="gptContextualReady_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">Contextual URL</td><td><div class="gptContextualURL_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">Contextual Initial Segments</td><td><div class="gptContextualInit_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">Contextual Used Segments</td><td><div class="gptContextualResult_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">AdUnit</td><td><div class="gptSite_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">SubAdUnit</td><td><div class="gptPage_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">Custom Targeting</td><td><div class="gptCustomTargeting_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">Ad Events</td><td><div class="gptEvent_Debug"></div></td></tr> <tr><td class="gpt-DebugTitle">Invalid Ad Sizes</td><td><div class="gptInvalidAdSize_Debug"></div></td></tr> </tbody> </table> </div> </div> </div> </div> <style type="text/css"> .gpt-DebugBtn { display: inline-block; text-align: center; text-decoration: none; margin: 2px 0; border: solid 1px #122b40; border-radius: 0.4em; padding: 0.5em 1em; color: #ffffff; background-color: #204d74; font-weight: normal; font-size: 12px; } .gpt-DebugDiv { margin: 5px; } .gpt-DebugTitle { font-weight: bold; } .gpt-DebugTable { border: 1px solid #dddddd; border-collapse: collapse; margin: 5px; font-size: 0.9em; font-family: sans-serif; min-width: 400px; margin-bottom: 25px; } .gpt-DebugTable td:first-child { padding: 5px; } .gpt-DebugTable thead tr th { background-color: #d9edf7; color: #ffffff; text-align: left; padding: 5px; border-bottom: 2px solid #204d74; } .gpt-DebugTable th, .gpt-DebugTable td { padding: 5px; } .gpt-DebugTable tbody tr { border-bottom: 1px solid #dddddd; } .gpt-DebugTable tbody tr:nth-of-type(even) { background-color: #f3f3f3; } .gpt-DebugTable tbody tr:last-of-type { border-bottom: 2px solid #204d74; } .gpt-InScrollLabel { background-color: rgb(0, 0, 0); color: rgb(255, 255, 255); position: relative; max-width: 100vw; left: 50%; right: 50%; margin-left: -50vw; margin-right: -50vw; padding: 0; text-align: center; width: 100vw !important; font-size: 10px; } .gpt-InScrollBox { height: 100vh !important; width: 100vw; cursor: pointer; max-width: 100vw; position: relative; left: 50%; right: 50%; margin-left: -50vw; margin-right: -50vw; margin-bottom: 50px; padding-top: 5px; padding-bottom: 5px; z-index: 10001; background-color: white; } .gpt-InScrollBox .gpt-InScrollContainer { height: 95vh !important; position: relative; } .gpt-InScrollBox .gpt-InScrollWrapper { position: absolute !important; top: 0 !important; left: 0; width: 100%; height: 95vh !important; border: 0 !important; margin: 0 !important; padding: 0 !important; clip: rect(0, auto, auto, 0) !important; -webkit-clip-path: polygon(0px 0px, 100% 0px, 100% 100%, 0px 100%) !important; clip-path: polygon(0px 0px, 100% 0px, 100% 100%, 0px 100%) !important; } .gpt-InScrollBox .gpt-InScrollInner { position: fixed !important; top: 0 !important; left: 0 !important; bottom: 0; margin: auto; width: 100vw; height: 95vh; -webkit-transform: translateZ(0) !important; display: -webkit-box; display: -ms-flexbox; display: flex; -webkit-box-orient: vertical; -webkit-box-direction: normal; -ms-flex-direction: column; flex-direction: column; -webkit-box-align: center; -ms-flex-align: center; align-items: center; -webkit-box-pack: center; -ms-flex-pack: center; justify-content: center; text-align: center; } </style> <script class="optanon-category-4" type="text/plain"> //-------------------------------------------------------------------------- var gpt_Version = '04072023'; var gpt_AdFile = $('#gpt-Variables').data('adfile'); var gpt_MaxMOATInitAttempts = 11; var gpt_MaxMOATReadyAttempts = 11; var gpt_MaxContextualReadyAttempts = 21; var gpt_MOATInitAttempts = 0; var gpt_MOATReadyAttempts = 0; var gpt_ContextualReadyAttempts = 0; var gpt_IsProd = true; var gpt_StartTime; var gs_channels = 'DEFAULT'; var gpt_ContextualReady = false; var gpt_ContextualURL = 'Not enabled outside of Production'; var gpt_DisableAdsFlagFound = false; var gptEvent_Debug = ''; var gptCustomTargeting_Debug = ''; var gptSite_Debug = ""; var gptPage_Debug = ""; var gptContextualEnabled = false; var gptContextualResult_Debug = ''; var gptContextualInit_Debug = ''; var gptContextualURL_Debug = ''; var gptContextualReady_Debug = ''; var gptMoatReady_Debug = ''; var gptMoatInit_Debug = ''; var gptTestENV_Debug = ''; var gptDisableAds_Debug = ''; var gptStatus_Debug = 'GPT Still Loading - Refresh Values'; var gptInvalidAdSize_Debug = ''; //-------------------------------------------------------------------------- $(document).ready(function () { //Check to see if we are on a development env gpt_CheckEnvironment(); if (!gpt_DisableAdsFlagFound) { gpt_StartTime = new Date(); if (gpt_AdFile !== "PU") { //Once the dom has loaded, begin the process to init the ads. gpt_InitScripts(); gpt_CheckForMOATInit(); } } }); </script> <script type="text/javascript"> function gpt_CheckEnvironment() { if ($('#gptDisableAds').length > 0) { gptDisableAds_Debug = 'Flag Detected - Ads Disabled'; gpt_DisableAdsFlagFound = true; } else { gptDisableAds_Debug = 'Ads Enabled'; gpt_DisableAdsFlagFound = false; } switch (gpt_AdFile) { case 'PU': gptContextualEnabled = true; if ($("#Environment").val() !== "Prod") { gpt_IsProd = false; gptTestENV_Debug = 'Test Environment Detected'; } else { gptTestENV_Debug = 'Production Environment Detected'; } break; case 'JBS': case 'LANCET': gptContextualEnabled = true; if ($(".gpt-ad").length > 0) { //Other ads exist, allow the Prestitial $(document.body).append("<div class='gpt-ad' data-adsize='prestitial'></div>"); } if (window.location.hostname.toLowerCase().indexOf('.literatumonline.') >= 0) { gpt_IsProd = false; gptTestENV_Debug = 'Test Environment Detected'; } else { gptTestENV_Debug = 'Production Environment Detected'; } break; default: //Cell gptContextualEnabled = false; if (window.location.hostname.toLowerCase().indexOf('.literatumonline.') >= 0) { gpt_IsProd = false; gptTestENV_Debug = 'Test Environment Detected'; } else { gptTestENV_Debug = 'Production Environment Detected'; } break; } } //-------------------------------------------------------------------------- function gpt_GetNonCMEArticle() { var url; $('.articleCitation').each(function (i, obj) { if ($(obj).find('.toc__item__detials').length > 0) { var itemDetails = $(obj).first().find('.toc__item__detials'); var cmeFound = 0; if (cmeFound == 0) { cmeFound = $(itemDetails).find("img[title~='CME']").length; } if (cmeFound == 0) { cmeFound = $(itemDetails).find("img[title~='(CME)']").length; } if (cmeFound == 0) { cmeFound = $(itemDetails).find("img[title~='Education']").length; } if (cmeFound == 0) { cmeFound = $(itemDetails).find("img[title~='education']").length; } if (cmeFound == 0) { var itemLink = $(itemDetails).first().find('.toc__item__title'); var linkCount = $(itemLink).first().find("a:contains('CME')").length; if (linkCount == 0) { url = $(itemLink).first().find('a').attr('href'); return false; //Found a URL, break the loop } } } }); return url; } //-------------------------------------------------------------------------- function gpt_GetElapsedTime() { var endTime = new Date(); var timeDiff = endTime - gpt_StartTime; //in ms // strip the ms timeDiff /= 1000; // get seconds return seconds = Math.round(timeDiff) + " seconds"; } //-------------------------------------------------------------------------- function gpt_CheckSSC() { //Check for ScreenShot Client var gpt_SSC = gpt_GetQuerystring('gpt_ssc', ''); if (gpt_SSC == '1') { //it's the ScreenShotClient, disable MOAT IVT Detection $('#gpt-Variables').append('<input type="hidden" id="gptm_data">'); $('#gptm_data').val(0); gpt_Target("m_data"); } } //-------------------------------------------------------------------------- function gpt_LocalSS(adTypeId) { window.scrollTo(0, 0); gpt_Sleep(1000); gpt_ScrollToAd(adTypeId); gpt_PreTearsheet(adTypeId); } //-------------------------------------------------------------------------- function gpt_InitScripts() { if (gpt_AdFile == "PU") { if (gpt_IsProd) { gpt_ContextualURL = gpt_CheckForNULL($('#canonicalurl').val()); if (gpt_ContextualURL === null) { gpt_ContextualURL = window.location.href.split('?')[0].split('#')[0]; } else { gpt_ContextualURL = window.location.origin + gpt_ContextualURL; } var useSSL = 'https:' == document.location.protocol; var gs = document.createElement('script'); gs.async = true; gs.type = 'text/javascript'; gs.src = (useSSL ? 'https:' : 'http:') + '//elsevier.gscontxt.net/multizone/channels.cgi?url=' + encodeURIComponent(gpt_ContextualURL); gs.onload = function () { gpt_ContextualReady = true; }; document.head.appendChild(gs); } } else { //JBS-Cell-Lancet var useSSL = 'https:' == document.location.protocol; var gads = document.createElement('script'); gads.async = true; gads.type = 'text/javascript'; gads.src = (useSSL ? 'https:' : 'http:') + '//securepubads.g.doubleclick.net/tag/js/gpt.js'; document.head.appendChild(gads); if (gpt_IsProd && gptContextualEnabled) { gpt_ContextualURL = window.location.href.split('?')[0].split('#')[0]; var gs = document.createElement('script'); gs.async = true; gs.type = 'text/javascript'; gs.src = (useSSL ? 'https:' : 'http:') + '//elsevier.gscontxt.net/multizone/channels.cgi?url=' + encodeURIComponent(gpt_ContextualURL); gs.onload = function () { gpt_ContextualReady = true; }; document.head.appendChild(gs); } var elsevierMoatHeader = document.createElement('link'); elsevierMoatHeader.rel = 'preload'; elsevierMoatHeader.as = 'script'; elsevierMoatHeader.src = (useSSL ? 'https:' : 'http:') + '//z.moatads.com/elsevierheader150204004183/moatheader.js'; document.head.appendChild(elsevierMoatHeader); var elsevierMoatAds = document.createElement('link'); elsevierMoatAds.rel = 'preconnect'; elsevierMoatAds.src = (useSSL ? 'https:' : 'http:') + '//mb.moatads.com'; document.head.appendChild(elsevierMoatAds); var moatHeader = document.createElement('script'); moatHeader.async = true; gads.type = 'text/javascript'; moatHeader.src = (useSSL ? 