The 19th Workshop on Recent Issues in Bioanalysis (19th WRIB) took place in New Orleans, LA, USA on April 7-11, 2025. Over 1200 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 19th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on "Implementation Practice for the Newest ELN/LIMS Systems" and on "Vaccine Cell-Based/Functional & Molecular Assays as part of the harmonization of vaccine clinical assays global initiative" were the special features of the 19th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and Regulatory Agency experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2025 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2025 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers in the Part 1A the recommendations on Mass Spectrometry Assays and Regulated Bioanalysis/BMV and in Part 1B the Regulatory Inputs on these topics. Part 2 (Biomarkers/BAV, IVD/CDx, Ligand-Binding Assays and Cell-Based Assays) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 18 of Bioanalysis, issues 1 and 2 (2026), respectively.

The 18(th) Workshop on Recent Issues in Bioanalysis (18(th) WRIB) took place in San Antonio, TX, USA on May 6-10, 2024. Over 1100 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 18th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on "IVDR Implementation in EU & Changes for LDT in the US" and on "Harmonization of Vaccine Clinical Assays Validation" were the special features of the 18(th) edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2024 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2024 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers in Part 1A the Recommendations on Mass Spectrometry Assays and Regulated Bioanalysis/BMV and in Part 1B the Regulatory Inputs on these topics. Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) and Part 2 (Biomarkers/BAV, IVD/CDx, LBA and Cell-Based Assays) are published in volume 17 of Bioanalysis, issues 3 and 4 (2025), respectively.

AIM:Oral peptide therapeutics typically have short half-lives due to rapid degradation by digestive enzymes. Systematic peptide engineering and formulation optimization led to the development of a clinical candidate MEDI7219, an orally bioavailable glucagon-like peptide 1 (GLP-1) peptide, with greater stability than wild-type GLP-1 or semaglutide:~60% of MEDI7219 remained intact after 2 h in vitro incubation with simulated intestinal fluid. This study further investigates proteolytic stability by elucidating biotransformation products of MEDI7219 using liquid chromatography-mass spectrometry (LC-MS) methods. METHOD:Peptide metabolism was assessed using in vitro pancreatin assay followed by analysis utilizing liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID) and electron-activated dissociation (EAD) approaches. RESULTS:We have confidently identified 13 metabolites. Time course profiles of parent and metabolite peaks are consistent with sequential enzymatic cleavage pattern. The 13 metabolites mapped to 8 cleavage sites. Most of these cleavage sites can be explained by the specificity of digestive enzymes, e.g. trypsin, pepsin, and elastase. However, α-methyl-L-phenylalanine appeared to be well protected from chymotrypsin and pepsin digestion since no cleavage peptides ending with α-methyl-L-phenylalanine were observed. CONCLUSION:These study results provide further structural details explaining previously published stability data and provide new insights into potential GLP1 proteolytic liabilities for future engineering.

Surface plasmon resonance; In vitro cytotoxicity assays; In vitro proliferation assays; Internalization assays; Subcellular localization microscopy; Immunoblotting; Statistical analysis

Supplementary Table S1. Primary antibodies used for immunoblotting; Supplementary Table S2. Characterization of ADCs evaluated in this study; Supplementary Table S3. B7-H4 expression in human normal tissue; Supplementary Table S4. Binding affinity of anti-B7-H4 antibody INT016 Fab to human, cynomolgus monkey, and mouse B7-H4, measured by surface plasmon resonance.

Permeation enhancers (PEs) are excipients used in oral biotherapeutic formulations to facilitate the transport of bioactive compounds across the intestinal barrier and prevent their degradation. Concerns associated with the chronic use of PEs demand comprehensive approaches to elucidate their potential toxicity mechanisms. A recent publication from our group reported nephrotoxicity in beagles after daily administration of enteric-coated (EC) tablets containing propyl gallate (PG) as a PE. To further characterize EC-PG-mediated nephrotoxicity mechanisms, we conducted a longitudinal mass spectrometry (MS)-based multiomics analysis of the dog plasma lipidome and proteome. Time-course analyses revealed elevation across multiple lipid classes and, in particular, species containing arachidonic acid, which may reflect EC-PG treatment-induced inflammation. At the protein level, alterations in biological processes associated with coagulation, complement activation, protein degradation and metabolism, and lipid transport and metabolism were observed. Integrative multiomics analyses provided additional insights into toxicity mechanisms at the interface between lipids and proteins. This holistic approach highlighted lipid transport and metabolism, oxidative stress, and inflammation as altered biological processes by EC-PG administration. Altogether, longitudinal multiomics profiling and integrative analysis provided additional mechanistic hypotheses for EC-PG induced renal toxicity, demonstrating the value of such an approach to investigate mechanisms relevant to drug safety.

