Journal of Chromatography B最新文献

{"title":"Significant improvements in targeted UHPLC-ESI-MS/MS analysis of the reactive aldehydes 4-hydroxy-2(E)-nonenal and 4-hydroxy-2(E)-hexenal and application to rat serum.","authors":"S Chevolleau, C Orlandi, L Mervant, R Vuillaume, I Jouanin, N Naud, F Pierre, F Gueraud, L Debrauwer","doi":"10.1016/j.jchromb.2025.124747","DOIUrl":"10.1016/j.jchromb.2025.124747","url":null,"abstract":"<p><p>The elevated risk of colorectal cancer (CRC) induced by red or processed meat rich diets is now established. Those haem‑iron rich diets induce luminal lipid peroxidation, one of the most recognised hypotheses explaining CRC promotion. Due to their known toxic properties, quantification of reactive aldehydes such as 4-hydroxy-2(E)-nonenal (HNE) and 4-hydroxy-2(E)-hexenal (HHE) as lipid peroxidation end-products in biological fluids is of upmost importance. Following previous works on faecal waters, an UHPLC-ESI-MS/MS method has been developed and validated for HNE and HHE quantification in rat serum, using deuterated internal standards (ISs). After protein precipitation (PP) and solid phase extraction (SPE), LC-ESI-MS/MS analysis was achieved by MRM. The use of a brominated derivatisation reagent allowed using the bromine isotopes for selective detection of both HNE and HHE based on diagnostic transitions. This new method was validated according to the European Medicines Agency (EMA) guidelines. Our method proved to efficiently determine HNE and HHE serum concentrations with the required sensitivity (nM range) in serum of rats fed diets rich or not in red meat and different fatty acid compositions.</p>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1265 ","pages":"124747"},"PeriodicalIF":2.8,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144811470","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrating network pharmacology and metabolomics to elucidate the mechanism of action of Yangluan formula for treating of diminished ovarian reserve.","authors":"Yang Wang, Panwei Hu, Hua Yan, Yuanyuan Wu, Ping Yin, Dongyi Shen, Xiaole Zhang, Cong Qi, Qinhua Zhang","doi":"10.1016/j.jchromb.2025.124749","DOIUrl":"10.1016/j.jchromb.2025.124749","url":null,"abstract":"<p><strong>Objectives: </strong>The Yangluan Formula (YLF) influences the outcomes of in vitro fertilization-embryo transfer (IVF-ET) in infertile women with diminished ovarian reserve (DOR); however, the potential mechanisms by which YLF ameliorates DOR have not yet been elucidated. The aim of this study was to examine the effects of YLF on IVF-ET outcomes in infertile women with DOR, and to elucidate the potential mechanisms by which YLF addresses DOR.</p><p><strong>Methods: </strong>Non-targeted metabolomics studies were conducted on follicular fluid specimens procured from individuals with DOR treated with or without YLF, and from patients with normal ovarian reserve who underwent IVF-ET treatment. Distinct metabolites were identified using untargeted metabolomics, and MetaboAnalyst was used to examine metabolic pathways. After applying network pharmacology (NP), the target of YLF acting on DOR was determined. Cytoscape software was used to develop compound-reaction-enzyme-gene networks, and molecular docking (MD) simulations were conducted to confirm the link between YLF and crucial targets.</p><p><strong>Results: </strong>Patients with DOR showed a notable reduction in the number of oocytes retrieved, incidence of 2PN fertilization, and number of cleaved embryos (P < 0.001). Additionally, the DOR cohort exhibited a markedly reduced quantity of high-quality embryos on day 3 compared to the CON cohort (P < 0.005). In contrast to the DOR cohort, the YLF cohort exhibited notably superior outcomes in terms of 2PN fertilization rates, cleavage-stage embryo development, and the number of high-grade embryos on day 3 (P < 0.05). The proportion of 2PN fertilization observed in YLF subjects substantially exceeded that in DOR individuals (81.2 % vs. 64.3 %, P < 0.05). Combined analysis of metabolomics and NP, focusing on five key targets for the action of YLF (monoamine oxidase A, monoamine oxidase B, myeloperoxidase, xanthine dehydrogenase, and phosphodiesterase 3A), four key metabolites (pelargonic acid, 1-(5-Phospho-D-ribosyl)-5-amino-4-imidazolecarboxylate, isokobusone, 5-O-(1-Carboxyvinyl)-3-phosphoshikimate), and two related pathways (glycine, serine, alanine, and threonine metabolism).