{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T14:08:58Z","timestamp":1791209338761,"version":"4.1.0"},"reference-count":40,"publisher":"American Chemical Society (ACS)","issue":"10","license":[{"start":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T00:00:00Z","timestamp":1790121600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,5]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Visible particles were observed for a therapeutic monoclonal antibody at pH 5.5. This pH was considered optimal for this antibody during preclinical development. To minimize the particle formation, the pH of the final drug substance was increased to 6.5. There were no visible particles at pH 6.5 initially, but visible particles started to form after 2 months. To minimize impact to the clinical program, a lyophilized formulation presentation was chosen. Subsequently, due to the desire for patient administration through subcutaneous injection for this antibody, a stable, high concentration solution formulation was needed. Thus, the pH was further increased to 7.2 to mitigate the visible particle formation. LC-MS-based approaches, including reduced LC\u2212MS analysis and cathepsin D-targeted LC\u2212MS Lys-C peptide map analysis, were developed to identify the root cause of the visible particle formation of this antibody at pH 5.5 to ensure the successful development of a stable solution formulation at pH 7.2. Results from studies supported by LC-MS-based analyses indicated that visible particles observed in the antibody protein A capture main pool sample at pH 5.0 were associated with cathepsin D clipping of the antibody. In contrast, visible particles observed in the purified antibody drug substance samples at pH 5.5 and pH 6.5 and in the antibody in-process samples (from the low pH viral inactivation, anion exchange chromatography, hydrophobic interaction chromatography, and tangential flow filtration unit operations) were associated with succinimide intermediate formation at an Asp residue with an Asp\u2212Ser sequence motif in one of the complementarity-determining regions of the light chain. Understanding the root cause of the particle formation of this antibody at pH 5.5 enabled us to perform targeted experiments to rapidly develop a stable high concentration solution formulation for this antibody and meet project timelines.<\/jats:p>","DOI":"10.1021\/acs.molpharmaceut.6c00548","type":"journal-article","created":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T16:01:44Z","timestamp":1790179304000},"page":"4960-4973","source":"Crossref","is-referenced-by-count":0,"title":["Identification of the Root Cause of Visible Particle Formation in a Therapeutic Monoclonal Antibody by LC-MS-Based Approaches"],"prefix":"10.1021","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3646-3229","authenticated-orcid":true,"given":"Amareth","family":"Lim","sequence":"first","affiliation":[{"name":"Eli Lilly and Company , , ,","place":["Indianapolis, Indiana,, United States, 46285"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew G.","family":"Werner","sequence":"additional","affiliation":[{"name":"Eli Lilly and Company , , ,","place":["Indianapolis, Indiana,, United States, 46285"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brandon L.","family":"Doyle","sequence":"additional","affiliation":[{"name":"Eli Lilly and Company , , ,","place":["Indianapolis, Indiana,, United States, 46285"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William D.","family":"Holmes","sequence":"additional","affiliation":[{"name":"Eli Lilly and Company , , ,","place":["Indianapolis, Indiana,, United States, 46285"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew W.","family":"Carr","sequence":"additional","affiliation":[{"name":"Eli Lilly and Company , , ,","place":["Indianapolis, Indiana,, United States, 46285"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chi A.","family":"Nguyen","sequence":"additional","affiliation":[{"name":"Eli Lilly and Company , , ,","place":["Indianapolis, Indiana,, United States, 46285"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suzanne E.","family":"Stone","sequence":"additional","affiliation":[{"name":"Eli Lilly and Company , , ,","place":["Indianapolis, Indiana,, United States, 46285"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elisabeth","family":"Krug","sequence":"additional","affiliation":[{"name":"Eli Lilly and Company , , ,","place":["Indianapolis, Indiana,, United States, 46285"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,23]]},"reference":[{"issue":"7","key":"2026100509324305000_cit1","doi-asserted-by":"publisher","first-page":"491","DOI":"10.1038\/d41573-021-00079-7","article-title":"FDA approves 100th monoclonal antibody product","volume":"20","author":"Mullard","year":"2021","journal-title":"Nat. Rev. Drug Discov."