During nonclinical development of an oral formulation for a glucagon-like peptide-1 (GLP-1) receptor agonist, MEDI7219, toxicology studies revealed that propyl gallate (PG), when administered in enteric-coated (EC) tablets, led to nephrotoxicity in beagles. While PG has been widely used in food and cosmetics as an antioxidant, understanding of its toxicology, metabolism, and pharmacokinetics has been rarely discussed. To elucidate the nephrotoxicity observed after administration of PG in an EC tablet formulation, we employed dog and human renal proximal tubule epithelial cells (RPTECs). We observed greater cytotoxicity to PG in dog RPTECs compared to human cells and greater increases in response to PG treatment of glutathione in human cells compared to dog cells. Glutathione elevation is a common response to detoxify xenobiotics, especially ones that produce free radicals such as PG. We hypothesize that glutathione in human RPTECs was elevated to detoxify PG, but not in dog RPTECs, leading to greater cytotoxicity for dog RPTECs. However, a subsequent study in dogs demonstrated that the oral administration of PG in a non-EC capsule did not result in renal toxicity, suggesting the physiological response to PG is modulated by the mode of absorption and a blunted glutathione response may not completely explain the PG-related renal toxicity observed in dogs. Furthermore, to characterize the pharmacokinetics and metabolism of PG we developed a 10-plex, highly sensitive and robust LC-MS/MS-based quantification method for PG and its phase-I and phase-II metabolites. The methods were employed to support preclinical dog studies and clinical study (NCT03362593).

Supplementary Figure S11. In vitro cytotoxicity of the TOP1i warhead AZ’0132 to HT29 and HT29-huB7-H4 cells; Supplementary Figure S12. Study design for pharmacodynamic and pharmacokinetic analysis of AZD8205 in HT29 and HT29-huB7-H4 mouse xenograft models; Supplementary Figure S13. DNA damage response signaling in HT29-huB7-H4 and MX-1 cells treated with AZD8205 or the TOP1i warhead AZ’0132; Supplementary Figure S14. Accumulation of AZD8205 and changes in epithelial cell density in HT29 and HT29-huB7-H4 tumor xenografts over time; Supplementary Figure S15. Correlation of B7-H4 mean OD and gene expression in TNBC PDX models; Supplementary Figure S16. Impact of homologous recombination deficiency (HRD) on sensitivity to the TOP1i warhead AZ’0132 in vitro with SK-OV-3 ovarian cancer isogenic cells; Supplementary Figure S17. SLFN11 expression in PDX models; Supplementary Figure S18. Analysis of SLFN11 expression and AZD8205 response in PDX.

Permeation enhancers (PEs) are excipients used in oral biotherapeutic formulations to facilitate the transport of bioactive compounds across the intestinal barrier and prevent their degradation. Concerns associated with the chronic use of PEs demand comprehensive approaches to elucidate potential toxicity mechanisms. A recent publication from our group reported nephrotoxicity in beagles after daily administration of enteric-coated (EC) tablets containing propyl gallate (PG) as PE. To further characterize EC-PG mediated nephrotoxicity mechanisms, we conducted a longitudinal mass spectrometry (MS)-based multi-omics analysis of dog plasma lipidome and proteome. Time-course analyses revealed elevation across multiple lipid classes and in particular species containing arachidonic acid, which may reflect EC-PG treatment-induced inflammation. At the protein level, alterations in biological processes associated with coagulation, complement activation, protein degradation and metabolism, and lipid transport and metabolism were observed. Integrative multi-omics analyses provided additional insight into toxicity mechanisms at the interface of lipids and proteins. This holistic approach highlighted lipid transport and metabolism, oxidative stress, and inflammation as altered biological processes by EC-PG administration. Altogether, longitudinal multi-omics profiling and integrative analysis provided additional mechanistic hypotheses of EC-PG induced renal toxicity demonstrating the value of such approach to investigate mechanisms relevant to drug safety.