'https:' : 'http:') + '//z.moatads.com/elsevierheader150204004183/moatheader.js'; document.head.appendChild(moatHeader); } } //-------------------------------------------------------------------------- async function gpt_CheckForMOATInit() { //Check every 1/10 of a second until MOAT is Loaded or 1 second timeout if (gpt_MOATInitAttempts < gpt_MaxMOATInitAttempts) { gpt_MOATInitAttempts += 1; if (typeof window.moatPrebidApi === "object") { //MOAT LOADED gptMoatInit_Debug = '0.0' + gpt_MOATInitAttempts + ' seconds'; gpt_CheckForMOATReady(); } else { setTimeout(() => { gpt_CheckForMOATInit(); }, 100); } } else { //TIMEOUT: MOAT Not LOADED gptMoatInit_Debug = 'Timeout Expired'; var googletag = googletag || {}; googletag.cmd = googletag.cmd || []; gpt_CheckForContextualReady(); } } //-------------------------------------------------------------------------- function gpt_CheckForMOATReady() { //Check every 1/10 of a second until MOAT is Ready or 1 second timeout if (gpt_MOATReadyAttempts < gpt_MaxMOATReadyAttempts) { gpt_MOATReadyAttempts += 1; if (window.moatPrebidApi.safetyDataAvailable()) { //MOAT Ready gptMoatReady_Debug = '0.0' + gpt_MOATReadyAttempts + " seconds"; gpt_CheckForContextualReady(); } else { setTimeout(() => { gpt_CheckForMOATReady(); }, 100); } } else { //TIMEOUT: MOAT Not Ready gptMoatReady_Debug = 'Timeout Expired'; gpt_CheckForContextualReady(); } } //-------------------------------------------------------------------------- function gpt_CheckForContextualReady() { //Check every 1/10 of a second until Contextual is Ready or 2 second timeout if (gpt_IsProd && gptContextualEnabled) { //Enabled if (gpt_ContextualReadyAttempts < gpt_MaxContextualReadyAttempts) { gpt_ContextualReadyAttempts += 1; if (gpt_ContextualReady) { //Contextual Ready gptContextualURL_Debug = gpt_ContextualURL; gptContextualReady_Debug = '0.0' + gpt_ContextualReadyAttempts + " seconds"; gptContextualInit_Debug = gs_channels; gpt_InitAds(); } else { setTimeout(() => { gpt_CheckForContextualReady(); }, 100); } } else { //LOG TIMEOUT error //REMOVE FOR NOW (SHOULD LOG TO A DIFF LOCATION) //TIMEOUT: Contextual Not Ready $('#gpt_GrapeShotDebugBtn').remove(); gptContextualURL_Debug = gpt_ContextualURL; gptContextualReady_Debug = 'Timeout Expired'; gptContextualInit_Debug = ""; gpt_InitAds(); } } else { //Not Enabled $('#gpt_GrapeShotDebugBtn').remove(); switch (gpt_AdFile) { case 'PU': case 'LANCET': case 'JBS': gptContextualURL_Debug = 'Not enabled outside of Production'; gptContextualReady_Debug = 'Not enabled outside of Production'; gptContextualInit_Debug = 'Not enabled outside of Production'; break; case "CELLJOURNAL": case "CELLBUCKET": gptContextualReady_Debug = 'Not enabled on Cell'; gptContextualInit_Debug = 'Not enabled on Cell'; gptContextualURL_Debug = 'Not enabled on Cell'; break; } gpt_InitAds(); } } //-------------------------------------------------------------------------- function GPT_GetPageByURL() { var host = window.location.host.toLocaleLowerCase() var localpath = window.location.pathname.toLocaleLowerCase(); ///Check for Cell.com if (host.indexOf('cell.com') >= 0) { return null; } ///Check for Cell.com (marlin-stag) if (host.indexOf('www-cell-com') >= 0) { return null; } ///Check for Lancet if (host.indexOf('thelancet') >= 0) { return null; } if (localpath.indexOf('/article/') >= 0) { return 'article.fulltext'; } if (localpath.indexOf('/action/dosearch') >= 0) { return 'search.searchResults'; } if (localpath.indexOf('/content/') >= 0) { return 'periodical.editorial'; } return 'periodical.home'; } //-------------------------------------------------------------------------- function gpt_CheckForNULL(x, valueifnull) { if (typeof valueifnull == "undefined") { valueifnull = null; } if (typeof x == 'undefined') { return valueifnull; } if (x == null) { return valueifnull; } if (x == "null") { return valueifnull; } if ($.trim(x) == '') { return valueifnull; } return x; } //-------------------------------------------------------------------------- function gpt_GetAdditionalTargeting() { //adds page level targeting of custom variables //the id (minus gpt) and the value will be used as a key-value pair //eg: <input type="hidden" id="gptissuepii" value="x" class="gpt-Target"> //eg: target issuepii=x $(".gpt-Target").each(function (index) { var gptTargetId = $(this).attr('id'); gptTargetId = gptTargetId.replace('gpt', '').toLowerCase(); if (gptTargetId !== '') { gpt_Target(gptTargetId); } }) } //-------------------------------------------------------------------------- function gpt_GetQuerystring(key, default_) { //function to read querystring variables if (default_ === null) default_ = ''; key = key.replace(/[\[]/, "\\\[").replace(/[\]]/, "\\\]"); var regex = new RegExp("[\\?&]" + key + "=([^&#]*)"); var qs = regex.exec(window.location.href); if (qs === null) return default_; else return qs[1]; } //-------------------------------------------------------------------------- function gpt_GetDFPTestId() { //Get any request for a test id from querystring and set custom targeting parameter var gptDFPTestId = gpt_GetQuerystring('dfptestid', ''); if (gptDFPTestId !== '') { $('#gpt-Variables').append('<input type="hidden" id="gptDFPTestId">'); $('#gptDFPTestId').val(gptDFPTestId); gpt_Target("DFPTestId"); } } //-------------------------------------------------------------------------- function gpt_Target(targetName) { //Set page level target if value is found if ($('#gpt' + targetName).length > 0) { var targetValue = $('#gpt' + targetName).val(); if (targetValue !== '') { targetValue = targetValue.replace(/[^-a-z0-9]/ig, ''); //Only Allow a-z, 0-9 googletag.pubads().setTargeting(targetName.toLowerCase(), targetValue.toLowerCase()); gptCustomTargeting_Debug = gptCustomTargeting_Debug + targetName.toLowerCase() + ' = ' + targetValue.toLowerCase() + '<br>'; } } } //-------------------------------------------------------------------------- function gpt_TargetAccessType() { var currentAccessType = $('#gptAccessType').val(); if (typeof currentAccessType == "undefined") { currentAccessType = ""; } if (currentAccessType.indexOf('ANON_IP') >= 0) { googletag.pubads().setTargeting("loggedin", "yes"); } else { gpt_Target("LoggedIn"); } } //-------------------------------------------------------------------------- async function gpt_PreTearsheet(adTypeId) { if (typeof adTypeId === "undefined") { adTypeId = 0; } //Remove Elsevier Cookie Policy Notice if ($("#onetrust-consent-sdk").length > 0) { $("#onetrust-consent-sdk").remove(); } //Mobile Nav $('#skip').remove(); $('#skipNavigationLink').remove(); //JBS Modals if ($('.leadinModal').length > 0) { $('.leadinModal').remove(); } if ($('.leadinModal').length > 0) { $('.leadinModal').remove(); } if ($('.usabilla__overlay').length > 0) { $('.usabilla__overlay').remove(); } //Pendo if ($('._pendo-step-container').length > 0) { $('._pendo-step-container').remove(); } if ($('._pendo-guide-tt_').length > 0) { $('._pendo-guide-tt_').remove(); } if ($('._pendo-image').length > 0) { $('._pendo-image').remove(); } if ($('._pendo-badge-image').length > 0) { $('._pendo-badge-image').remove(); } switch (gpt_AdFile) { case "CELLJOURNAL": case "CELLBUCKET": //Mobile Bug Fix on Cell if (parseInt(adTypeId) == 5) { $('.widget-horizontalAd').css('position', 'static'); $('.body-horizontalAd').css('display', 'block'); } break; case "JBS": case "LANCET": objLeaderboard = ".container-leaderboard"; offset = 53; switch (parseInt(adTypeId)) { case 3: //MPU case 4: //SkyScraper //BUGFIX: Show Sidebar ad for JBS //skyscraper & mpu if ($('.widget-verticalAd').length > 0) { $('.widget-verticalAd').css('display', 'block'); } $('#backgroundLayer').css('display', 'none'); //Hide any Prestitial break; case 6: //Prestitial $('#backgroundLayer').css('display', 'block'); break; default: $('#backgroundLayer').css('display', 'none'); //Hide any Prestitial break; } break; } } //-------------------------------------------------------------------------- function gpt_Sleep(ms) { return new Promise(resolve => setTimeout(resolve, ms)); } //-------------------------------------------------------------------------- async function gpt_ScrollToAd(adTypeId) { if (typeof adTypeId === "undefined") { adTypeId = 0; } var adId = ""; var offset = 0; var objLeaderboard = ""; var gpt_ScrollToAd = true; switch (gpt_AdFile) { case "PU": //PracticeUpdate var hideOtherAds = true; $('#modalPrestitial').modal('hide'); $('.jumbotron-welcome').hide(); objLeaderboard = ".container-leaderboard"; offset = 53; switch (parseInt(adTypeId)) { case 2: //LeaderBoard, no Scroll Needed gpt_ScrollToAd = false; adId = "adSlot1"; break; case 3: //MPU adId = "adSlot2"; break; case 4: //SkyScraper adId = "adSlot3"; $('.most-read').hide(); break; case 5: //Mobile, no Scroll Needed gpt_ScrollToAd = false; adId = "adSlot1"; break; case 6: //Interstitial gpt_ScrollToAd = false; adId = "adSlot15"; await gpt_Sleep(1000); $(objLeaderboard).hide(); $('#modalPrestitial').unbind(); $('#modalPrestitial').modal(); break; case 7: //Native Ad break; case 10: //Master Companion hideOtherAds = false; gpt_ScrollToAd = false; break; default: //Unknown Ad Type gpt_ScrollToAd = false; adId = "adSlot1"; break; } if (hideOtherAds) { //Hide all other ads $('.ad-container').each(function (i, obj) { if (this.id !== adId) { var tempParent = this.id; tempParent = tempParent.replace("adSlot", "adParent"); $("#" + tempParent).addClass('hide'); } }); } break; case "JBS": objLeaderboard = ".js__adToHide"; offset = 80; switch (parseInt(adTypeId)) { case 2: //Leaderboard gpt_ScrollToAd = false; $(".widget-verticalAd").hide(); break; case 5: //Mobile //Scroll to Top gpt_ScrollToAd = false; break; case 3: //MPU case 4: //SkyScraper $(objLeaderboard).hide(); adId = $('.gpt-ad-defined[data-adsize="sidebar"').attr('id'); break; case 10: //MasterCompanion gpt_ScrollToAd = false; break; case 13: //Phone - Portrait case 14: //Phone - Landscape $(objLeaderboard).hide(); offset = 60; adId = 'gpt-Interscroller'; gpt_ScrollToAd = true; break; default: //Unknown Ad Type gpt_ScrollToAd = false; break; } break; case "CELLJOURNAL": case "CELLBUCKET": objLeaderboard = ".js__adToHide"; offset = 260; switch (parseInt(adTypeId)) { case 2: //Leaderboard gpt_ScrollToAd = false; $('.body-verticalAd').hide(); break; case 5: //Mobile //Scroll to Top gpt_ScrollToAd = false; break; case 3: //MPU case 4: //SkyScraper $(objLeaderboard).hide(); adId = $('.gpt-ad-defined[data-adsize="boombox"').attr('id'); break; case 10: //MasterCompanion gpt_ScrollToAd = false; break; default: //Unknown Ad Type gpt_ScrollToAd = false; break; } //Hide the second ad if found var tCount = 0; $(".widget.literatumAd.none.GPT-ad-placeholder.widget-horizontalAd.widget-compact-all").each(function (index) { tCount += 1; if (tCount !== 1) { $(this).hide(); } }); break; case "LANCET": objLeaderboard = ".js__adToHide"; offset = 80; switch (parseInt(adTypeId)) { case 2: //Leaderboard gpt_ScrollToAd = false; $('.body-verticalAd').hide(); break; case 5: //Mobile //Scroll to Top gpt_ScrollToAd = false; break; case 3: //MPU case 4: //SkyScraper $(objLeaderboard).hide(); adId = $('.gpt-ad-defined[data-adsize="skyscraper"').attr('id'); break; case 10: //MasterCompanion gpt_ScrollToAd = false; break; default: //Unknown Ad Type gpt_ScrollToAd = false; break; } break; } //Scroll to Top var gpt_container = $("html,body"); gpt_container.animate({ scrollTop: 0, scrollLeft: 0 }); if (gpt_ScrollToAd) { //If Adexists, scroll to it var gpt_scrollTo = $('#' + adId); if (gpt_scrollTo.length > 0) { //Scroll to the Ad var gpt_topPosition = gpt_scrollTo.offset().top - gpt_container.offset().top + gpt_container.scrollTop(); if (gpt_topPosition > 0) { gpt_container.animate({ scrollTop: gpt_topPosition, scrollLeft: 0 }); if (gpt_topPosition - offset > 0 && offset > 0) { gpt_container.animate({ scrollTop: gpt_topPosition - offset, scrollLeft: 0 }); } } } } } //-------------------------------------------------------------------------- async function gpt_CheckForCookiePolicy() { var returnVal = false; if ($('#onetrust-accept-btn-handler').length > 0) { $('#onetrust-accept-btn-handler').click(); returnVal = true; } return returnVal; } //-------------------------------------------------------------------------- function gpt_InitAds() { var gpt_ResponsiveHomepage = false; switch (gpt_AdFile) { case "JBS": case "CELLJOURNAL": case "CELLBUCKET": //Responsive Check if ($('#hdrHomeBrand').length > 0) { gpt_ResponsiveHomepage = true; } break; } //InterScroller Check if (gpt_AdFile === "JBS") { var gpt_InterScrollerEnabled = false; var gpt_InterScrollerInsertObj; if ($("#articleHeader").length > 0) { //It's an article Page if ($('.article__sections > section').length > 0) { gpt_InterScrollerEnabled = true; gpt_InterScrollerInsertObj = $(".article__sections > section"); } } else { if ($('.toc__section').length > 0) { //It's a TOC Page gpt_InterScrollerEnabled = true; gpt_InterScrollerInsertObj = $(".toc__section").first(); } } //Add the Interscroller if (gpt_InterScrollerEnabled) { gpt_InterScrollerInsertObj.first().append($("#gpt-Interscroller").clone()); $("#gpt-Interscroller").remove(); $(".gpt-adinterscroller").addClass("gpt-ad"); } } googletag.cmd.push(function () { var gptBoomBoxSizeMapping; var gptFluidMapping; var gptHomePageSidebarMapping; var gptCellHomePageSidebarMapping; var gptInterscrollerMapping; var