},{"issue":"1","key":"2026100509324305000_cit2","doi-asserted-by":"publisher","first-page":"2443538","DOI":"10.1080\/19420862.2024.2443538","article-title":"Antibodies to watch in 2025","volume":"17","author":"Crescioli","year":"2025","journal-title":"mAbs"},{"issue":"1\u22122","key":"2026100509324305000_cit3","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1016\/s0378-5173(03)00376-4","article-title":"Shifting paradigms: biopharmaceuticals versus low molecular weight drugs","volume":"266","author":"Crommelin","year":"2003","journal-title":"Int. J. Pharm."},{"key":"2026100509324305000_cit4","doi-asserted-by":"publisher","first-page":"301","DOI":"10.1016\/j.jconrel.2018.08.001","article-title":"Subcutaneous delivery of monoclonal antibodies: How do we get there?","volume":"286","author":"Viola","year":"2018","journal-title":"J. Control Release"},{"issue":"3","key":"2026100509324305000_cit5","doi-asserted-by":"publisher","first-page":"56","DOI":"10.3390\/antib11030056","article-title":"Alternative routes of administration for therapeutic antibodies-state of the art","volume":"11","author":"Pitiot","year":"2022","journal-title":"Antibodies"},{"issue":"5","key":"2026100509324305000_cit6","doi-asserted-by":"publisher","first-page":"425","DOI":"10.1007\/s40259-018-0295-0","article-title":"Subcutaneous administration of biotherapeutics: an overview of current challenges and opportunities","volume":"32","author":"Bittner","year":"2018","journal-title":"BioDrugs"},{"issue":"6","key":"2026100509324305000_cit7","doi-asserted-by":"publisher","first-page":"1390","DOI":"10.1002\/jps.20079","article-title":"Challenges in the development of high protein concentration formulations","volume":"93","author":"Shire","year":"2004","journal-title":"J. Pharm. Sci."},{"issue":"8","key":"2026100509324305000_cit8","doi-asserted-by":"publisher","first-page":"2255","DOI":"10.1016\/j.xphs.2016.05.029","article-title":"Subcutaneous injection volume of biopharmaceuticals--pushing the boundaries","volume":"105","author":"Mathaes","year":"2016","journal-title":"J. Pharm. Sci."},{"issue":"8","key":"2026100509324305000_cit9","doi-asserted-by":"publisher","first-page":"2268","DOI":"10.1002\/jps.24054","article-title":"Ester hydrolysis of polysorbate 80 in mAb drug product: evidence in support of the hypothesized risk after the observation of visible particulate in mAb formulations","volume":"103","author":"Labrenz","year":"2014","journal-title":"J. Pharm. Sci."},{"issue":"9","key":"2026100509324305000_cit10","doi-asserted-by":"publisher","first-page":"2877","DOI":"10.1007\/s11095-015-1670-x","article-title":"Identification of subvisible particles in biopharmaceutical formulations using raman spectroscopy provides insight into polysorbate 20 degradation pathway","volume":"32","author":"Saggu","year":"2015","journal-title":"Pharm. Res."},{"issue":"5","key":"2026100509324305000_cit11","doi-asserted-by":"publisher","first-page":"1633","DOI":"10.1016\/j.xphs.2016.02.022","article-title":"Polysorbates 20 and 80 degradation by group XV lysosomal phospholipase A2 isomer X1 in monoclonal antibody formulations","volume":"105","author":"Hall","year":"2016","journal-title":"J. Pharm. Sci."},{"issue":"1\u22122","key":"2026100509324305000_cit12","doi-asserted-by":"publisher","first-page":"422","DOI":"10.1016\/j.ijpharm.2018.10.008","article-title":"Polysorbate degradation in biotherapeutic formulations: identification and discussion of current root causes","volume":"552","author":"Dwivedi","year":"2018","journal-title":"Int. J. Pharm."},{"issue":"1","key":"2026100509324305000_cit13","doi-asserted-by":"publisher","first-page":"633","DOI":"10.1016\/j.xphs.2019.10.069","article-title":"Comparison of polysorbate 80 hydrolysis and oxidation on the aggregation of a monoclonal antibody","volume":"109","author":"Larson","year":"2020","journal-title":"J. Pharm. Sci."},{"issue":"9","key":"2026100509324305000_cit14","doi-asserted-by":"publisher","first-page":"3313","DOI":"10.1016\/j.xphs.2021.05.012","article-title":"Polysorbate degradation and particle formation in a high concentration mAb: formulation strategies to minimize effect of enzymatic polysorbate degradation","volume":"110","author":"Roy","year":"2021","journal-title":"J. Pharm. Sci."},{"key":"2026100509324305000_cit15","doi-asserted-by":"publisher","first-page":"100202","DOI":"10.1016\/j.ijpx.2023.100202","article-title":"Oxidation of polysorbates - an underestimated degradation pathway?","volume":"6","author":"Weber","year":"2023","journal-title":"Int. J. Pharm. X."