Research into antibody-drug conjugates (ADCs) is currently at an inflection point due to recent clinical impact. ADC biotransformation analysis is key for understanding the structural integrity of ADCs in vivo and is a critical aspect of drug development, especially at the lead selection stage. Data analysis of biotansformed products is hindered by the manual and time-consuming analyte identification process oftentimes taking days to weeks. We developed a streamlined data analysis workflow enabling more automated peak identification using several commercial software tools that significantly improve data processing efficiency. A linker-payload biotransformation library was created for each new molecule and combined with antibody sequence information for peak matching. As a proof of concept, we tested this workflow across different payload and linker types, acquired using different mass spectrometers: an example using a topoisomerase I inhibitor-conjugated ADC (SCIEX ZenoTOF 7600) and a comparison to a published in vivo ADC biotransformation data set for a pyrrolobenzodiazepine-conjugated ADC (ThermoFisher QE HF-X). Using this more automated workflow, we rapidly identified major biotransformation species that were previously found manually including loss of linker-payload, thiosuccinimide ring hydrolysis, cysteinylation at the deconjugation site(s), and partial linker-payload cleavage. This improved data-analysis workflow has demonstrated superb effectiveness in streamlining overall ADC biotransformation identification and enabled quantification that was highly comparable to previously obtained results. Broadening application of advanced analytical techniques to study biotherapeutic biotransformation can now more effectively impact drug development by enabling faster design-test-analyze cycle times, critical in early drug discovery settings, opening new avenues for more effective collaboration between analytical chemists and bioconjugate engineers.

AIM:Airway mucins in sputum are promising respiratory disease biomarkers, despite posing substantial analytical challenges due to their physicochemical properties and rare and heterogenous nature of the matrix. We aimed to identify a suitable sputum collection and processing method, and qualify a bioanalytical method for MUC5AC and MUC5B quantification in clinical samples. METHOD:Mucins were quantified in induced and spontaneous sputum collected from the same COPD patients, following various sample processing procedures. LC-MS/MS method used truncated recombinant mucins as surrogate analytes in surrogate matrix. RESULTS:Frozen spontaneous sputum was found to be a suitable and convenient matrix for mucin quantification and fit-for-purpose method qualification was performed. CONCLUSION:Our methodology provides accurate and reliable MUC5AC and MUC5B quantification and facilitates multi-site clinical sputum collection.

Supplementary Figure S1. Analytical characterization of INT016-ADCs; Supplementary Figure S2. Validation of B7-H4 antibodies for use in IHC; Supplementary Figure S3. Relationship between B7-H4 expression level and heterogeneity in the studied human triple-negative cohort; Supplementary Figure S4. Binding of antibody INT016 to HEK 293 cells stably expressing human, mouse, or cynomolgus monkey B7-H4; Supplementary Figure S5. Binding of antibody INT016 to human breast cancer cell lines and to HT29 cells stably expressing human B7-H4; Supplementary Figure S6. Internalization and lysosomal trafficking of INT016; Supplementary Figure S7. In vitro stability of INT016-ADCs in mouse and cynomolgus monkey serum; Supplementary Figure S8. In vitro cytotoxicity of INT016-ADCs in HT29 and HT29-huB7-H4 isogenic cell lines; Supplementary Figure S9. In vivo efficacy of INT016-ADCs in the MX-1 xenograft model; Supplementary Figure S10. Growth rate analysis of MX-1 in vivo efficacy study comparing INT016-ADCs from days 14–40.

Background Endothelial lipase (EL) promotes high-density lipoproteins (HDL) phospholipid degradation, increases catabolism of HDL and is an attractive target for the potential treatment for cardiovascular disease. Inhibition of EL using a monoclonal neutralizing antibody, MEDI5884, demonstrated increased quantity and function of HDL. Determinants of anti-atherosclerotic function of HDL comprise the interplay of various components of HDL structure-activity relationship: size, shape and composition (lipid and protein). Previous studies have shown that single doses of MEDI5884 administered to healthy nonhuman primates (NHPs) and healthy subjects resulted in a dose- dependent increase in plasma phospholipids (PL) and that plasma PI levels in placebo treated healthy subjects are significantly increased relative to CAD subjects participating in clinical trials [NCT03001297][1] and [NCT03351738][2], respectively. Methods Herein, we characterized using LC-MS/MS the plasma lipidome of NHPs, heathy subjects and subjects with coronary artery disease (CAD) following MEDI5884 administration. Results MEDI5884 treated NHPs resulted in a prominent increase in phosphatidylinositols (PI) and cholesteryl esters (CE). Treatment with MEDI5884 restores near-normal levels of PI in CAD patients. PI increases in both healthy subjects and CAD patients were dose-dependent, correlated with exposure and saturated at approximately 200 mg MEDI5884 subcutaneous (SC) dose in CAD patients. Comparison of pharmacodynamic (PD) effects of repeat SC 200 mg doses of MEDI5884 in CAD patients revealed greater and more rapid increases in PI levels compared to HDL-C and HDL phospholipid (HDL-PL). The increase in PI species was inversely correlated with decreases in free EL mass levels. Conclusions PI has previously been shown to possess anti-atherosclerotic properties and led to increases in HDL cholesterol (HDL-C) and reverse cholesterol transport (RCT). The mechanism by which CE levels increase as the result of MEDI5884 administration can be attributed to the observed increase in both substrates of the lecithin-cholesterol acyltransferase (LCAT) reaction: phosphatidylcholine/phosphatidylethanolamine (PC/PE) and cholesterol as the consequence of EL inhibition. Further characterization of the underlying biological mechanisms responsible for the decrease of the PI biomarker in CAD patient population relative to healthy subjects as well as in conjunction with pharmacological intervention by MEDI5884 may reveal more information on this clinically-relevant biomarker and potential role in CAD. ### Competing Interest Statement The authors are or were employees of AstraZeneca at the time this work was conducted and may hold stock ownership and/or stock options or interests in the company. This study was funded by AstraZeneca. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03001297&atom=%2Fbiorxiv%2Fearly%2F2024%2F06%2F03%2F2024.05.30.596497.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03351738&atom=%2Fbiorxiv%2Fearly%2F2024%2F06%2F03%2F2024.05.30.596497.atom