gptLeaderboardSizeMapping; var gptMobileSizeMapping; var gptPrestitialMapping; var gptRectangleShortSizeMapping; var gptRectangleSizeMapping; var gptSideBarSizeMapping; var gptTallSizeMapping; var gptWideSizeMapping; var gptWideRectangleSizeMapping; //*********************************************** //Start Define Size mapping based on client Viewport //*********************************************** if (gpt_AdFile === 'PU') { gptTallSizeMapping = googletag.sizeMapping(). addSize([992, 0], [[300, 600], [160, 600], [120, 600]]). addSize([0, 0], []). build(); gptRectangleSizeMapping = googletag.sizeMapping(). addSize([992, 0], [[300, 250]]). addSize([0, 0], []). build(); gptRectangleShortSizeMapping = googletag.sizeMapping(). addSize([992, 0], [[300, 250], [320, 50], [300, 50]]). addSize([0, 0], [[300, 250], [320, 50], [300, 50]]). build(); gptWideSizeMapping = googletag.sizeMapping(). addSize([992, 0], [[728, 90]]). addSize([760, 0], [[728, 90], [320, 50], [300, 50]]). addSize([0, 0], [[320, 50], [300, 50]]). build(); gptWideRectangleSizeMapping = googletag.sizeMapping(). addSize([992, 0], []). addSize([760, 0], [[320, 50], [300, 50], [300, 250]]). addSize([0, 0], [[320, 50], [300, 50], [300, 250]]). build(); gptMobileSizeMapping = googletag.sizeMapping(). addSize([0, 0], [[320, 50], [300, 50]]). build(); gptFluidMapping = googletag.sizeMapping(). addSize([0, 0], ['fluid']). build(); gptPrestitialMapping = googletag.sizeMapping(). addSize([760, 0], [[1, 1]]). build(); } else { //JBS-CELL-LANCET gptLeaderboardSizeMapping = googletag.sizeMapping(). addSize([992, 0], [[728, 90]]). // Desktop addSize([768, 0], [[728, 90]]). // Tablet Landscape addSize([0, 0], [[320, 50], [300, 50]]). // Phone & Tablet Portrait build(); gptHomePageSidebarMapping = googletag.sizeMapping(). addSize([2090, 795], [[300, 600], [300, 250], [160, 600], [120, 600]]). addSize([1910, 795], [[160, 600], [120, 600]]). addSize([0, 0], []). build(); gptCellHomePageSidebarMapping = googletag.sizeMapping(). addSize([2090, 795], [[300, 600], [300, 250], [336, 280], [160, 600], [120, 600]]). addSize([1910, 795], [[160, 600], [120, 600]]). addSize([0, 0], []). build(); gptSideBarSizeMapping = googletag.sizeMapping(). addSize([1140, 795], [[300, 600], [160, 600], [120, 600], [300, 250]]). // SkyScraper - Desktop (Require min 630 vertical) addSize([1140, 445], [[300, 250]]). // BoomBox Fall Back - Desktop (Require min 280 vertical) addSize([0, 0], []). // Phone build(); gptBoomBoxSizeMapping = googletag.sizeMapping(). addSize([768, 0], [[300, 250], [336, 280]]). // Desktop & Tablet Landscape addSize([0, 0], []). // Phone build(); gptFluidMapping = googletag.sizeMapping(). addSize([0, 0], ['fluid']). build(); gptPrestitialMapping = googletag.sizeMapping(). addSize([600, 700], [[1, 1]]). // Desktop & Tablet Landscape addSize([0, 0], []). // Phone build(); gptInterscrollerMapping = googletag.sizeMapping(). addSize([1140, 445], []). addSize([1024, 768], [[1024, 768]]). addSize([768, 1024], [[768, 1024]]). addSize([480, 320], [[480, 320]]). addSize([320, 480], [[320, 480]]). build(); } //*********************************************** //End Define Size mapping based on client Viewport //*********************************************** //*********************************************** //Start detection for ad slots on page //*********************************************** var gptMasterPage = gpt_CheckForNULL($('#gptPage').val()); var gptMasterSite; if (gpt_AdFile === 'PU') { gptMasterSite = $('#gptSite').val(); } else { //JBS-CELL-LANCET gptMasterSite = "dev"; if (gpt_IsProd) { gptMasterSite = gpt_CheckForNULL($('#gptSite').val()); //If there is no master site, check for a defaultsite if (gptMasterSite == null) { gptMasterSite = gpt_CheckForNULL($('#gptDefaultSite').val()); } } else { //Dev ENV //If the page has no gptSite Value, assume no value which will disable ads if (gpt_CheckForNULL($('#gptSite').val()) === null) { gptMasterSite = null; } } //If there is no master Page, check for a Page using the URL if (gptMasterPage === null) { gptMasterPage = GPT_GetPageByURL(); } } $(".gpt-ad").each(function (index) { var gptPage = ''; var adSizeMapping = ''; var gptPOS; var gptTC; var gptNative; var adId; var gptSite; var tempSlot = null; if (gpt_IsProd) { //Check if the ad slot has it's own site defined if (typeof $(this).data('site') === 'undefined') { gptSite = gptMasterSite; } else { gptSite = gpt_CheckForNULL($(this).data('site')); } } else { gptSite = gptMasterSite; } if (gpt_AdFile === 'PU') { adId = $(this).attr('id'); } else { //JBS-CELL-LANCET adId = 'gpt-ad-' + index + 1; } if (gpt_CheckForNULL(gptSite) !== null) { gptSite_Debug = gptSite; gptPOS = parseInt($(this).data('pos')) || -1; if (gptPOS === -1) { gptPOS = index + 1; } //Check if the ad slot has it's own page defined gptPage = $(this).data('page'); if (gpt_CheckForNULL(gptPage) == null) { //Use the Master Page gptPage = gptMasterPage; } //If the page is not null, preceed it with a slash if (gptPage !== null) { gptPage = '/' + gptPage; } else { gptPage = ''; } gptPage_Debug = gptPage; gptTC = $(this).data('tc'); if (typeof gptTC === "undefined") { gptTC = ''; } gptNative = $(this).data('native'); if (typeof gptNative === "undefined") { gptNative = ''; } //Determine sizemapping based on data-adsize if (gpt_AdFile === 'PU') { switch ($(this).data('adsize')) { case 'tall': adSizeMapping = gptTallSizeMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[120, 600], [160, 600], [300, 600]], adId); break; case 'rectangle': adSizeMapping = gptRectangleSizeMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[300, 250]], adId); break; case 'rectangleshort': adSizeMapping = gptRectangleShortSizeMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[300, 250], [320, 50], [300, 50]], adId); break; case 'wide': adSizeMapping = gptWideSizeMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[728, 90], [320, 50], [300, 50]], adId); break; case 'widerectangle': adSizeMapping = gptWideRectangleSizeMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[320, 50], [300, 50], [300, 250]], adId); break; case 'mobile': adSizeMapping = gptMobileSizeMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[320, 50], [300, 50]], adId); break; case 'fluid': adSizeMapping = gptFluidMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, 'fluid', adId); break; case 'prestitial': adSizeMapping = gptPrestitialMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[1, 1]], adId); break; } } else { //JBS-CELL-LANCET switch ($(this).data('adsize')) { case 'cell-leaderboard': case 'leaderboard': adSizeMapping = gptLeaderboardSizeMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[728, 90], [320, 50], [300, 50]], adId); break; case 'bottom': if (gpt_ResponsiveHomepage) { adSizeMapping = gptHomePageSidebarMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[120, 600], [160, 600], [300, 600], [300, 250]], adId); } else { adSizeMapping = gptBoomBoxSizeMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[300, 250]], adId); } break; case 'boombox': if (gpt_AdFile === "CELLJOURNAL" || gpt_AdFile == "CELLBUCKET") { if (gpt_ResponsiveHomepage) { adSizeMapping = gptCellHomePageSidebarMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[120, 600], [160, 600], [300, 600], [300, 250], [336, 280]], adId); } else { adSizeMapping = gptBoomBoxSizeMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[300, 250], [336, 280]], adId); } } else { adSizeMapping = gptBoomBoxSizeMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[300, 250]], adId); } break; case 'interscroller': adSizeMapping = gptInterscrollerMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[320, 480], [480, 320], [768, 1024], [1024, 768]], adId); break; case 'sidebar': case 'skyscraper': adSizeMapping = gptSideBarSizeMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, [[120, 600], [160, 600], [300, 600], [300, 250]], adId); break; case 'native': case 'fluid': adSizeMapping = gptFluidMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, 'fluid', adId); break; case 'prestitial': adSizeMapping = gptPrestitialMapping; tempSlot = googletag.defineSlot('/6053/els.' + gptSite + gptPage, 'fluid', adId); break; } } } //Ignore any invalid adsizes that could be on the page if (adSizeMapping !== '') { $(this).addClass('gpt-ad-defined'); if (gpt_AdFile !== 'PU') { $(this).attr('id', adId); } tempSlot.defineSizeMapping(adSizeMapping).addService(googletag.pubads()).setTargeting("pos", gptPOS); if (gptTC !== '') { tempSlot.setTargeting("tc", gptTC); } if (gptNative !== '') { tempSlot.setTargeting("native", gptNative); } gptEvent_Debug = gptEvent_Debug + $(this).data('adsize') + ' Requested<br>'; } else { gptInvalidAdSize_Debug = gptInvalidAdSize_Debug + "Invalid Size Found, Id: " + $(this).prop('id') + ', adSize: ' + $(this).data('adsize') + '<br>'; $(this).remove(); } }); if (gpt_CheckForNULL(gptInvalidAdSize_Debug) === null) { gptInvalidAdSize_Debug = "None Detected"; } //*********************************************** //End detection for ad slots on page //*********************************************** //*********************************************** //Start page level targeting //*********************************************** if (gpt_AdFile !== 'PU') { //Logged in or IP Access gpt_TargetAccessType(); //Check for Screenshot Client gpt_CheckSSC(); } //Check for DFP Test gpt_GetDFPTestId(); //Check for Additional Targeting gpt_GetAdditionalTargeting(); if (gpt_IsProd && gptContextualEnabled) { var gpt_ContextualRetry = false; //Check for Contextual Targeting if (gs_channels !== "DEFAULT") { var gpt_ContextualCategories = Array.from(gs_channels); for (var i = 0; i < gpt_ContextualCategories.length; i++) { if (gpt_ContextualCategories[i] == 'gx_retry') { gpt_ContextualRetry = true; } if (!gpt_ContextualCategories[i].startsWith("custom_")) { //Remove any tag that is not custom gpt_ContextualCategories.splice(i, 1); i--; } else { //Strip the 'custom_' from the tag gpt_ContextualCategories[i] = gpt_ContextualCategories[i].replace("custom_", "") } } if (gpt_ContextualCategories.length > 0) { googletag.pubads().setTargeting("gs_cat", gpt_ContextualCategories); } else { gpt_ContextualCategories = 'none'; } if (gpt_ContextualRetry) { gpt_ContextualCategories = "*Indexing*"; } gptContextualResult_Debug = gpt_ContextualCategories; } else { gptContextualResult_Debug = 'No segments found'; } } else { //Not Prod gptContextualResult_Debug = 'Not enabled'; var gpt_DevSite = gpt_CheckForNULL($('#gptSite').val(), "dev"); $('#gpt-Variables').append('<input type="hidden" id="gptEnvironment" value="' + gpt_DevSite + '">'); gpt_Target("Environment"); } //*********************************************** //End page level targeting //*********************************************** //Enable google dfp services if (parseInt($('#gptLazyLoading').val()) === 1) { googletag.pubads().enableLazyLoad({ fetchMarginPercent: 200, renderMarginPercent: 0, mobileScaling: 2.0 }); } else { googletag.pubads().enableSingleRequest(); } //Add Listener for RenderEnded googletag.pubads().addEventListener('slotRenderEnded', function (event) { if (!event.isEmpty) { if (typeof LocalAdLoaded === "function") { LocalAdLoaded(event); } if (gpt_ResponsiveHomepage) { if (typeof positionAd === "function") { positionAd(); } } if (event.size[0] == "480" || event.size[1] == "480") { $('.gpt-InScrollBox').css('display', 'block'); gptEvent_Debug = gptEvent_Debug + 'Interscroller ' + event.size + ' rendered: ' + gpt_GetElapsedTime() + '<br>'; } else { gptEvent_Debug = gptEvent_Debug + event.size + ' rendered: ' + gpt_GetElapsedTime() + '<br>'; } } }); if (gpt_AdFile !