},{"issue":"5","key":"2026100509324305000_cit16","doi-asserted-by":"publisher","first-page":"1360","DOI":"10.1002\/btpr.2150","article-title":"Trace levels of the CHO host cell protease cathepsin D caused particle formation in a monoclonal antibody product","volume":"31","author":"Bee","year":"2015","journal-title":"Biotechnol. Prog."},{"issue":"10","key":"2026100509324305000_cit17","doi-asserted-by":"publisher","first-page":"5436","DOI":"10.1021\/acs.analchem.7b00304","article-title":"A novel sample preparation for shotgun proteomics characterization of HCPs in antibodies","volume":"89","author":"Huang","year":"2017","journal-title":"Anal. Chem."},{"issue":"11","key":"2026100509324305000_cit18","doi-asserted-by":"publisher","first-page":"6164","DOI":"10.1016\/s0021-9258(18)83327-0","article-title":"Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins","volume":"264","author":"Stephenson","year":"1989","journal-title":"J. Biol. Chem."},{"issue":"5","key":"2026100509324305000_cit19","doi-asserted-by":"publisher","first-page":"751","DOI":"10.1023\/a:1018944800691","article-title":"Chemical pathways of peptide degradation. VI. Effect of the primary sequence on the pathways of degradation of aspartyl residues in model hexapeptides","volume":"11","author":"Oliyai","year":"1994","journal-title":"Pharm. Res."},{"issue":"6","key":"2026100509324305000_cit20","doi-asserted-by":"publisher","first-page":"1145","DOI":"10.1007\/s11095-007-9241-4","article-title":"Accumulation of succinimide in a recombinant monoclonal antibody in mildly acidic buffers under elevated temperatures","volume":"24","author":"Chu","year":"2007","journal-title":"Pharm. Res."},{"issue":"10","key":"2026100509324305000_cit21","doi-asserted-by":"publisher","first-page":"3509","DOI":"10.1002\/jps.21655","article-title":"Succinimide formation at Asn 55 in the complementarity determining region of a recombinant monoclonal antibody IgG1 heavy chain","volume":"98","author":"Yan","year":"2009","journal-title":"J. Pharm. Sci."},{"issue":"1","key":"2026100509324305000_cit22","doi-asserted-by":"publisher","first-page":"2151075","DOI":"10.1080\/19420862.2022.2151075","article-title":"Restoring the biological activity of crizanlizumab at physiological conditions through a pH-dependent aspartic acid isomerization reaction","volume":"15","author":"Bickel","year":"2023","journal-title":"mAbs"},{"issue":"6","key":"2026100509324305000_cit23","doi-asserted-by":"publisher","first-page":"1411","DOI":"10.1007\/s11095-022-03462-0","article-title":"Discovery and control of succinimide formation and accumulation at aspartic acid residues in the complementarity-determining region of a aherapeutic monoclonal antibody","volume":"40","author":"VanAernum","year":"2023","journal-title":"Pharm. Res."},{"issue":"1","key":"2026100509324305000_cit24","doi-asserted-by":"publisher","first-page":"2215364","DOI":"10.1080\/19420862.2023.2215364","article-title":"Identification and characterization of an unexpected isomerization motif in CDRH2 that affects antibody activity","volume":"15","author":"Yi","year":"2023","journal-title":"mAbs"},{"key":"2026100509324305000_cit25","doi-asserted-by":"publisher","first-page":"145","DOI":"10.1016\/0076-6879(77)47017-4","article-title":"Cleavage at aspartyl-prolyl bonds","volume":"47","author":"Landon","year":"1977","journal-title":"Methods Enzymol."},{"issue":"1","key":"2026100509324305000_cit26","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1016\/j.jpba.2009.03.020","article-title":"Factors affecting cleavage at aspartic residues in model decapeptides","volume":"50","author":"Li","year":"2009","journal-title":"J. Pharm. Biomed. Anal."},{"issue":"3","key":"2026100509324305000_cit27","doi-asserted-by":"publisher","first-page":"253","DOI":"10.4161\/mabs.3.3.15608","article-title":"Fragmentation of monoclonal antibodies","volume":"3","author":"Vlasak","year":"2011","journal-title":"mAbs"},{"issue":"1","key":"2026100509324305000_cit28","doi-asserted-by":"publisher","first-page":"2213365","DOI":"10.1080\/19420862.2023.2213365","article-title":"Technical advancement and practical considerations of LC-MS\/MS-based methods for host cell protein identification and quantitation to support process development","volume":"15","author":"Guo","year":"2023","journal-title":"mAbs"},{"key":"2026100509324305000_cit29","doi-asserted-by":"publisher","first-page":"235","DOI":"10.1007\/978-1-0716-4104-0_15","article-title":"Analytical tools for HCP detection, identification, and quantitation","volume":"2853","author":"Kanwal","year":"2025","journal-title":"Methods Mol. Biol."