Raman spectroscopy for monitoring free sulfhydryl formation during monoclonal antibody manufacturing
During production, harvested cell culture fluid (HCCF) can degrade due to reductases breaking interchain disulfide bonds, forming low molecular weight (LMW) impurities that contain free sulfhydryl and high molecular weight (HMW) impurities through disulfide shuffling. Thus, detecting and quantifying...
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container_title | Journal of pharmaceutical and biomedical analysis |
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creator | Wang, Zhenshu Hsieh, Andrew Rose, Patricia Zhou, George Battle, Sonja Raymond, Kelly Haley, Monica Cote, Aaron Bennun, Sandra Ahuja, Sanjeev |
description | During production, harvested cell culture fluid (HCCF) can degrade due to reductases breaking interchain disulfide bonds, forming low molecular weight (LMW) impurities that contain free sulfhydryl and high molecular weight (HMW) impurities through disulfide shuffling. Thus, detecting and quantifying the free sulfhydryl increase in HCCF is critical. Herein, Raman spectroscopy is implemented as a process analytical technology, and multivariate data analysis is applied to characterize and quantify sulfhydryl formation in HCCF with disulfide-containing indicator molecules. Raman spectra qualitatively probe the presence or absence of disulfide bond breakage in antibodies, consistent with offline non-reduced capillary electrophoresis sodium dodecyl sulfate results. Between two antibodies studied, mAb A was identified for a higher risk of antibody reduction where sulfhydryl formation was observed within 16 h, while mAb B did not show similar concerns even after 1 week. The offline measurement of redox potential is below –100 mV in HCCF for mAb A, while the stable mAb B HCCF shows redox potentials above +20 mV. A multivariate partial least squares (PLS) model for quantification is developed using an offline free sulfhydryl assay, applying Raman spectra to predict free sulfhydryl concentration with high accuracy (R2 > 0.98) and expected mean error of 0.677 mM from the offline Ellman’s Assay. This work confirms the use of Raman PAT to monitor real-time disulfide reduction, enabling improvements to process understanding and product quality.
[Display omitted]
•Raman spectroscopy is developed as a PAT tool to monitor antibody reduction.•The qualitative antibody reduction result is verified with non-reduced CE-SDS.•Disulfide bond contained indicators help boost Raman signals for quantitation.•The quantitative PLS model can detect free sulfhydryl with 0.677 mM RMSECV. |
doi_str_mv | 10.1016/j.jpba.2024.116530 |
format | Article |
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[Display omitted]
•Raman spectroscopy is developed as a PAT tool to monitor antibody reduction.•The qualitative antibody reduction result is verified with non-reduced CE-SDS.•Disulfide bond contained indicators help boost Raman signals for quantitation.•The quantitative PLS model can detect free sulfhydryl with 0.677 mM RMSECV.</description><identifier>ISSN: 0731-7085</identifier><identifier>ISSN: 1873-264X</identifier><identifier>EISSN: 1873-264X</identifier><identifier>DOI: 10.1016/j.jpba.2024.116530</identifier><identifier>PMID: 39447421</identifier><language>eng</language><publisher>England: Elsevier B.V</publisher><subject>Animals ; Antibodies, Monoclonal - chemistry ; CHO Cells ; Cricetulus ; Disulfide reduction ; Disulfides - analysis ; Disulfides - chemistry ; Least-Squares Analysis ; Multivariate Analysis ; Oxidation-Reduction ; PAT ; PLS ; Raman spectroscopy ; Redox ; Spectrum Analysis, Raman - methods ; Sulfhydryl Compounds - analysis ; Sulfhydryl Compounds - chemistry</subject><ispartof>Journal of pharmaceutical and biomedical analysis, 2025-01, Vol.252, p.116530, Article 116530</ispartof><rights>2024</rights><rights>Copyright © 2024. Published by Elsevier B.V.