== 'PU') { googletag.pubads().collapseEmptyDivs(); } googletag.enableServices(); //Populate Defined Ad Slots $(".gpt-ad-defined").each(function (index) { var adId = $(this).attr('id'); googletag.cmd.push(function () { googletag.display(adId); }); }); gptStatus_Debug = 'GPT Complete'; }); } //-------------------------------------------------------------------------- function gpt_DisplayDebug() { //Clone the template and append to Body $(document.body).append($('#gpt-DebugDiv').clone()); $('#gpt-DebugDiv').remove(); //Populate the Values gpt_LoadDebugValues(); //Display and scroll to debug section $('.gpt-DebugDiv').css('display', 'block'); window.scrollTo(0, document.body.scrollHeight); } //-------------------------------------------------------------------------- function gpt_LoadDebugValues() { $('.gpt-DebugBtn').blur(); $('.gptVersion_Debug').text(gpt_Version); $('.gptAdFile_Debug').html(gpt_AdFile); $('.gptEvent_Debug').html(gptEvent_Debug); $('.gptCustomTargeting_Debug').html(gptCustomTargeting_Debug); $('.gptSite_Debug').text(gptSite_Debug); $('.gptContextualResult_Debug').text(gptContextualResult_Debug); $('.gptContextualInit_Debug').text(gptContextualInit_Debug); $('.gptContextualURL_Debug').text(gptContextualURL_Debug); $('.gptContextualReady_Debug').text(gptContextualReady_Debug); $('.gptMoatReady_Debug').text(gptMoatReady_Debug); $('.gptMoatInit_Debug').text(gptMoatInit_Debug); $('.gptTestENV_Debug').text(gptTestENV_Debug); $('.gptDisableAds_Debug').text(gptDisableAds_Debug); $('.gptStatus_Debug').text(gptStatus_Debug); $('.gptPage_Debug').text(gptPage_Debug); $('.gptInvalidAdSize_Debug').html(gptInvalidAdSize_Debug); } //-------------------------------------------------------------------------- function gpt_GrapeShotDebug() { $('.gpt-DebugBtn').blur(); window.open("http://admin-elsevier.grapeshot.co.uk/custom/bookmarklet.cgi?blocking=1&vn=2&url=" + encodeURIComponent(document.location), "GRAPESHOT_POPUP", "width=650,height=600,scrollbars=yes,screenX=40,screenY=50,menubar=no,toolbar=no,resizable=yes", "Custom").focus(); } //-------------------------------------------------------------------------- function gpt_ShowConsole() { $('.gpt-DebugBtn').blur(); googletag.openConsole(); } 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class="page-body-flag"></div></div> </div> </div> </div> <div data-widget-id="cce513c3-ede2-454b-83a2-b235c17a37ed" data-widget-name="UX3HTMLWidget"></div> <div class="widget axelPublicationContent none widget-none" id="922bea2a-77ad-4d06-a464-4510160d2279"> <div class="wrapped " > <div class="widget-body body body-none "><link id="build-style-article" rel="stylesheet" type="text/css" href="/products/marlin/ha/releasedAssets/css/build-article-45520a2122a8fee68553.css"/><article xmlns="http://www.w3.org/1999/xhtml" data-design="pill" data-has="right-rail long-title" vocab="http://schema.org/" typeof="ScholarlyArticle" data-pii="S2472-5552(24)00049-2" lang="en" dir="ltr"><div data-core-wrapper="header"><header data-extent="frontmatter"><div class="core-container"><div data-article-access="full" data-article-access-type="open" class="meta-panel"><span class="meta-panel__left"><span class="meta-panel__type"><span>Full Length Article</span></span></span><span class="meta-panel__middle"><span class="meta-panel__volumeIssue"><a href="/issue/S2472-5552(24)X0008-8"><span>Volume 29</span>, <span>Issue 7</span></a></span><span class="meta-panel__number"><span>100187</span></span><span class="meta-panel__onlineDate">October 2024</span></span><span class="meta-panel__right"><span class="meta-panel__access meta-panel__access--open"><span>Open access</span></span></span></div><h1 property="name">A mechanism of action-reflective, dual cell-based bioassay for determining the bioactivity of sclerostin-neutralizing antibodies</h1><div class="contributors"><span class="authors"><span role="list"><span property="author" typeof="Person" role="listitem"><a href="#au0001"><span property="givenName">Suzhen</span> <span property="familyName">Wei</span></a><sup class="xref"><a href="#aff0001" property="affiliation" typeof="Organization" role="doc-noteref">a</a></sup></span> ∙ <span property="author" typeof="Person" role="listitem"><a href="#au0002"><span property="givenName">Qiang</span> <span property="familyName">Wu</span></a><sup class="xref"><a href="#aff0002" property="affiliation" typeof="Organization" role="doc-noteref">b</a></sup></span> ∙ <span property="author" typeof="Person" role="listitem"><a href="#au0003"><span property="givenName">Chunlai</span> <span property="familyName">Cao</span></a><sup class="xref"><a href="#aff0001" property="affiliation" typeof="Organization" role="doc-noteref">a</a></sup></span><span data-displayed-on="all"> ∙ … </span><span data-hidden-on="all"> ∙ <span property="author" typeof="Person" role="listitem"><a href="#au0004"><span property="givenName">Zhuoni</span> <span property="familyName">Yang</span></a><sup class="xref"><a href="#aff0001" property="affiliation" typeof="Organization" role="doc-noteref">a</a></sup></span></span><span data-hidden-on="all"> ∙ <span property="author" typeof="Person" role="listitem"><a href="#au0005"><span property="givenName">Jianrui</span> <span property="familyName">Shi</span></a><sup class="xref"><a href="#aff0001" property="affiliation" typeof="Organization" role="doc-noteref">a</a></sup></span></span><span data-hidden-on="all"> ∙ <span property="author" typeof="Person" role="listitem"><a href="#au0006"><span property="givenName">Jingqun</span> <span property="familyName">Huang</span></a><sup class="xref"><a href="#aff0001" property="affiliation" typeof="Organization" role="doc-noteref">a</a></sup></span></span> ∙ <span property="author" typeof="Person" role="listitem"><a href="#au0007"><span property="givenName">Hua</span> <span property="familyName">He</span></a><sup class="xref"><a href="#aff0001" property="affiliation" typeof="Organization" role="doc-noteref">a</a></sup></span> ∙ <span property="author" typeof="Person" role="listitem" class="corresponding-author"><a href="#au0008"><span property="givenName">Yongjie</span> <span property="familyName">Lai</span></a><sup class="xref"><a href="#aff0003" property="affiliation" typeof="Organization" role="doc-noteref">c</a></sup> <a id="ead0001" href="mailto:laiyongjie121@163.com" property="email" title="Send email to Yongjie Lai">laiyongjie121@163.com</a></span> ∙ <span property="author" typeof="Person" role="listitem" class="corresponding-author"><a href="#au0009"><span property="givenName">Jing</span> <span property="familyName">Li</span></a><sup class="xref"><a href="#aff0001" property="affiliation" typeof="Organization" role="doc-noteref">a</a>,<a href="#aff0002" property="affiliation" typeof="Organization" role="doc-noteref">b</a></sup> <a id="ead0002" href="mailto:lijvica@163.com" property="email" title="Send email to Jing Li">lijvica@163.com</a></span><span data-displayed-on="all"> … <span><span data-action="reveal">Show more</span></span> </span><span data-displayed-on="none" data-on-display="all" aria-hidden="true"> <span data-action="hide">Show less</span></span></span></span></div><div class="core-self-citation"><div id="core-affiliations-notes"><div class="affiliations"><div property="affiliation" typeof="Organization"><span class="label">a</span><span id="aff0001" property="name">Zhuhai United Biopharma Co., Ltd, 399 Airport West Road, Zhuhai, Guangdong, China</span></div><div property="affiliation" typeof="Organization"><span class="label">b</span><span id="aff0002" property="name">Zhuhai United Laboratories Co., Ltd, 2428 Anji Road, Zhuhai, Guangdong, China</span></div><div property="affiliation" typeof="Organization"><span class="label">c</span><span id="aff0003" property="name">Department of Microbiology and Immunology, Zunyi Medical University (Zhuhai Campus), 368 Golden Coast Avenue, Zhuhai, Guangdong, China</span></div></div></div><div id="core-content-info"><div class="history"><div>Publication History:</div><div><span>Received July 27, 2024</span>; <span>Revised October 1, 2024</span>; <span>Accepted October 7, 2024</span>; <span>Published online October 7, 2024</span></div></div><div class="core-links"><span class="doi">DOI: <a href="https://doi.org/10.1016/j.slasd.2024.100187" property="sameAs" target="_blank">10.1016/j.slasd.2024.100187</a></span><span class="science-direct">Also available on <a href="https://www.sciencedirect.com/science/article/pii/S2472555224000492" target="_blank">ScienceDirect</a></span></div><div role="paragraph">Copyright: © 2024 The Author(s). 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role="list"><div id="celistitem0001" role="listitem"><div class="label">•</div><div class="content"><div id="para0001" role="paragraph">Sclerostin is an inhibitor of the Wnt1 ligand, which acts in a juxtacrine manner to activate the Wnt signaling pathway in osteoblast cells and promote bone formation.</div></div></div><div id="celistitem0002" role="listitem"><div class="label">•</div><div class="content"><div id="para0002" role="paragraph">The mechanism of action of sclerostin-neutralizing antibodies is not reflected in any of the cell-based bioassays currently used to determine the bioactivity of these antibodies.</div></div></div><div id="celistitem0003" role="listitem"><div class="label">•</div><div class="content"><div id="para0003" role="paragraph">We present a dual cell-based bioassay in which Wnt1 is produced and exerts its function in a juxtacrine fashion, and the bioactivity of sclerostin-neutralizing antibodies was determined by antagonizing sclerostin induced suppression of the Wnt1 juxtacrine signaling.</div></div></div></div></div></section><section id="author-abstract" property="abstract" typeof="Text" role="doc-abstract"><h2 property="name">Abstract</h2><div id="spara008" role="paragraph">Osteoporosis is a major threat to the elderly worldwide. The Wnt signaling pathway plays a critical role in bone development and homeostasis. Sclerostin, a Wnt ligand inhibitor, competes with Wnt ligands for low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) on osteoblasts, thereby suppressing bone formation. Sclerostin-neutralizing monoclonal antibodies (mAbs) have emerged as a potential bone-forming therapy for osteoporosis. A cell-based bioassay which determines the relative activity of a product, related to its mechanism of action, is of great importance from drug discovery to quality control and batch release. Currently used cell-based bioassays for sclerostin-neutralizing mAbs usually use Wnt1 or Wnt3a to stimulate the Wnt pathway; sclerostin is a direct inhibitor of Wnt1 but not Wnt3a. Wnt1 is a highly hydrophobic protein that binds to the producing cell membrane and acts in a juxtacrine manner to stimulate the Wnt pathway in neighboring cells. Bioassays for drugs that induce Wnt1 signaling should be performed in a juxtacrine manner. Here, we present a mechanism of action-reflective, dual cell-based reporter gene assay. In this assay, Wnt1 producer cells are co-cultured with cells containing the Wnt reporter genes, Wnt1 on the producer cells activates the Wnt signaling pathway in the reporter cells that are in direct cell-to-cell contact, and sclerostin-neutralizing mAbs specifically and effectively antagonize the sclerostin-mediated Wnt reporter gene suppression. This bioassay demonstrates good specificity, accuracy, linearity, and precision and is suitable for quality control, stability testing, batch release, and biosimilarity assessment of sclerostin-neutralizing mAbs.