{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T07:58:05Z","timestamp":1790927885108,"version":"4.1.0"},"reference-count":29,"publisher":"Wiley","issue":"21","license":[{"start":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T00:00:00Z","timestamp":1787529600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"},{"start":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T00:00:00Z","timestamp":1787529600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Rapid Comm Mass Spectrometry"],"published-print":{"date-parts":[[2026,11,15]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Rationale<\/jats:title>\n                    <jats:p>Non\u2010reduced trypsin digestion of therapeutic antibodies can generate large disulfide\u2010linked peptide complexes that exhibit poor chromatographic and mass spectrometric performance. This limits the confident characterization of posttranslational modifications (PTMs) in specific antibody regions, such as the heavy\u2010chain constant region 1 (CH1) domain, where conventional digestion produces 8\u2010kDa complexes with poor fragmentation.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>We developed automated multistep and multienzyme digestion workflows using immobilized trypsin, chymotrypsin, and proteinase K on magnetic beads. These workflows were implemented on a robotic platform and evaluated for their ability to characterize engineered cysteine residues in the CH1 domain of antibody\u2013drug conjugate (ADC) intermediates. Analysis was performed using liquid chromatography\u2013tandem mass spectrometry (LC\u2013MS\/MS).<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>The multienzyme workflows generated highly orthogonal peptide pools; for instance, selected workflows using trypsin only, trypsin combined with chymotrypsin, and trypsin combined with proteinase K yielded 13%, 14%, and 34% unique peptide identifications, respectively. In the challenging CH1 region, the multienzyme approach identified six distinct cysteine adducts at engineered Cys152. These included cysteinylation, homocysteinylation, and an unexpected, putative process\u2010related impurity, thioglycolic acid capping, which was validated with unambiguous MS and MS\/MS evidence using selected multienzyme workflows. Relative extracted\u2010ion current\u2010based levels of these cappings were observed and found to be similar across selected workflows; for instance, relative levels of thioglycolic acid capping from 12% to 15% were observed across selected workflows.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>The presented automated multienzyme workflows serve as complementary tools in an analytical toolbox, which can be strategically deployed for targeting challenging regions of biopharmaceutical products. We demonstrate significantly improved precursor signal quality and MS\/MS sequence coverage compared to trypsin\u2010only protocols. The presented workflows are fast (36\u201392\u2009min total runtime) and utilize commercially available reagents and instrumentation, making them suitable for direct implementation in biopharmaceutical analytical development and quality control laboratories.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/rcm.70165","type":"journal-article","created":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T10:21:26Z","timestamp":1787566886000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Automated Multienzyme Digestion Workflows for Confident Characterization of Challenging Posttranslational Modifications in Therapeutic Antibodies by LC\u2013MS\/MS"],"prefix":"10.1002","volume":"40","author":[{"given":"Nanna Sofie","family":"Eskesen","sequence":"first","affiliation":[{"name":"Servier Symphogen  Ballerup Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yann","family":"Leblanc","sequence":"additional","affiliation":[{"name":"Structural Biophysics and Protein Sciences Servier R&amp;D Center  Gif\u2010sur\u2010Yvette France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Justine","family":"Lempereur","sequence":"additional","affiliation":[{"name":"Structural Biophysics and Protein Sciences Servier R&amp;D Center  Gif\u2010sur\u2010Yvette France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martin","family":"\u00d8rgaard","sequence":"additional","affiliation":[{"name":"Servier Symphogen  Ballerup Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeta","family":"Madzharova","sequence":"additional","affiliation":[{"name":"Servier Symphogen  Ballerup Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Trine M.","family":"Sloth","sequence":"additional","affiliation":[{"name":"Servier Symphogen  Ballerup Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simon","family":"Krabbe","sequence":"additional","affiliation":[{"name":"Servier Symphogen  Ballerup Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandra K.","family":"Rebak","sequence":"additional","affiliation":[{"name":"Servier Symphogen  Ballerup Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Magali","family":"Andr\u00e9","sequence":"additional","affiliation":[{"name":"Structural Biophysics and Protein Sciences Servier R&amp;D Center  Gif\u2010sur\u2010Yvette France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pernille F.","family":"Jensen","sequence":"additional","affiliation":[{"name":"Servier Symphogen  Ballerup Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5491-1665","authenticated-orcid":false,"given":"Dan B.","family":"Kristensen","sequence":"additional","affiliation":[{"name":"Servier Symphogen  Ballerup Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,8,24]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1080\/19420862.2023.2197668"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1080\/10408347.2023.2238058"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1080\/19420862.2024.2341641"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1080\/19420862.2015.1069454"},{"key":"e_1_2_7_6_1","doi-asserted-by":"publisher","DOI":"10.3390\/antib8010018"},{"key":"e_1_2_7_7_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00216\u2010018\u20100848\u20106"},{"key":"e_1_2_7_8_1","doi-asserted-by":"publisher","DOI":"10.1080\/19420862.2018.1553476"},{"key":"e_1_2_7_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ab.2015.11.010"},{"key":"e_1_2_7_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.5b02800"},{"key":"e_1_2_7_11_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac801560k"},{"key":"e_1_2_7_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jpba.2020.113251"},{"key":"e_1_2_7_13_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac200750u"},{"key":"e_1_2_7_14_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00216\u2010020\u201002809\u2010z"},{"key":"e_1_2_7_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jpba.2021.113988"},{"key":"e_1_2_7_16_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.3c01554"},{"key":"e_1_2_7_17_1","unstructured":"A.Farrell J.Bones K.Cook S.Patel A.Schwahn andJ.Bardsley \u201cHigh\u2010Precision Automated Peptide Mapping of Proteins \u201d Thermo Scientific Application Note 21682. 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