Aims: AZD7442 is a combination SARS-CoV-2 therapy comprising two co-dosed monoclonal antibodies. Materials & methods: The authors validated a hybrid ligand-binding assay-LC-MS/MS method for pharmacokinetic assessment of AZD7442 in human serum with nominal concentration range of each analyte of 0.300-30.0 mu g/ml. Results: Validation results met current regulatory acceptance criteria. The validated method supported three clinical trials that spanned more than 17 months and >= 720 analytical runs (similar to 30,000 samples and similar to 3000 incurred sample reanalyses per analyte). The data generated supported multiple health authority interactions, across the globe. AZD7442 (EVUSHELD) was approved in 12 countries for pre-exposure prophylaxis of COVID-19. Conclusion: The results reported here demonstrate the robust, high-throughput capability of the hybrid ligand binding assay-LC-MS/MS approach being employed to support-next generation versions of EVUSHELD, AZD3152. The measurement of antibodies in human body fluids (e.g., blood, serum) has historically been tied to laboratory tests that may face operational limitations, including susceptibility to interference from other blood components and a reliance on unique reagents that can take months to produce. As such, there is a pursuit of alternative analytical methods to more accurately detect and measure antibody drugs from complex matrices. In the method, the authors describe different techniques that once combined were used to capture, separate, filter, fragment and then detect and measure the co-dosed antibody drugs. This method has been validated in accordance with current health authority guidelines and has been used to support three clinical trials that spanned more than 17 months; that is, the validated method was used to analyze nearly 30,000 serum samples from more than 2000 patients. Collectively, the results reported here demonstrate the robustness and high-throughput capability of this analytical approach.

Abstract AZD4573 is a highly potent and selective Cyclin-Dependent Kinase 9 inhibitor. The AZD4573 First-in-Human (FIH) study revealed variable alanine transaminase (ALT) and/or bilirubin elevation and a clear correlation to dose or PK exposure could not be defined. While concurrent ALT elevation ≥3x upper limit of normal (ULN) and bilirubin elevation ≥2x ULN (potential Hy’s law) is a biochemical finding indicating potential drug-induced liver injury, quantitative modeling disconnected ALT and bilirubin elevation demonstrating that the observations were not Hy’s Law cases. Furthermore, we implemented a targeted metabolomics workflow to investigate the ALT and bilirubin elevation mechanism and identify predictive biomarkers to better understand and/or predict any hepatic stress liabilities associated with AZD4573 clinical development. We implemented an established metabolomics methodology to 359 samples from 15 patients yielding 47,000 total data points. Data was separated into 98 different dosing intervals from the 15 patients to identify the ALT and bilirubin-associated metabolic changes conserved across all patients while incorporating metabolic differences in individual patents with ALT and bilirubin elevation in only a fraction of multiple doses. Orthogonal Partial Least Squares modeling identified several metabolites and pathways altered as a function of maximum ALT or bilirubin levels. Post-dose conjugated bile acids were elevated when ALT and bilirubin were elevated. Pre-dose creatine levels were decreased when ALT and bilirubin elevation occurred, and pre-dose glutamate levels were elevated when ALT elevation occurred. Pre-dose decreased total carnitines and kynurenine pathway activation were observed when ALT and bilirubin elevation occurred, but the effect did not reach statistical significance. Elevated bile acids are an established characteristic of cholestasis and suggest cholestasis as a probable mechanism for AZD4573-induced reversible liver stress. Interestingly, decreased creatine, glutamate elevation, decreased total carnitine, and kynurenine pathway activation are all associated with some form of liver stress or disfunction which may explain the irregular ALT and bilirubin elevations observed in patients receiving AZD4573; while ALT itself is a biomarker of liver damage, these pre-dose biochemical changes may illuminate potential preceding liver stress. Dosing patients already experiencing higher liver stress levels may be more susceptible to drug-induced ALT and bilirubin elevation, and patients with increasing in-study liver stress may only present at later doses. This work demonstrates metabolomics utility to elucidate possible mechanisms of liver toxicity as well as to identify potential predictive biomarkers for further investigation to explore individual patient risk in clinical studies. Citation Format: John K. Meissen, Kevin Contrepois, Sophie Regan, Jennifer Tan, Alison J. Foster, Jiaqi Yuan, Shringi Sharma, Dominic Williams, Anton I. Rosenbaum. Clinical metabolomics reveals baseline biomarkers of hepatic dysfunction correlating with irregular drug-induced ALT and bilirubin elevations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7094.