</div></section><section id="graphical-abstract" property="abstract" typeof="Text" role="doc-abstract"><h2 property="name">Graphical abstract</h2><div id="spara009" role="paragraph"><div class="figure-wrap"><figure id="fig0005" class="graphic"><a class="icon-full-screen" href="/cms/10.1016/j.slasd.2024.100187/asset/7c5c964f-c4b5-42c0-a6f4-90c4585a3079/main.assets/ga1_lrg.jpg" target="_blank" title="View full size image in a new tab"><img alt="Image, graphical abstract" src="/cms/10.1016/j.slasd.2024.100187/asset/db3adad0-afb1-4595-8b10-f37263c39220/main.assets/ga1.jpg" height="200" width="317" data-viewer-src="/cms/10.1016/j.slasd.2024.100187/asset/7c5c964f-c4b5-42c0-a6f4-90c4585a3079/main.assets/ga1_lrg.jpg" loading="lazy" /></a></figure></div></div></section></div></div><div class="core-linked-content"></div><section id="keywords" property="keywords"><h2>Keywords</h2><ol><li><a href="/action/doSearch?AllField="Bioassay"&journalCode=slasd" alt="Search on this keyword">Bioassay</a></li><li><a href="/action/doSearch?AllField="Bioactivity"&journalCode=slasd" alt="Search on this keyword">Bioactivity</a></li><li><a href="/action/doSearch?AllField="Sclerostin"&journalCode=slasd" alt="Search on this keyword">Sclerostin</a></li><li><a href="/action/doSearch?AllField="Neutralizing"&journalCode=slasd" alt="Search on this keyword">Neutralizing</a></li><li><a href="/action/doSearch?AllField="Antibody"&journalCode=slasd" alt="Search on this keyword">Antibody</a></li></ol></section><div class="core-linked-content"></div><div class="core-linked-content"></div><div class="core-linked-content"></div><section id="bodymatter" data-extent="bodymatter" property="articleBody" typeof="Text"><div class="core-container"><section id="sec-1"><h2>1 Introduction</h2><div id="para0004" role="paragraph">Osteoporosis is a skeletal condition characterized by the reduction in density (mass/volume) of normally mineralized bone. On a cellular level, osteoporosis arises from osteoclastic bone resorption that is not compensated by osteoblastic bone formation [<a id="crf0013" href="#bib0001" role="doc-biblioref" data-xml-rid="bib0001">1</a>]. One of the pathways that plays a crucial role during skeletal development and bone homeostasis is the Wnt signaling pathway [<a id="crf0014" href="#bib0002" role="doc-biblioref" data-xml-rid="bib0002">2</a>]. Wnt ligands are a family of secreted glycoproteins which undergo post-translational modification with palmitoylation. These lipid-modified Wnt ligands are highly hydrophobic and have strong membrane binding affinity, making it difficult for them to diffuse freely into the extracellular space [<a id="crf0015" href="#bib0003" role="doc-biblioref" data-xml-rid="bib0003">3</a>,<a id="crf0016" href="#bib0004" role="doc-biblioref" data-xml-rid="bib0004">4</a>]. Wnt1, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt7b, Wnt9b, and Wnt10b have been identified in bone tissue and are implicated in new bone formation [<a id="crf0017" href="#bib0005" role="doc-biblioref" data-xml-rid="bib0005 bib0006 bib0007">5-7</a>]. Wnt signaling is activated by the binding of a Wnt ligand to a coupled receptor complex consisting of LRP5/6 and Frizzled receptors. Upon ligand binding, the signal is transduced to ß-catenin, which accumulates in the cytoplasm, subsequently translocates to the nucleus, and forms a complex with the TCF/LEF transcription factors. The transcriptional complex binds to the TCF/LEF response element and induces transcription of osteogenic genes [<a id="crf0019" href="#bib0008" role="doc-biblioref" data-xml-rid="bib0008 bib0009 bib0010">8-10</a>].</div><div id="para0005" role="paragraph">Sclerostin is a glycoprotein secreted by osteocytes that binds to the extracellular domains of LRP5/6 to inhibit Wnt ligand binding, thereby inhibiting canonical Wnt signaling and bone formation in osteoblast-lineage cells [<a id="crf0021" href="#bib0011" role="doc-biblioref" data-xml-rid="bib0011">11</a>]. Both sclerostin and Wnt ligands bind to the LRP5/6, which contain four tandem YWTD-type β-propellers, termed E1 to E4, spanning from N- to C-terminus of extracellular domain [<a id="crf0022" href="#bib0012" role="doc-biblioref" data-xml-rid="bib0012">12</a>,<a id="crf0023" href="#bib0013" role="doc-biblioref" data-xml-rid="bib0013">13</a>]. It has been demonstrated that Wnt1, Wnt2, Wnt6, Wnt8a, Wnt9b, and Wnt10b interact with the E1E2 domain, whereas Wnt3 and Wnt3a prefer the E3E4 domain. Sclerostin effectively antagonizes Wnt1, Wnt2, and Wnt9b signaling, but not Wnt3a signaling, through competition for the E1E2 domain of LRP5/6 [<a id="crf0024" href="#bib0003" role="doc-biblioref" data-xml-rid="bib0003">3</a>,<a id="crf0025" href="#bib0011" role="doc-biblioref" data-xml-rid="bib0011">11</a>,<a id="crf0026" href="#bib0014" role="doc-biblioref" data-xml-rid="bib0014 bib0015 bib0016 bib0017">14-17</a>]. Several mAbs that specifically antagonize sclerostin-mediated inhibition of the Wnt signaling pathway have been developed for the treatment of osteoporosis and osteogenesis imperfecta, including SHR-1222, setrusumab, blosozumab and romosozumab [<a id="crf0028" href="#bib0018" role="doc-biblioref" data-xml-rid="bib0018 bib0019 bib0020 bib0021 bib0022 bib0023 bib0024">18-24</a>]. Of these, romosozumab, which was developed jointly by Amgen and UCB, has received approval from regulatory authorities in the US, EU, and Japan. Romosozumab is a novel bone anabolic treatment option for osteoporosis that works by directly neutralizing sclerostin and re-activating the Wnt signaling pathway in osteoblasts [<a id="crf0030" href="#bib0018" role="doc-biblioref" data-xml-rid="bib0018">18</a>].</div><div id="para0006" role="paragraph">Cell-based in vitro bioassays serve as valuable tools for drug discovery, development, quality control, and batch release. The classic cell-based bioassay for evaluating the bioactivity of sclerostin-neutralizing mAbs is the osteoblast mineralization assay using the pre-osteoblastic MC3T3-E1 cell line [<a id="crf0031" href="#bib0025" role="doc-biblioref" data-xml-rid="bib0025">25</a>,<a id="crf0032" href="#bib0026" role="doc-biblioref" data-xml-rid="bib0026">26</a>]. Sclerostin suppresses MC3T3-E1 mineralization, which is supposed to be caused by sclerostin mediated inhibition of Wnt signaling. Sclerostin-neutralizing mAbs antagonize the mineralization suppressing effect of sclerostin. However, this assay has several limitations: 1) the assay is laborious and time consuming (12-20 days); 2) the mineralization process is affected by the type of base medium, the batch of fetal bovine serum, and the freshness of the medium [<a id="crf0033" href="#bib0025" role="doc-biblioref" data-xml-rid="bib0025">25</a>,<a id="crf0034" href="#bib0027" role="doc-biblioref" data-xml-rid="bib0027">27</a>]; 3) no exogenous Wnt ligands are added in this assay; therefore, the only possible source of Wnt ligands is fetal bovine serum, which is subject to lot-to-lot variability [<a id="crf0035" href="#bib0026" role="doc-biblioref" data-xml-rid="bib0026">26</a>,<a id="crf0036" href="#bib0028" role="doc-biblioref" data-xml-rid="bib0028">28</a>]. The inability of this assay to generate a dose-response curve and EC50 renders it unsuitable for quality control, particularly for batch release.</div><div id="para0007" role="paragraph">The C2C12 trans-differentiation and alkaline phosphatase (ALP) activity assay is additionally employed to evaluate the bioactivity of sclerostin-neutralizing mAbs. C2C12, a murine myoblast cell line, is thought to differentiate into osteoblasts when stimulated with Wnt3a in conjunction with bone morphogenetic protein 2 (BMP2) or BMP4 [<a id="crf0037" href="#bib0029" role="doc-biblioref" data-xml-rid="bib0029">29</a>,<a id="crf0038" href="#bib0030" role="doc-biblioref" data-xml-rid="bib0030">30</a>]. However, the underlying mechanism seems paradoxical. It has been demonstrated that BMP2 and BMP4 can independently induce the differentiation of C2C12 cells into osteoblasts in the absence of Wnt ligands [<a id="crf0039" href="#bib0031" role="doc-biblioref" data-xml-rid="bib0031">31</a>,<a id="crf0040" href="#bib0032" role="doc-biblioref" data-xml-rid="bib0032">32</a>]. Sclerostin does not act as a BMP antagonist, nor does it directly suppress the Wnt3a pathway [<a id="crf0041" href="#bib0015" role="doc-biblioref" data-xml-rid="bib0015">15</a>,<a id="crf0042" href="#bib0017" role="doc-biblioref" data-xml-rid="bib0017">17</a>,<a id="crf0043" href="#bib0033" role="doc-biblioref" data-xml-rid="bib0033">33</a>]. Furthermore, activation of BMP signaling pathway induces sclerostin expression, resulting in the inhibition of the Wnt pathway [<a id="crf0044" href="#bib0034" role="doc-biblioref" data-xml-rid="bib0034">34</a>,<a id="crf0045" href="#bib0035" role="doc-biblioref" data-xml-rid="bib0035">35</a>]. On the other side, the Wnt pathway induced by Wnt3a antagonizes the function of BMP2 and induces the differentiation of C2C12 towards myogenic cells [<a id="crf0046" href="#bib0036" role="doc-biblioref" data-xml-rid="bib0036">36</a>]. Although BMP2 and Wnt3a are able to induce significant expression of ALP in C2C12 cells, the morphological properties indicate that the C2C12 cells are more prone to differentiate into myocytes.</div><div id="para0008" role="paragraph">The reporter gene assay was also developed to evaluate the bioactivity of sclerostin-neutralizing mAbs. In the methodologies described by Novartis and Chugai Pharmaceutical, a Wnt1 expression vector and a vector containing a luciferase gene under the control of the TCF/LEF response element were transiently transfected into HEK293 cells. The bioactivity of sclerostin-neutralizing mAbs was determined by adding the mixture of sclerostin and sclerostin-neutralizing mAbs after plasmid transfection [<a id="crf0047" href="#bib0037" role="doc-biblioref" data-xml-rid="bib0037">37</a>,<a id="crf0048" href="#bib0038" role="doc-biblioref" data-xml-rid="bib0038">38</a>]. In another reporter gene assay, HEK293T cells were engineered to harbor an integrated TCF/LEF response element-luciferase reporter gene and stably express Wnt1. The bioactivity of sclerostin-neutralizing mAbs was determined using this stable cell line [<a id="crf0049" href="#bib0039" role="doc-biblioref" data-xml-rid="bib0039">39</a>]. Wnt1 is a pivotal Wnt ligand in human bone homeostasis. Sclerostin directly competes with Wnt1 for the E1E2 domain of LRP5/6, thereby making Wnt1 an appropriate stimulus for the Wnt pathway in these assays. From a physiological perspective, Wnt1 functions in a strictly juxtacrine manner to induce osteoblast differentiation [<a id="crf0050" href="#bib0010" role="doc-biblioref" data-xml-rid="bib0010">10</a>]. However, Wnt1 does not function in a strictly juxtacrine manner in these reporter gene assays. Bioassays are used to evaluate the potency of the drug and may predict clinical efficacy. To this end, the assay should reflect or mimic the product's known or intended mechanism of action. The mechanism of action of sclerostin neutralizing mAbs is the restoration of Wnt1′s juxtacrine signaling pathway. The main difference between juxtacrine and paracrine is that juxtacrine signaling requires close contact of cells, whereas paracrine signaling produces signals that induce changes in nearby cells [<a id="crf0051" href="#bib0040" role="doc-biblioref" data-xml-rid="bib0040">40</a>]. The use of a soluble ligand (either purified or contained in conditioned medium) and co-culture studies are the most common methods to distinguish between these two types of interactions.</div><div id="para0009" role="paragraph">In this study, we developed a dual cell-based reporter gene assay, in which the Wnt1 producer cells (HEK293T cells stably expressing Wnt1) are co-cultured with the Wnt reporter cells (HEK293T cells harboring the TCF/LEF response element-luciferase reporter gene). Wnt1 tethered to the producer cell membrane induces activation of Wnt signaling pathway in the neighboring reporter cells as reflected by the luciferase activity. The presence of sclerostin suppresses Wnt signaling by competing for the LRP5/6 receptor. Addition of sclerostin-neutralizing mAbs antagonizes sclerostin and re-activates the Wnt pathway. This method was systematically optimized and validated to demonstrate its potency for the determination of bioactivity of sclerostin-neutralizing mAbs.