</p><p><strong>Conclusion: </strong>We elucidated the mode of action of YLF in DOR treatment by integrating metabolomics and NP. YLF can effectively improve IVF outcomes in patients with DOR. This study provides new perspectives on the mechanism by which YLF improves ovarian function.</p>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1265 ","pages":"124749"},"PeriodicalIF":2.8,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144797780","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Screening biomarkers for diagnosis of COPD by multiplex-high-resolution mass spectrometry based pseudotargeted lipidomics","authors":"Yunfan Zhao ,&nbsp;Yang Xie ,&nbsp;Hulei Zhao ,&nbsp;Jinyan Wu ,&nbsp;Xuemei Xu ,&nbsp;Liuyue Yin ,&nbsp;Yanmin Shi ,&nbsp;Jianya Yang ,&nbsp;Peng Zhao ,&nbsp;Qingzhou Guan ,&nbsp;Yan Du ,&nbsp;Suyun Li ,&nbsp;Jiansheng Li ,&nbsp;Xinguang Liu","doi":"10.1016/j.jchromb.2025.124809","DOIUrl":"10.1016/j.jchromb.2025.124809","url":null,"abstract":"<div><div>Triple quadrupole (QQQ) MS based pseudotargeted lipidomics combines high coverage and quantitative accuracy, is commonly established by selecting the most responsive lipid ion pairs identified from high-resolution mass spectrometry (HRMS), then sending them to QQQ MS for quantification by multiple reaction monitoring. Due to the low resolution of QQQ MS, it may result in faulty peak identification in integration and method transition from HRMS to QQQ MS, thus, directly establish pseudotargeted lipidomics on HRMS is needed to improve the accuracy of present methods. We propose a method for rapidly screening high-resolution ion pairs for constructing multiplex-HRMS-based pseudotargeted lipidomics (MHPL). Firstly, high-resolution precursor and product ions for lipid quantification were obtained by HRMS. To solve the problem of lower acquisition speed in HRMS, we used the multiplex mode to simultaneously fragment co-eluting lipid precursor ions within one isolation window, scan the MS/MS mass spectra, and quantify the unique product ions (UPI) of co-eluting lipid (defined as Quan-PIs). Meanwhile, we provide a group of scripts for searching for lipid Quan-PIs in multiplex mode. The proposed MHPL strategy could ensure accurate quantification of 460 lipids within 5 injections, and was applied in serum differential lipid discovery for chronic obstructive pulmonary disease (COPD) patients. As a result, 47 differential lipids were found to comprise a potential biomarker panel for COPD diagnosis. The lipid identification and quantification in this work were conducted in the same instrument, and faulty peak identification was considerably reduced in integration and when transitioning methods from HRMS to QQQ MS.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1267 ","pages":"Article 124809"},"PeriodicalIF":2.8,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145217305","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Validation and sample bioanalysis of protoporphyrin IX in rat plasma by the surrogate matrix approach","authors":"Aliz Széles ,&nbsp;Károly Schöll ,&nbsp;Gábor Hirka ,&nbsp;Katalin Monostory ,&nbsp;Tibor Renkecz","doi":"10.1016/j.jchromb.2025.124799","DOIUrl":"10.1016/j.jchromb.2025.124799","url":null,"abstract":"<div><div>Protoporphyrin IX (PPIX) plays a pivotal role in the heme biosynthesis pathway and serves as both a valuable biomarker in clinical diagnostics and a photosensitizer in photodynamic applications. Despite its physiological importance, accurate quantification of endogenous PPIX in biological matrices remains challenging due to the lack of an analyte-free authentic control matrix and inherent baseline variability. This study describes the development and validation of a high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) for PPIX quantification in rat plasma, applying a surrogate matrix strategy in full compliance with the International Council for Harmonisation M10 guideline.