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c237t-c5d154f8d221b1a9fb840f76b6e62ea2b9389f785292a48ed563ae8abd14763f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jpba.2024.116530$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/39447421$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wang, Zhenshu</creatorcontrib><creatorcontrib>Hsieh, Andrew</creatorcontrib><creatorcontrib>Rose, Patricia</creatorcontrib><creatorcontrib>Zhou, George</creatorcontrib><creatorcontrib>Battle, Sonja</creatorcontrib><creatorcontrib>Raymond, Kelly</creatorcontrib><creatorcontrib>Haley, Monica</creatorcontrib><creatorcontrib>Cote, Aaron</creatorcontrib><creatorcontrib>Bennun, Sandra</creatorcontrib><creatorcontrib>Ahuja, Sanjeev</creatorcontrib><title>Raman spectroscopy for monitoring free sulfhydryl formation during monoclonal antibody manufacturing</title><title>Journal of pharmaceutical and biomedical analysis</title><addtitle>J Pharm Biomed Anal</addtitle><description>During production, harvested cell culture fluid (HCCF) can degrade due to reductases breaking interchain disulfide bonds, forming low molecular weight (LMW) impurities that contain free sulfhydryl and high molecular weight (HMW) impurities through disulfide shuffling. Thus, detecting and quantifying the free sulfhydryl increase in HCCF is critical. Herein, Raman spectroscopy is implemented as a process analytical technology, and multivariate data analysis is applied to characterize and quantify sulfhydryl formation in HCCF with disulfide-containing indicator molecules. Raman spectra qualitatively probe the presence or absence of disulfide bond breakage in antibodies, consistent with offline non-reduced capillary electrophoresis sodium dodecyl sulfate results. Between two antibodies studied, mAb A was identified for a higher risk of antibody reduction where sulfhydryl formation was observed within 16 h, while mAb B did not show similar concerns even after 1 week. The offline measurement of redox potential is below –100 mV in HCCF for mAb A, while the stable mAb B HCCF shows redox potentials above +20 mV. A multivariate partial least squares (PLS) model for quantification is developed using an offline free sulfhydryl assay, applying Raman spectra to predict free sulfhydryl concentration with high accuracy (R2 > 0.98) and expected mean error of 0.677 mM from the offline Ellman’s Assay. This work confirms the use of Raman PAT to monitor real-time disulfide reduction, enabling improvements to process understanding and product quality.
[Display omitted]
•Raman spectroscopy is developed as a PAT tool to monitor antibody reduction.•The qualitative antibody reduction result is verified with non-reduced CE-SDS.•Disulfide bond contained indicators help boost Raman signals for quantitation.•The quantitative PLS model can detect free sulfhydryl with 0.677 mM RMSECV.</description><subject>Animals</subject><subject>Antibodies, Monoclonal - chemistry</subject><subject>CHO Cells</subject><subject>Cricetulus</subject><subject>Disulfide reduction</subject><subject>Disulfides - analysis</subject><subject>Disulfides - chemistry</subject><subject>Least-Squares Analysis</subject><subject>Multivariate Analysis</subject><subject>Oxidation-Reduction</subject><subject>PAT</subject><subject>PLS</subject><subject>Raman spectroscopy</subject><subject>Redox</subject><subject>Spectrum Analysis, Raman - methods</subject><subject>Sulfhydryl Compounds - analysis</subject><subject>Sulfhydryl Compounds - chemistry</subject><issn>0731-7085</issn><issn>1873-264X</issn><issn>1873-264X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2025</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kEtr3DAUhUVJ6Uwm_QNZFC-z8URPS4ZuQkjawEChtJCdkKWrRoNtuZJd8L-P59Ess7qL-50D50PomuAtwaS63W_3Q2O2FFO-JaQSDH9Aa6IkK2nFny_QGktGSomVWKHLnPcYY0Fq_gmtWM255JSskftpOtMXeQA7pphtHObCx1R0sQ9jTKH_U_gEUOSp9S-zS3N7eHdmDLEv3HQEFjbaNvamLUw_hia6uVhKJ2_seCSu0Edv2gyfz3eDfj8-_Lr_Xu5-fHu6v9uVljI5llY4IrhXjlLSEFP7RnHsZdVUUFEwtKmZqr1UgtbUcAVOVMyAMo0jXFbMsw26OfUOKf6dII-6C9lC25oe4pQ1IxSLWhCpFpSeULuszgm8HlLoTJo1wfpgV-_1wa4-2NUnu0voy7l_ajpwb5H_Ohfg6wmAZeW_AElnG6C34EJaBGsXw3v9r6EfjcU</recordid><startdate>20250101</startdate><enddate>20250101</enddate><creator>Wang, Zhenshu</creator><creator>Hsieh, Andrew</creator><creator>Rose, Patricia</creator><creator>Zhou, George</creator><creator>Battle, Sonja</creator><creator>Raymond, Kelly</creator><creator>Haley, Monica</creator><creator>Cote, Aaron</creator><creator>Bennun, Sandra</creator><creator>Ahuja, Sanjeev</creator><general>Elsevier B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20250101</creationdate><title>Raman spectroscopy for monitoring free sulfhydryl formation during monoclonal antibody manufacturing</title><author>Wang, Zhenshu ; Hsieh, Andrew ; Rose, Patricia ; Zhou, George ; Battle, Sonja ; Raymond, Kelly ; Haley, Monica ; Cote, Aaron ; Bennun, Sandra ; Ahuja, Sanjeev</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c237t-c5d154f8d221b1a9fb840f76b6e62ea2b9389f785292a48ed563ae8abd14763f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2025</creationdate><topic>Animals</topic><topic>Antibodies, Monoclonal - chemistry</topic><topic>CHO Cells</topic><topic>Cricetulus</topic><topic>Disulfide reduction</topic><topic>Disulfides - analysis</topic><topic>Disulfides - chemistry</topic><topic>Least-Squares Analysis</topic><topic>Multivariate Analysis</topic><topic>Oxidation-Reduction</topic><topic>PAT</topic><topic>PLS</topic><topic>Raman spectroscopy</topic><topic>Redox</topic><topic>Spectrum Analysis, Raman - methods</topic><topic>Sulfhydryl Compounds - analysis</topic><topic>Sulfhydryl Compounds - chemistry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wang, Zhenshu</creatorcontrib><creatorcontrib>Hsieh, Andrew</creatorcontrib><creatorcontrib>Rose, Patricia</creatorcontrib><creatorcontrib>Zhou, George</creatorcontrib><creatorcontrib>Battle, Sonja</creatorcontrib><creatorcontrib>Raymond, Kelly</creatorcontrib><creatorcontrib>Haley, Monica</creatorcontrib><creatorcontrib>Cote, Aaron</creatorcontrib><creatorcontrib>Bennun, Sandra</creatorcontrib><creatorcontrib>Ahuja, Sanjeev</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of pharmaceutical and biomedical analysis</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wang, Zhenshu</au><au>Hsieh, Andrew</au><au>Rose, Patricia</au><au>Zhou, George</au><au>Battle, Sonja</au><au>Raymond, Kelly</au><au>Haley, Monica</au><au>Cote, Aaron</au><au>Bennun, Sandra</au><au>Ahuja, Sanjeev</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Raman spectroscopy for monitoring free sulfhydryl formation during monoclonal antibody manufacturing</atitle><jtitle>Journal of pharmaceutical and biomedical analysis</jtitle><addtitle>J Pharm Biomed Anal</addtitle><date>2025-01-01</date><risdate>2025</risdate><volume>252</volume><spage>116530</spage><pages>116530-</pages><artnum>116530</artnum><issn>0731-7085</issn><issn>1873-264X</issn><eissn>1873-264X</eissn><abstract>During production, harvested cell culture fluid (HCCF) can degrade due to reductases breaking interchain disulfide bonds, forming low molecular weight (LMW) impurities that contain free sulfhydryl and high molecular weight (HMW) impurities through disulfide shuffling. Thus, detecting and quantifying the free sulfhydryl increase in HCCF is critical. Herein, Raman spectroscopy is implemented as a process analytical technology, and multivariate data analysis is applied to characterize and quantify sulfhydryl formation in HCCF with disulfide-containing indicator molecules. Raman spectra qualitatively probe the presence or absence of disulfide bond breakage in antibodies, consistent with offline non-reduced capillary electrophoresis sodium dodecyl sulfate results. Between two antibodies studied, mAb A was identified for a higher risk of antibody reduction where sulfhydryl formation was observed within 16 h, while mAb B did not show similar concerns even after 1 week. The offline measurement of redox potential is below –100 mV in HCCF for mAb A, while the stable mAb B HCCF shows redox potentials above +20 mV. A multivariate partial least squares (PLS) model for quantification is developed using an offline free sulfhydryl assay, applying Raman spectra to predict free sulfhydryl concentration with high accuracy (R2 > 0.98) and expected mean error of 0.677 mM from the offline Ellman’s Assay. This work confirms the use of Raman PAT to monitor real-time disulfide reduction, enabling improvements to process understanding and product quality.
[Display omitted]
•Raman spectroscopy is developed as a PAT tool to monitor antibody reduction.•The qualitative antibody reduction result is verified with non-reduced CE-SDS.•Disulfide bond contained indicators help boost Raman signals for quantitation.•The quantitative PLS model can detect free sulfhydryl with 0.677 mM RMSECV.</abstract><cop>England</cop><pub>Elsevier B.V</pub><pmid>39447421</pmid><doi>10.1016/j.jpba.2024.116530</doi></addata></record> |
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subjects | Animals Antibodies, Monoclonal - chemistry CHO Cells Cricetulus Disulfide reduction Disulfides - analysis Disulfides - chemistry Least-Squares Analysis Multivariate Analysis Oxidation-Reduction PAT PLS Raman spectroscopy Redox Spectrum Analysis, Raman - methods Sulfhydryl Compounds - analysis Sulfhydryl Compounds - chemistry |
title | Raman spectroscopy for monitoring free sulfhydryl formation during monoclonal antibody manufacturing |
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