</div></section><section id="sec-2"><h2>2 Materials and methods</h2><section id="sec-2-1"><h3>2.1 Cell culture and reagents</h3><div id="para0010" role="paragraph">HEK293T cells (CRL3216, ATCC) were cultured in DMEM medium (11965092, Gibco) containing 10% (v/v) fetal bovine serum (10099141C, Gibco) in a humidified incubator with 5% CO<sub>2</sub>. The TurboFect™ transfection reagent (R0531) was a product of Thermo-Fisher Scientific. The Bright-Glo<sup>TM</sup> Luciferase Assay System (E2650) was a product of Promega. Recombinant human sclerostin (HST-H5245) was purchased from the ACRO Biosystems. Romosozumab and denosumab were obtained from Amgen Inc. Dupilumab was obtained from Regeneron Pharmaceuticals, Inc. Tocilizumab was obtained from Genentech, Inc. Siltuximab was obtained from Janssen Biotech, Inc.</div></section><section id="sec-2-2"><h3>2.2 Lentiviral vector construction, lentivirus production and stable cell line generation</h3><div id="para0011" role="paragraph">The lentiviral transfer plasmids, pLenti-Wnt1, pLenti-Wnt3a, and pLenti-TCF/LEF-RE-luc were generated by subcloning Wnt1, Wnt3a, and TCF/LEF-response element-luciferase reporter gene into a self-inactivating lentiviral vector, pLenti. All constructs were verified by DNA sequencing. For lentiviral packaging, HEK293T cells were grown to 95-100% confluence in T25 flasks (430639, Corning). The transfer plasmids (pLenti-Wnt1, pLenti-Wnt3a, or pLenti-TCF/LEF-RE-luc) were transfected into HEK293T, together with pMD2.G and psPAX2 plasmids, using TurboFect™ transfection reagent. Forty-eight hours later, the packaged lentiviruses in the supernatant were harvested and filtered through 0.45 μm filters. The lentiviruses were then used to transduce HEK293T cells to generate stable cell lines, including Wnt1 producer cells (HEK293T stably expressing Wnt1), Wnt3a producer cells (HEK293T stably expressing Wnt3a), and Wnt reporter cells (HEK293T harboring TCF/LEF response element-luciferase reporter gene). Single cell clones were generated by limiting dilution in 96-well plates.</div></section><section id="sec-2-3"><h3>2.3 Conditioned medium preparation</h3><div id="para0012" role="paragraph">The HEK293T, Wnt1 producer cells and Wnt3a producer cells were cultured in DMEM medium supplemented with10% (v/v) fetal bovine serum for 4 days without medium change. Afterward, the cell culture supernatant was collected and filtered through 0.22 μM sterile filters, aliquoted, and stored at -80°C.</div></section><section id="sec-2-4"><h3>2.4 Dual cell-based reporter gene assay</h3><div id="para0013" role="paragraph">Wnt producer cells and Wnt reporter cells were trypsinized, mixed, and plated at 100 μL/well in a white-bottomed 96-well plate (3610, Corning) to achieve working densities of 2-6 × 10<sup>3</sup> cells/well for producer cells and 1-3 × 10<sup>4</sup> cells/well for reporter cells. Serially diluted antibody was preincubated with 1.25, 2.5, or 5.0 μg/mL sclerostin for 1 h at room temperature and the antibody/antigen mixture were added to the co-culture immediately after cell seeding. Twenty hours later, the cells were lysed for luciferase assay.</div></section><section id="sec-2-5"><h3>2.5 Method optimization</h3><div id="para0014" role="paragraph">The parameters influencing the assay performance were systematically optimized, encompassing cell density, producer-to-reporter cell (P/R) ratio, working concentration of sclerostin, and assay incubation time, as detailed in <a id="crf0052" href="#tbl0001">Table 1</a>.</div><div class="figure-wrap"><figure id="tbl0001" class="table" data-figureviewer-ignore="true"><div class="table-wrap"><table data-figureviewer-ignore="true"><thead><tr class="rowsep" data-xml-align="left" data-xml-valign="top"><th id="en0001">Parameters</th><th id="en0002" colspan="3"><span>Design of experiments</span></th></tr></thead><tbody><tr><td id="en0003" rowspan="4"><b>Cell density and P/R ratio</b></td><td id="en0004">Producer cell<br />(× 10<sup>3</sup>cells/well)</td><td id="en0005">Reporter cell<br />(× 10<sup>3</sup> cells/well)</td><td id="en0006">P/R ratio</td></tr><tr><td id="en0008">0.25, 0.333, 0.5, 1, 2, 4</td><td id="en0009">10</td><td id="en0010" rowspan="3">1:2.5, 1:5, 1:10, 1:20, 1:30, 1:40</td></tr><tr><td id="en0012">0.5, 0.667, 1, 2, 4, 8</td><td id="en0013">20</td></tr><tr><td id="en0016">0.75, 1, 1.5, 3, 6, 12</td><td id="en0017">30</td></tr><tr><td id="en0019"><b>Sclerostin working concentration</b></td><td id="en0020" colspan="3">40, 20, 10, 5, 2.5, 1.0, 0.4, 0.16, 0.064, 0.0256, 0.01, and 0.004 μg/mL</td></tr><tr><td id="en0021"><b>Assay incubation time</b></td><td id="en0022" colspan="3">4, 6, 8, 16, 20, and 24 hours</td></tr></tbody></table></div><figcaption><span class="heading">Table 1</span><div id="spara005" role="paragraph">Assay development and optimization</div><div class="core-table-tools"><ul><li><a class="core-table-show-action" href="/action/showFullTableHTML?isHtml=true&tableId=tbl0001&pii=S2472-5552%2824%2900049-2" target="_blank">Open table in a new tab</a></li></ul></div></figcaption></figure></div></section><section id="sec-2-6"><h3>2.6 Method validation</h3><div id="para0015" role="paragraph">Specificity was evaluated by comparing the biological response of romosozumab with respect to other mAbs not targeting sclerostin, including dupilumab (targeting IL-4Ra), denosumab (targeting RANKL), tocilizumab (targeting IL-6R), and siltuximab (targeting IL-6). Additionally, the obtained response was compared to the formulation buffer of romosozumab. All data were analyzed using a four-parameter logistic curve equation. The established acceptance criterion was the expected biological response with romosozumab while mAbs not targeting sclerostin or the formulation buffer did not show biological effect.</div><div id="para0016" role="paragraph">For accuracy validation, romosozumab was adjusted to 50%, 75%, 100%, 125%, and 150% of the activity of the reference (set at 100%). The EC50 value of each sample was determined using this assay. The percentage recovery was reported as (the mean of EC50/the theoretical value) × 100. Linearity was determined by plotting the measured potency values for each concentration versus the expected potency values on a linear scale and performing a linear regression analysis. The predefined acceptance criteria for acceptable linearity were R<sup>2</sup>≥ 0.90 and slope in the range of 0.80 to 1.25.</div><div id="para0017" role="paragraph">Precision is assessed at two levels: repeatability and intermediate precision. For repeatability, romosozumab corresponding to 100% activity of the reference was run in six experiments by a single analyst under identical assay conditions. For intermediate precision, the romosozumab sample was evaluated in nine independent experiments performed by two analysts on three separate days. EC50 values were determined and the relative standard deviation (RSD) for both repeatability and intermediate precision was calculated using the equation: (standard deviation/mean) × 100. The predefined acceptance criterion was that the RSD between replicates should be ≤ 20% at all levels evaluated.</div></section><section id="sec-2-7"><h3>2.7 Data analysis</h3><div id="para0018" role="paragraph">A four-parameter model was used to analyze the corresponding relationship between relative luciferase units (RLU) and the logarithm of sample concentrations. The four-parameter equation is described as follows: Y = (a - d) / [1 + (x / c)^b] + d. The concentration resulting in 50% of the maximal effect (EC50) of sclerostin-neutralizing mAbs was utilized to assess their bioactivity. The signal-to-noise ratio was calculated as the ratio of the upper asymptote to the lower asymptote. Data analysis was performed using GraphPad Prism 6.0 (La Jolla, CA).</div></section></section><section id="sec-3"><h2>3 Results</h2><section id="sec-3-1"><h3>3.1 Wnt1 and Wnt3a have distinct modes of action</h3><div id="para0019" role="paragraph">The current cell-based bioassays for determining the bioactivity of sclerostin-neutralizing mAbs usually use Wnt1 or Wnt3a to activate the Wnt signaling pathway. We first demonstrated that Wnt1 and Wnt3a have different modes of action. As shown in <a id="crf0053" href="#fig0001">Fig. 1</a>, conditioned medium from Wnt1 producer cells did not induce Wnt reporter activity in Wnt reporter cells. The conditioned media from Wnt3a producer cells induced significant luciferase activity in Wnt reporter cells. The co-culture of Wnt1 or Wnt3a producer cells with Wnt reporter cells resulted in activation of the Wnt pathway. These data demonstrate that secreted Wnt3a can diffuse into the medium and activate the Wnt pathway. However, Wnt1 only stimulates the Wnt signaling pathway in the co-culture of producer and reporter cells. These data also suggest that secreted Wnt1 binds to the cell membrane and induce Wnt signaling in the Wnt reporter cells in a juxtacrine manner. Next, we determined the suppressive effect of sclerostin on the Wnt1 and Wnt3a signaling pathways. The use of 1.25 µg/mL sclerostin was observed to effectively suppress the Wnt1 signaling pathway, while no discernible effect was noted on the Wnt3a signaling pathway. This is consistent with previous reports that sclerostin functions as an inhibitor of Wnt1, rather than Wnt3a [<a id="crf0054" href="#bib0015" role="doc-biblioref" data-xml-rid="bib0015">15</a>,<a id="crf0055" href="#bib0017" role="doc-biblioref" data-xml-rid="bib0017">17</a>].</div><div class="figure-wrap"><figure id="fig0001" class="graphic"><a class="icon-full-screen" href="/cms/10.1016/j.slasd.2024.100187/asset/61d48269-4e94-4c10-ac7e-91cf75b82d28/main.assets/gr1_lrg.jpg" target="_blank" title="View full size image in a new tab"><img alt="Fig. 1" src="/cms/10.1016/j.slasd.2024.100187/asset/b152f008-dfd6-4799-830d-e14c9d6e1d73/main.assets/gr1.jpg" height="397" width="395" data-viewer-src="/cms/10.1016/j.slasd.2024.100187/asset/61d48269-4e94-4c10-ac7e-91cf75b82d28/main.assets/gr1_lrg.jpg" loading="lazy" /></a><figcaption class="figure__caption__body"><div id="fig0001-title" class="figure__title"><span class="label figure__label">Fig. 1</span> <span class="figure__title__text"><b>Wnt1 and Wnt3a have distinct modes of action</b>. The Wnt signaling pathway was induced either by conditioned medium (CM) or by co-culture of Wnt producer cells and reporter cells. Wnt1 CM did not activate the Wnt pathway, but Wnt1 producer cells significantly activated the Wnt pathway. Both Wnt3a CM and Wnt3a producer cells activated the Wnt pathway. Sclerostin inhibited Wnt1 induced Wnt pathway activation, but did not inhibit Wnt3a induced Wnt pathway activation.</span></div></figcaption></figure></div></section><section id="sec-3-2"><h3>3.2 Method development and optimization</h3><div id="para0020" role="paragraph">The mechanism of action of sclerostin-neutralizing mAbs is that the mAbs antagonize the inhibitory effects of sclerostin on the Wnt1 juxtacrine signaling. Cell-based bioassays are inherently complex and variable. To minimize this variability, a stepwise approach is used to develop and optimize the assay. The bioassay is based on the co-culture of producer and reporter cells, and parameters that potentially influence assay performance include cell seeding density, producer to reporter cell (P/R) ratio, working concentration of sclerostin, and assay incubation time (<a id="crf0056" href="#tbl0001">Table 1</a>). Single clone cell lines were employed for subsequent experiments due to their genetic uniformity, which ensures more consistent results.