</div><div>Endogenous PPIX was removed from rat plasma by visible light-induced analyte stripping, enabling the in-house preparation of a surrogate matrix, for accurate calibration sample generation. The method showed appropriate selectivity, excellent linearity over the range of 10 and 700 ng/mL (<em>r</em> ≥ 0.995), satisfactory precision and accuracy across all validation levels. The lower limit of quantification was established at 10 ng/mL. The stability of both the analyte and internal standard was confirmed under various conditions, including 3 freeze–thaw cycles, short- and long-term storage, and autosampler residence. The method was successfully applied in an <em>in vivo</em> study in which male rats were treated with aminolevulinic acid to induce PPIX formation, thereby confirming its suitability for study sample analysis.</div><div>This fluorescence-based HPLC method offers a practical and cost-effective solution for monitoring PPIX in plasma samples. The bioanalytical method validation using a surrogate matrix approach for endogenous PPIX quantification fully aligned with current international regulatory standards may set a precedent for future method development in this field.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1267 ","pages":"Article 124799"},"PeriodicalIF":2.8,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145155300","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enabling faster and greener peptide purification in discovery chemistry","authors":"Cangming Yang ,&nbsp;Zachary Z. Brown ,&nbsp;Tao Meng ,&nbsp;John R. Frost ,&nbsp;Ashwin Rao ,&nbsp;Xiaohong Zhu ,&nbsp;Natalya Pissarnitski ,&nbsp;Tianying Jian ,&nbsp;Jianping Pan ,&nbsp;Min Liu","doi":"10.1016/j.jchromb.2025.124795","DOIUrl":"10.1016/j.jchromb.2025.124795","url":null,"abstract":"<div><div>Peptides are attracting broad interest across the pharmaceutical industry owing to their vast therapeutic potential for drugging high-value targets and flexible dosing and formulation options to improve patient access. As the industry increases focus on peptide-based discovery programs, fast, high-throughput, and green purification methods have become increasingly valuable. Due to the vast sequence and physicochemical diversity of peptides, expedited purification of large libraries has remained a challenge. Herein, we describe the development of a High-Throughput Purification (HTP) approach for peptides wherein each purification is achieved in 6 min or less, representing a substantial reduction in time and solvent consumption compared with conventional methods, all while maintaining high purity levels. This approach relies on Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC), leveraging a dual pump preparative system and short focused solvent gradients to reduce purification times and achieve highly efficient and accelerated high-throughput purification.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1267 ","pages":"Article 124795"},"PeriodicalIF":2.8,"publicationDate":"2025-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145155301","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a bispecific antibody-homodimer separation process by cation exchange chromatography based on mechanistic modelling","authors":"Zichen Wang ,&nbsp;Guohong Qin ,&nbsp;Xiaoying Liang,&nbsp;Qingquan He,&nbsp;Qian Li,&nbsp;Hongyang Zhao,&nbsp;Guozhu Li,&nbsp;Dan Xu","doi":"10.1016/j.jchromb.2025.124801","DOIUrl":"10.1016/j.jchromb.2025.124801","url":null,"abstract":"<div><div>During the recombinant production of IgG-like bispecific antibodies (bsAbs), homodimer formation, which stems from unbalanced chain expression and incorrect chain pairing, presents a major purification challenge. Despite its utility in addressing such charge-based separations, the development of cation exchange chromatography processes remains inherently complex due to the interplay of multiple parameters (e.g., salt concentration, loading conditions) that govern protein-resin interactions. For the separation of bsAb and homodimer, a mechanistic model of cation exchange chromatography was employed to predict the elution behavior, thereby facilitating the optimization of process conditions. The calibration processes and validation results of the extended Langmuir and steric mass action (SMA) two adsorption models were compared. The latter was selected for process optimization due to its streamlined calibration workflow, enabling efficient prediction of elution behavior. The optimal eluent concentration was determined to be 120 mM NaCl. Under this condition, the target bsAb yield reached 96.4 %, and homodimer 1 content was reduced from 26.4 % to 0.5 %. Deviations between the predicted and experimental results for both yield and impurity content were confined within 1.5 %, demonstrating the high accuracy and reliability of the established chromatographic mechanistic model for guiding process optimization.