},{"issue":"3","key":"2026100509324305000_cit30","doi-asserted-by":"publisher","first-page":"708","DOI":"10.1002\/btpr.2272","article-title":"Characterization of the co-elution of host cell proteins with monoclonal antibodies during protein A purification","volume":"32","author":"Zhang","year":"2016","journal-title":"Biotechnol. Prog."},{"issue":"1","key":"2026100509324305000_cit31","doi-asserted-by":"publisher","first-page":"140","DOI":"10.1002\/btpr.2397","article-title":"Identification of an IgG CDR sequence contributing to co-purification of the host cell protease cathepsin D","volume":"33","author":"Bee","year":"2017","journal-title":"Biotechnol. Prog."},{"key":"2026100509324305000_cit32","doi-asserted-by":"publisher","first-page":"500","DOI":"10.1016\/j.jpba.2019.06.021","article-title":"Identification and characterization of co-purifying CHO host cell proteins in monoclonal antibody purification process","volume":"174","author":"Liu","year":"2019","journal-title":"J. Pharm. Biomed. Anal."},{"issue":"3","key":"2026100509324305000_cit33","doi-asserted-by":"publisher","first-page":"553","DOI":"10.1016\/j.bbapap.2013.12.011","article-title":"Addition of negatively charged residues can reverse the decrease in the solubility of an acidic protein caused by an artificially introduced non-polar surface patch","volume":"1844","author":"Yagi","year":"2014","journal-title":"Biochim. Biophys. Acta Gen. Subj."},{"key":"2026100509324305000_cit34","doi-asserted-by":"publisher","first-page":"904","DOI":"10.1016\/j.ijbiomac.2019.09.181","article-title":"Changes in hydrophobicity mainly promotes the aggregation tendency of ALS associated SOD1 mutants","volume":"145","author":"Tompa","year":"2020","journal-title":"Int. J. Biol. Macromol."},{"issue":"2","key":"2026100509324305000_cit35","doi-asserted-by":"publisher","first-page":"177","DOI":"10.1021\/acs.biochem.7b00861","article-title":"Post-translational modifications of protein backbones: unique functions, mechanisms, and challenges","volume":"57","author":"Mu\u0308ller","year":"2018","journal-title":"Biochemistry"},{"issue":"2","key":"2026100509324305000_cit36","doi-asserted-by":"publisher","first-page":"413","DOI":"10.1006\/jmbi.1999.2768","article-title":"Induction of beta-sheet structure in amyloidogenic peptides by neutralization of aspartate: a model for amyloid nucleation","volume":"289","author":"Orpiszewski","year":"1999","journal-title":"J. Mol. Biol."},{"issue":"38","key":"2026100509324305000_cit37","doi-asserted-by":"publisher","first-page":"8057","DOI":"10.1021\/acs.jpcb.9b06958","article-title":"Promotion effect of succinimide on amyloid fibrillation of hen egg-white lysozyme","volume":"123","author":"Fan","year":"2019","journal-title":"J. Phys. Chem. B"},{"key":"2026100509324305000_cit38","doi-asserted-by":"publisher","first-page":"123867","DOI":"10.1016\/j.saa.2024.123867","article-title":"In situ raman spectral observation of succinimide intermediates in amyloid fibrillation kinetics","volume":"309","author":"Chen","year":"2024","journal-title":"Spectrochim. Acta A Mol. Biomol. Spectrosc."},{"key":"2026100509324305000_cit39","doi-asserted-by":"publisher","first-page":"438","DOI":"10.1016\/j.cocis.2014.10.002","article-title":"Protein aggregation, particle formation, characterization & rheology","volume":"19","author":"Amin","year":"2014","journal-title":"Curr. Opin. Colloid Interface Sci."},{"issue":"2","key":"2026100509324305000_cit40","doi-asserted-by":"publisher","first-page":"377","DOI":"10.1016\/j.xphs.2022.10.009","article-title":"Surface-induced protein aggregation and particle formation in biologics: current understanding of mechanisms, detection and mitigation strategies","volume":"112","author":"Kopp","year":"2023","journal-title":"J. Pharm. Sci."}],"container-title":["Molecular Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.molpharmaceut.6c00548","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/mpohbp\/article-pdf\/23\/10\/4960\/68622925\/mp6c00548.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/mpohbp\/article-pdf\/23\/10\/4960\/68622925\/mp6c00548.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T13:49:21Z","timestamp":1791208161000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/mpohbp\/article\/23\/10\/4960\/5436733\/Identification-of-the-Root-Cause-of-Visible"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,23]]},"references-count":40,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2026,9,23]]},"published-print":{"date-parts":[[2026,10,5]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.molpharmaceut.6c00548","relation":{},"ISSN":["1543-8384","1543-8392"],"issn-type":[{"value":"1543-8384","type":"print"},{"value":"1543-8392","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,23]]}}}