</div><div id="para0021" role="paragraph">When Wnt reporter cells were plated at densities of 1 × 10<sup>4</sup>, 2 × 10<sup>4</sup> or 3 × 10<sup>4</sup> cells/well in a 96-well plate, the producer cell were seeded at densities corresponding to P/R ratios of 1:2.5, 1:5, 1:10, 1:20, 1:30, and 1:40. The co-cultured producer and reporter cells were incubated for 20 hours, and the activation of the Wnt signaling pathway was assessed through a luciferase assay. As shown in <a id="crf0057" href="#fig0002">Fig. 2</a>a, luciferase activity increased as the number of reporter cells increased from 1 × 10<sup>4</sup> to 3 × 10<sup>4</sup> cells/well. A seeding density of 3 × 10<sup>4</sup> cells/well was chosen for the reporter cells. On the other hand, relatively higher luciferase activity was achieved at P/R ratios of 1:2.5, 1:5, or 1:10. The highest luciferase activity was observed when the producer and reporter cells were seeded at densities of 1.2 × 10<sup>4</sup> and 3 × 10<sup>4</sup> cells/well respectively, corresponding to a P/R ratio of 1:2.5. This ratio was not selected for further evaluation because the cells were overgrown. The P/R ratios of 1:5 and 1:10 were further assessed using the sclerostin inhibition assay. In this assay, serially diluted sclerostin was added immediately after cell seeding and the luciferase activity was determined 20 hours later. As shown in <a id="crf0058" href="#fig0002">Fig. 2</a>b, the IC50 values for sclerostin were comparable (0.2985 μg/mL for the P/R ratio of 1:5 and 0.2188 μg/mL for the P/R ratio of 1:10). The 1:5 P/R ratio (6 × 10<sup>3</sup> producer cells and 3 × 10<sup>4</sup> reporter cells/well) was chosen for subsequent experiments because of its significantly higher signal-to-noise ratio.</div><div class="figure-wrap"><figure id="fig0002" class="graphic"><a class="icon-full-screen" href="/cms/10.1016/j.slasd.2024.100187/asset/8e080ccc-928b-458a-ad06-fab92921b36b/main.assets/gr2_lrg.jpg" target="_blank" title="View full size image in a new tab"><img alt="Fig. 2" src="/cms/10.1016/j.slasd.2024.100187/asset/4374a882-1a71-4d82-a5dd-8bfe48061c5d/main.assets/gr2.jpg" height="432" width="781" data-viewer-src="/cms/10.1016/j.slasd.2024.100187/asset/8e080ccc-928b-458a-ad06-fab92921b36b/main.assets/gr2_lrg.jpg" loading="lazy" /></a><figcaption class="figure__caption__body"><div id="fig0002-title" class="figure__title"><span class="label figure__label">Fig. 2</span> <span class="figure__title__text"><b>Method generation and optimization</b>. (a). Cell seeding number and P/R ratio. Producer and reporter cells were co-cultured in a 96-well plate. The final density of reporter cells was set at 1 × 10<sup>4</sup>, 2 × 10<sup>4</sup>, or 3 × 10<sup>4</sup> cells/well and the corresponding P/R ratios of 1:2.5, 1:5, 1:10, 1:20, 1:30, and 1:40. Twenty hours after cell seeding, a luciferase assay was performed to detect the activation of Wnt signaling pathway. (b). Optimization of P/R ratio and preliminary determination of sclerostin concentration. 3 × 10<sup>4</sup> reporter cells were co-cultured with 6 × 10<sup>3</sup> (P/R ratio of 1:5) or 3 × 10<sup>3</sup> (P/R ratio of 1:10) producer cells per well in a 96-well plate. Sclerostin was initially set at a working concentration of 40 μg/mL and subsequently serially diluted 11 times. Serially diluted sclerostin was added immediately after cell seeding. After 20 hours of incubation, a luciferase assay was performed, and the resulting relative luciferase units obtained from different concentrations of sclerostin were fitted with a four-parameter logistic curve equation. (c). Optimization of sclerostin concentration. 3 × 10<sup>4</sup> reporter cells were plated with 6 × 10<sup>3</sup> producer cells (P/R ratio of 1:5) per well in a 96-well plate. Sclerostin at working concentrations of 1.25, 2.5, and 5 μg/mL, along with serially diluted romosozumab, were preincubated for 1 hour at room temperature and added immediately after cell seeding. The activation of the Wnt signaling pathway was assessed through a luciferase assay after 20 hours of incubation. (d). Optimization of assay incubation time. Serially diluted romosozumab was preincubated with sclerostin (final working concentration: 2.5 μg/mL) for 1 hour at room temperature. Subsequently, 3 × 10<sup>4</sup> reporter cells were plated with 6 × 10<sup>3</sup> producer cells (P/R ratio of 1:5) per well in a 96-well plate. The antibody/antigen mixture was added immediately after cell seeding, and luciferase assays were conducted after 4, 6, 8, 16, 20, and 24 hours of incubation.</span></div></figcaption></figure></div><div id="para0022" role="paragraph">To determine the optimal working concentration of sclerostin, concentrations of 1.25, 2.5, and 5 μg/mL were further evaluated with a sclerostin-neutralizing mAb, romosozumab. Romosozumab was serially diluted and incubated with sclerostin for 1 h at room temperature, then the antibody/antigen mixture was added immediately after the seeding of producer and reporter cells, and luciferase activity was determined 20 hours later. The data demonstrated that romosozumab neutralized sclerostin and activated the Wnt signaling pathway (<a id="crf0059" href="#fig0002">Fig. 2</a>c). The EC50 values for romosozumab were 2.045, 4.708, and 8.965 μg/mL when 1.25, 2.5, and 5.0 μg/mL of sclerostin were applied, yielding signal-to-noise ratios of 4.35, 10.86, and 12.94. Taking into account of the signal-to-noise ratio and the cost of recombinant sclerostin, a concentration of 2.5 μg/mL was selected for further assay optimization.</div><div id="para0023" role="paragraph">The assay incubation time was optimized by conducting a time course experiment at 4, 6, and 8 hours, as well as overnight (16, 20, and 24 hours). As depicted in <a id="crf0060" href="#fig0002">Fig. 2</a>d, 16, 20, and 24 hours of incubation resulted in higher luciferase activities and signal-to-noise ratios compared to 4, 6, and 8 hours of incubation. Therefore, the optimal incubation time was determined to be within the range of 16 to 24 hours, with 20 hours selected as the standard incubation time.</div><div id="para0024" role="paragraph">In summary, the optimal assay conditions were determined to be: 1) 6 × 10<sup>3</sup> cells/well for producer cells; 2) 3 × 10<sup>4</sup> cells/well for reporter cells; 3) a P/R ratio of 1:5; 4) sclerostin working concentration of 2.5 μg/mL; and 5) an assay incubation time of 20 h.</div></section><section id="sec-3-3"><h3>3.3 Validation of the dual cell-based reporter gene assay</h3><div id="para0025" role="paragraph">Validation parameters were established according to the ICH Q2(R1) guidelines, including specificity, accuracy (dilution linearity), and precision.</div><div id="para0026" role="paragraph">Specificity refers to the ability of an assay to selectively measure the analyte of interest. The specificity of the method was demonstrated by assessing the potency of romosozumab, dupilumab (targeting IL-4Ra), denosumab (targeting RANKL), tocilizumab (targeting IL-6R), siltuximab (targeting IL-6) and romosozumab formulation buffer in antagonizing sclerostin and activating the Wnt signaling pathway. As shown in <a id="crf0061" href="#fig0003">Fig. 3</a>, romosozumab induced significant luciferase activity, with an EC50 value of 5.385 μg/mL and R<sup>2</sup> of 0.9887. However, mAbs not targeting sclerostin and the formulation buffer did not elicit a measurable response in this experiment. The results suggest that this method is specific for the evaluation of the biological activity of sclerostin-neutralizing mAb.</div><div class="figure-wrap"><figure id="fig0003" class="graphic"><a class="icon-full-screen" href="/cms/10.1016/j.slasd.2024.100187/asset/c6146677-aacf-4ee9-98ea-5851b9ec3388/main.assets/gr3_lrg.jpg" target="_blank" title="View full size image in a new tab"><img alt="Fig. 3" src="/cms/10.1016/j.slasd.2024.100187/asset/6b817067-ef07-49d5-9595-5d51afbaf2ab/main.assets/gr3.jpg" height="218" width="489" data-viewer-src="/cms/10.1016/j.slasd.2024.100187/asset/c6146677-aacf-4ee9-98ea-5851b9ec3388/main.assets/gr3_lrg.jpg" loading="lazy" /></a><figcaption class="figure__caption__body"><div id="fig0003-title" class="figure__title"><span class="label figure__label">Fig. 3</span> <span class="figure__title__text"><b>Validation of assay specificity</b>. Romosozumab, dupilumab (targeting IL-4Ra), denosumab (targeting RANKL), tocilizumab (targeting IL-6R), siltuximab (targeting IL-6), and romosozumab formulation buffer were employed to assess the specificity of the assay. The data were analyzed using a four-parameter logistic curve equation.</span></div></figcaption></figure></div><div id="para0027" role="paragraph">Accuracy is employed to assess how closely the actual measured value aligns with the reference value. Accuracy was assessed by adjusting the initial concentration of romosozumab to 50%, 75%, 100%, 125%, and 150% of the reference activity level (set at 100%). Each of the prepared samples were subjected to three independent tests, and the EC50 values were subsequently calculated. Compared to the reference, the percentage recoveries from the different bioactivity levels were all within 100 ± 5% (<a id="crf0062" href="#tbl0002">Table 2</a>). Furthermore, a linear regression analysis was conducted to assess the relationship between the measured potency and the theoretical values. The R<sup>2</sup> was determined as 0.9993 and the slope as 1.0195 (<a id="crf0063" href="#fig0004">Fig. 4</a>).</div><div class="figure-wrap"><figure id="fig0004" class="graphic"><a class="icon-full-screen" href="/cms/10.1016/j.slasd.2024.100187/asset/00a49235-a07f-4a38-a9c6-0630be6950aa/main.assets/gr4_lrg.jpg" target="_blank" title="View full size image in a new tab"><img alt="Fig. 4" src="/cms/10.1016/j.slasd.2024.100187/asset/e0d20691-fc6c-4a83-9d97-ff63e82d1bf3/main.assets/gr4.jpg" height="253" width="339" data-viewer-src="/cms/10.1016/j.slasd.2024.100187/asset/00a49235-a07f-4a38-a9c6-0630be6950aa/main.assets/gr4_lrg.jpg" loading="lazy" /></a><figcaption class="figure__caption__body"><div id="fig0004-title" class="figure__title"><span class="label figure__label">Fig. 4</span> <span class="figure__title__text"><b>Assessment of linearity</b>. Romosozumab samples, diluted to 50%, 75%, 100%, 125%, and 150% of the reference activity, were subjected to three rounds of testing using the bioassay. Subsequently, a linear regression analysis was conducted to evaluate the relationship between the expected potency and the measured potency.</span></div></figcaption></figure></div><div class="figure-wrap"><figure id="tbl0002" class="table" data-figureviewer-ignore="true"><div class="table-wrap"><table data-figureviewer-ignore="true"><thead><tr class="rowsep" data-xml-align="left" data-xml-valign="top"><th id="en0023">Parameters</th><th id="en0024" colspan="4"><span>Results</span></th></tr></thead><tbody><tr><td id="en0025" rowspan="6"><b>Accuracy</b></td><td id="en0026">%RP</td><td id="en0027">Relative bioactivity (%RP)</td><td id="en0028">RSD (%)</td><td id="en0029">Recovery (%)</td></tr><tr><td id="en0031">50</td><td id="en0032">50.69 ± 5.42</td><td id="en0033">10.69</td><td id="en0034">101.38</td></tr><tr><td id="en0036">75</td><td id="en0037">74.70 ± 1.65</td><td id="en0038">2.21</td><td id="en0039">99.60</td></tr><tr><td id="en0041">100</td><td id="en0042">100.63 ± 1.02</td><td id="en0043">1.01</td><td id="en0044">100.63</td></tr><tr><td id="en0046">125</td><td id="en0047">124.95 ± 12.05</td><td id="en0048">9.64</td><td id="en0049">99.96</td></tr><tr><td id="en0051">150</td><td id="en0052">153.00 ± 17.06</td><td id="en0053">11.15</td><td id="en0054">102.00</td></tr><tr><td id="en0055"><b>Repeatability</b></td><td id="en0056" colspan="4">Repeatability (%RSD):<br />1.77% for analyst 1; 5.32% for analyst 2.