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1267 ","pages":"Article 124801"},"PeriodicalIF":2.8,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145153207","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrated GNPS, semi-preparative HPLC, and network pharmacology reveal active ingredients and potential machenisms of Paeonia suffruticosa Andr. leaves in treating endometriosis with anxiety/depression","authors":"Yun-Yuan Tian ,&nbsp;Meng-Zhen Fan ,&nbsp;Xin-Wen Huang ,&nbsp;Qian Luo ,&nbsp;Qian Yang ,&nbsp;Yao Li ,&nbsp;Yun-Yang Lu ,&nbsp;Si-Wang Wang","doi":"10.1016/j.jchromb.2025.124798","DOIUrl":"10.1016/j.jchromb.2025.124798","url":null,"abstract":"<div><div>Endometriosis (EMs) with anxiety/depression is a common comorbidity of EMs, yet effective treatments are still lacking. Leaves of <em>Paeonia suffruticosa</em> Andr. (PSL) have potential therapeutic effects on EMs and their associated psychiatric disorders. In this study, 57 chemical compounds were identified using Global Natural Products Social Molecular Networking (GNPS) and semi-preparative HPLC. Network pharmacology analysis revealed that these compounds shared 68 common targets with the disease, primarily enriched in the PI3K/AKT signaling pathway. Protein-protein interaction analysis identified TNF, IL-6, ESR1, AKT1, and TP53 as core targets. Molecular docking confirmed that apigenin, quercetin, kaempferol, luteolin, and isorhamnetin exhibited strong binding affinities with these core targets. In conclusion, PSL may exert beneficial effects in treating EMs with anxiety/depression by modulating the PI3K/AKT signaling pathway through key targets including TNF, IL-6, ESR1, AKT1, and TP53.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1267 ","pages":"Article 124798"},"PeriodicalIF":2.8,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145109158","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Simultaneous determination of six nitrosamines in different pharmaceutical dosage forms by GC–MS/MS with headspace module vs. autosampler solvent vent mode: Development and validation","authors":"Hayriye İçin ,&nbsp;Hasan Tünel ,&nbsp;Hakan Akbıyık ,&nbsp;Gül Gönül Kayar ,&nbsp;Nevin Öztekin","doi":"10.1016/j.jchromb.2025.124792","DOIUrl":"10.1016/j.jchromb.2025.124792","url":null,"abstract":"<div><div>The determination of N-nitrosamines is of significant importance in pharmaceutical drug products. An analytical method was devised and validated in this study to accurately and easily determine nitrosamines, thereby eliminating the interfering matrix effect in various pharmaceutical forms. N-nitrosodiethylamine (NDEA), <em>N</em>-nitrosodimethylamine (NDMA), N-nitrosodiisopropylamine (NDIPA), <em>N</em>-nitrosomethylphenylamine (NMPA), N-nitrosoisopropylethylamine (NEIPA), and N-nitrosodibutylamine (NDBA) were quantified in the suspension, emulsion, and tablet pharmaceutical forms. The concentration range was adjusted from the quantification limit (QL) to the upper limit of quantification (ULOQ). The limit concentration levels were calculated according to acceptable daily intake of each product and daily limits of N-nitrosamines which mentioned in FDA guidance. The concentration limits were defined to be 1.92 ng/mL, 0.53 ng/mL, 8.0 ng/mL, 30.0 ng/mL, 2.0 ng/mL and 0.53 ng/mL, respectively, for each N-nitrosamines. The method validation was successfully applied with an acceptable linearity (R² &gt; 0.99), accuracy (recovery range is 79.5–122.4 %) and precision (RSD &lt; 15.0 %). During the analysis, a Gas Chromatography (GC) system with an MS/MS detector and a Headspace (HS) module was employed, equipped with a VF-WAXMS capillary column (30 m × 0.25 mm × 1 μm). The results obtained were compared with a validated GC–MS/MS Autosampler method with solvent vent mode.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1267 ","pages":"Article 124792"},"PeriodicalIF":2.8,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145083285","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ADAMTS16 and TRPV5: New targets of Zhenwu decoction on renal fibrosis investigated by chemomics and transcriptomics","authors":"Xiaopeng Ren ,&nbsp;Meiqi Hong ,&nbsp;Lijing Du ,&nbsp;Yuanfang Sun ,&nbsp;Xin Huang ,&nbsp;Xiaoying Wang ,&nbsp;Shasha Li ,&nbsp;Xue Xiao","doi":"10.1016/j.jchromb.2025.124797","DOIUrl":"10.1016/j.jchromb.2025.124797","url":null,"abstract":"<div><div>Renal fibrosis (RF) plays a crucial role in the transition from different forms of CKD to ESRD, recognized as the primary pathological change in chronic kidney disorders. Previous study demonstrated that <em>Zhenwu</em> decoction (ZWD) is efficacious in the treatment of RF whether initiated in the early or the late stage. To elucidate the molecular mechanisms of ZWD on RF treatment and to propose novel potential targets for therapeutic intervention in RF treatment, qualitative chemomics strategy was conducted to search the potential active compounds of ZWD by UPLC-Q-TOF/MS. And transcriptomic analysis and pathway enrichment was utilized to identify key regulatory genes and signaling involved in medicine and RF conditions. As a result, aconite alkaloids and paeoniflorin were identified as the principal pharmacodynamic constituents, while <em>ADAMTS16</em> and <em>TRPV5</em> emerged as novel targets of ZWD in the context of RF. Importantly, the robust expression of <em>ADAMTS16</em> and <em>TRPV5</em> in the kidney highlights their potential as therapeutic targets for RF.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1267 ","pages":"Article 124797"},"PeriodicalIF":2.8,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145093392","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient host cell protein clearance: A study of membrane adsorbers and resins in biopharmaceutical processes","authors":"Ernest Šprager ,&nbsp;Veronika Reisinger ,&nbsp;Jonas Sommer ,&nbsp;Tinkara Lekić ,&nbsp;Nina Pucelj ,&nbsp;Tina Kljun ,&nbsp;Mojca Lunder ,&nbsp;Jožica Vašl ,&nbsp;Tomaž Bratkovič","doi":"10.1016/j.jchromb.2025.124796","DOIUrl":"10.1016/j.jchromb.2025.124796","url":null,"abstract":"<div><div>In recent years, biopharmaceutical purification processes have shifted towards more productive and cost-effective methods. Membrane technology has emerged as viable alternative to conventional resins, capable of delivering similar product quality while facilitating a simpler and more flexible downstream purification process. This study compares the impurity removal efficiency of three anion-exchange media types (quaternary amine-functionalized agarose resin (Q Sepharose™ Fast Flow), cellulose membrane adsorber (Sartobind® Q), and a hybrid purifier, composed of two complementary anion-exchange media, a quaternary ammonium functional non-woven and a guanidinium functional polyamide membrane (3M™ Polisher ST)) in a flow-through mode during a monoclonal antibody product purification. The content of residual major impurities—host cell proteins (HCPs) and aggregates—were investigated using a design of experiments (DoE) approach, varying pH, ionic strength, and loading densities. Membrane-based devices exhibited high impurity removal capacity, with 3M™ Polisher ST capable of reducing HCP levels from 8000 ppm down to as low as 10 ppm at competitive loading densities in this case study. The presence of critical HCPs, such as esterases capable of hydrolysing ester bonds in polysorbates, was monitored using high-throughput enzyme assays and liquid chromatography-tandem mass spectrometry. Orthogonal HCP analytical methods were required for more informed process development, as esterase presence did not follow the same trend as the general HCP population. All chromatographic media were further tested for their robustness by conducting breakthrough experiments at an optimal pH of 6.5 and conductivity of 4 mS/cm. An additional orthogonal polishing step was required as anion-exchange chromatography alone was insufficient at clearing aggregates. Among options evaluated, 3M™ Polisher ST demonstrated the greatest potential for simplifying the purification process and enhancing productivity.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1267 ","pages":"Article 124796"},"PeriodicalIF":2.8,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145103206","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}