</td></tr><tr><td id="en0057"><b>Intermediate precision</b></td><td id="en0058" colspan="4">Intermediate precision (%RSD): 2.87%.</td></tr><tr><td id="en0059"><b>Linearity</b></td><td id="en0060" colspan="4">Regression line (R<sup>2</sup>): 0.9993<br />Slope: 1.0195</td></tr></tbody></table></div><figcaption><span class="heading">Table 2</span><div id="spara006" role="paragraph">Assay validation</div><div class="core-table-tools"><ul><li><a class="core-table-show-action" href="/action/showFullTableHTML?isHtml=true&tableId=tbl0002&pii=S2472-5552%2824%2900049-2" target="_blank">Open table in a new tab</a></li></ul></div></figcaption></figure></div><div id="para0028" role="paragraph">Precision of the assay was evaluated at two levels: repeatability and intermediate precision. Repeatability refers to the plate-to-plate variability of the method operating over a short interval under similar conditions (e.g., same day setup, same analyst). Two analysts performed the assay independently three times in one day. As shown in <a id="crf0064" href="#tbl0002">Table 2</a>, the percentage RSD of repeatability was 1.77% and 5.32% for the data from analyst 1 and analyst 2, respectively. Intermediate precision refers to within-laboratory variations such as different days. In this case, the intermediate precision experiments were conducted by two analysts and repeated 9 times over three different days. The percentage RSD of the intermediate precision in this assay is 2.87%.</div><div id="para0029" role="paragraph">Taken together, the validation data demonstrate that all the parameters meet the predefined criteria. The method has excellent specificity, accuracy (dilution linearity), and precision.</div></section></section><section id="sec-4"><h2>4 Discussion</h2><div id="para0030" role="paragraph">Biological activity is a critical quality attribute for biopharmaceuticals, which should be accurately measured using an appropriate bioassay. Design strategies for bioassays are driven by the drug's intended physiological mechanism of action. The mechanism of action of sclerostin-neutralizing mAbs is to antagonize sclerostin-mediated inhibition of Wnt1 juxtacrine signaling. Wnt1 and Wnt3a are pivotal osteogenic factors and are frequently employed as Wnt pathway activators in cell-based assays for the determination of the biological activity of sclerostin-neutralizing mAbs. It has been demonstrated that exosomes are effective vehicles for the trafficking of Wnt proteins [<a id="crf0065" href="#bib0041" role="doc-biblioref" data-xml-rid="bib0041 bib0042 bib0043">41-43</a>]. However, Wnt1 and Wnt3a have different modes of action. As shown in <a id="crf0067" href="#fig0001">Fig. 1</a>, Wnt3a conditioned medium activated Wnt signaling in the reporter cells. In contrast, Wnt1 conditioned medium did not activate Wnt signaling in the reporter cells, indicating that Wnt1 couldn't diffuse into the medium or be transported by exosomes. The Wnt1 producer cells triggered the Wnt signaling cascade in the co-cultured Wnt reporter cells, thereby demonstrating that Wnt1 signaling requires direct cell-to-cell contact. This mode of action is classified as juxtacrine signaling, which involves molecules that induce functional changes in neighboring target cells while remaining associated with the plasma membrane of the producer cells, as opposed to acting in the fluid phase [<a id="crf0068" href="#bib0044" role="doc-biblioref" data-xml-rid="bib0044">44</a>]. A recent study demonstrated that Wnt1-overexpressing cells in the insert failed to induce differentiation of osteoblastic cells at the bottom of the well in a trans-well assay, ruling out the possibility of Wnt1 being transported by exosomes or free diffusion. The authors concluded that Wnt1 signals in a strictly juxtacrine manner to induce osteoblast differentiation [<a id="crf0069" href="#bib0010" role="doc-biblioref" data-xml-rid="bib0010">10</a>]. When sclerostin was added to the co-culture of Wnt producer and reporter cells, it suppressed the bioactivity of Wnt1, but not that of Wnt3a. These data are in accordance with previous reports indicating that Wnt1 directly competes with sclerostin for the E1E2 domain of LRP5/6, whereas Wnt3a binds to the E3E4 domain [<a id="crf0070" href="#bib0015" role="doc-biblioref" data-xml-rid="bib0015">15</a>,<a id="crf0071" href="#bib0017" role="doc-biblioref" data-xml-rid="bib0017">17</a>]. Based on these data, we concluded that Wnt1 should be employed in the assay and that a dual-cell system should be utilized to provide a physiological niche that mimics the in vivo situation in bone tissue, wherein Wnt1 is secreted by osteocytes and mesenchymal cells and acts on neighboring osteoblasts through direct cell-to-cell contact [<a id="crf0072" href="#bib0010" role="doc-biblioref" data-xml-rid="bib0010">10</a>,<a id="crf0073" href="#bib0014" role="doc-biblioref" data-xml-rid="bib0014">14</a>,<a id="crf0074" href="#bib0045" role="doc-biblioref" data-xml-rid="bib0045">45</a>,<a id="crf0075" href="#bib0046" role="doc-biblioref" data-xml-rid="bib0046">46</a>].</div><div id="para0031" role="paragraph">The main advantages of reporter gene assay are high sensitivity, reliability, convenience, dynamic range, and adaptability to high-throughput screening [<a id="crf0076" href="#bib0047" role="doc-biblioref" data-xml-rid="bib0047">47</a>]. Due to the shorter time required for reporter gene expression, 20 hours compared to 12-20 days for the standard mineralization assay, the reporter gene assay appears to be less affected by extraneous influences and is therefore less variable and more accurate. The dual cell-based bioassay was developed and optimized using a methodical stepwise approach with romosozumab as the model mAb, including optimization of the seeding density of producer and reporter cells, the producer to reporter cell (P/R) ratio, the working concentration of sclerostin, and the assay incubation time. An optimal standard assay condition has finally been established. However, the assay conditions are not universal and may necessitate further optimization for recombinant sclerostin proteins from different suppliers and sclerostin-neutralizing mAbs other than romosozumab. The co-culture of ligand producing cells and reporter cells can be applied to detect other clinically relevant signaling pathways that operate through juxtacrine signaling, such as the Notch, ephrinB2, and osteoprotegerin signaling pathways [<a id="crf0077" href="#bib0010" role="doc-biblioref" data-xml-rid="bib0010">10</a>,<a id="crf0078" href="#bib0048" role="doc-biblioref" data-xml-rid="bib0048">48</a>,<a id="crf0079" href="#bib0049" role="doc-biblioref" data-xml-rid="bib0049">49</a>].</div><div id="para0032" role="paragraph">This bioassay has been systematically validated, including assessment of specificity, accuracy, linearity, and precision, according to pharmaceutical guidelines and previous studies [<a id="crf0080" href="#bib0050" role="doc-biblioref" data-xml-rid="bib0050 bib0051 bib0052 bib0053 bib0054">50-54</a>]. As shown in <a id="crf0082" href="#fig0003">Fig. 3</a>, this assay specifically detected the bioactivity of mAb targeting sclerostin (romosozumab). The formulation buffer of romosozumab and mAbs not targeting sclerostin did not elicit a measurable response. Accuracy validation showed that the percentage recoveries of the different bioactivity levels of romosozumab compared to the reference were all within 100 ± 5% of the reference. The R<sup>2</sup> of a linear regression analysis was 0.9993 and the slope was 1.0195. These data suggest that this assay could accurately determine the biological activity of sclerostin-neutralizing mAbs. For repeatability, the percentage RSD of repeatability was 1.77% and 5.32% for two analysts. The percentage RSD of intermediate precision in this assay is 2.87%. All the parameters meet the predefined standard.</div><div id="para0033" role="paragraph">Another crucial issue that needs to be considered is the suitability of the HEK293T cell line for this assay. For a cell-based bioassay, the cell line should respond well to the drug; more specifically, the cell line must express biologically relevant signaling molecules and pathways to recapitulate the in vivo functional response activated or inhibited by the drug. The wild type HEK293T cells express endogenous LRP5/6 and Frizzled co-receptors, respond to Wnt stimulation and have no known mutations in the proteins associated with Wnt signaling [<a id="crf0083" href="#bib0055" role="doc-biblioref" data-xml-rid="bib0055">55</a>,<a id="crf0084" href="#bib0056" role="doc-biblioref" data-xml-rid="bib0056">56</a>]. This was further confirmed in this study, wherein Wnt1 expressed from producer cells induced activation of the Wnt pathway in the reporter cells. Therefore, HEK293T cells are suitable for assessing drugs that modulate the Wnt signaling pathway. However, HEK293T could not reflect the physiological phenotype of bone formation. Co-culture of Wnt1 producer cells with pre-osteoblast cells, such as MC3T3-E1, should be able to reflect the physiological phenotype of bone formation. The reporter gene assay and the mineralization assay of MC3T3-E1 induced by Wnt1 producer cells can be used as orthogonal methods.</div></section><section id="sec-5"><h2>5 Conclusion</h2><div id="para0034" role="paragraph">Compared to the MC3T3-E1 mineralization assay, C2C12 trans-differentiation and ALP activity assay and single cell-based reporter gene assay, the dual cell-based bioassay demonstrated in this study is mechanism of action reflective. In addition, this bioassay is accurate, precise, time-saving, and easy to perform. It could serve for bioactivity determination including antibody discovery, characterization, quality control, batch release, and biosimilarity assessment.</div></section><section id="sec-6"><h2>CRediT authorship contribution statement</h2><div id="para0034a" role="paragraph"><b>Suzhen Wei:</b> Writing – original draft, Methodology, Formal analysis, Data curation, Conceptualization. <b>Qiang Wu:</b> Methodology, Data curation. <b>Chunlai Cao:</b> Validation. <b>Zhuoni Yang:</b> Methodology. <b>Jianrui Shi:</b> Methodology. <b>Jingqun Huang:</b> Validation. <b>Hua He:</b> Validation. <b>Yongjie Lai:</b> Writing – original draft, Investigation, Funding acquisition, Conceptualization. <b>Jing Li:</b> Writing – review & editing, Supervision, Investigation, Funding acquisition, Data curation, Conceptualization.</div></section><section id="conflict" class="core-conflict"><h2>Conflicts of Interest</h2><div id="para0035" role="paragraph">All authors except for Yongjie Lai are employees of Zhuhai United Biopharma Co., Ltd or Zhuhai United Laboratories Co., Ltd. The authors are aware of no affiliations, memberships, funding, or financial interests that might be perceived to affect the objectivity of this work.</div></section><section id="acknowledgments" role="doc-acknowledgments"><h2>Acknowledgments</h2><div id="para0036" role="paragraph">This work was supported by Zhuhai United Laboratories Co., Ltd, Zhuhai United Biopharma Co., Ltd, and the Natural Science Foundation of Guizhou Province (Grant Number: 20201Y297, ZK-YIBAN2023510). 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A mechanism of action-reflective, dual cell-based bioassay for determining the bioactivity of sclerostin-neutralizing antibodies - SLAS Discovery