Permeability plays a major role in oral biotherapeutic delivery and permeation enhancers can improve the intestinal permeability of poorly absorbed active pharmaceutical ingredients such as peptides. As part of nonclinical development of an oral formulation for a glucagon-like peptide-1 (GLP-1) receptor agonist, MEDI7219, toxicology studies revealed that one of the formulation excipients, propyl gallate (PG), when administered in enteric-coated tablets, led to nephrotoxicity in beagles. While PG has been widely used in food and cosmetics as an anti-oxidant, understanding of its toxicology, metabolism and disposition has been rarely discussed. To elucidate the nephrotoxicity observed after administration of PG in an enteric coated tablet formulation, we employed dog and human renal proximal tubule epithelial cells (RPTEC). We observed greater cytotoxicity to PG in dog RPTEC compared to human cells. We also observed greater increases in response to PG treatment of glutathione in human cells compared to dog cells. Glutathione elevation is a common response to detoxify xenobiotics, especially ones that produce free radicals such as PG. Thus, we hypothesize that glutathione in human RPTECs was elevated to detoxify PG, but not in dog RPTECs, leading to greater cytotoxicity for dog RPTECs. Furthermore, to characterize disposition and metabolism of PG in both humans and dogs we developed a 10-plex, highly sensitive and robust LC-MS/MS-based quantification method of PG and its phase-I and phase-II metabolites in dog and human plasma. The methods were employed to support clinical study (NCT03362593) and preclinical dog studies to evaluate safety, pharmacokinetics and tolerability of PG to support its use in an oral formulation for MEDI7219.

Antibody-drug conjugate (ADC) is a therapeutic modality that aims to improve payload delivery specificity and reduce systemic toxicity. Considering the complex structure of ADCs, various bioanalytical methods by liquid chromatography coupled with mass spectrometry (LC-MS), ligand binding assay (LBA) and hybrid LBA-LC-MS approaches have been established for ADC characterization and quantification. LCMS-based assays enable drug-antibody ratio (DAR) sensitive quantification of the conjugated payload. Typically, for quantitative, DAR-sensitive, assessment by LC-MS/MS,the conjugated payload is enzymatically liberated and quantified. Despite recent advances in ADC bioanalytical methods, the DAR-sensitive quantification of noncleavable linker ADCs by LC-MS/MS remains challenging. Thus, we developed a novel digestion-free middle-down mass spectrometry (DF-MDMS) using a collision-induced dissociation approach for absolute quantification of conjugated payload from four different ADCs in a biological matrix with minimum sample preparation. These results demonstrate that ADCs with different linker-payload structures can be quantified, including a noncleavable linker ADC, trastuzumab emtansine. It also shows that the assay sensitivity is comparable to the conventional ADC quantification method by linker-payload cleavage using enzyme, while the assay dynamic range depends on factors including payload ionization and dissociation efficiency, DAR and its distribution, and species abundance. By demonstrating absolute quantification of both cleavable and noncleavable linker ADCs, this novel middle-down ADC approach demonstrates its potential application in bioanalysis and analytical characterization, especially for early discovery where high-throughput screening is required as the new approach saves time and resources by not requiring enzymatic digestion for cleavable ADCs or development of anti-payload antibodies for noncleavable linker ADCs.

Antibody-drug conjugates (ADC) are a promising drug modality experiencing substantial expansion in both discovery space and clinical development, due to its targeted delivery and potentially improved therapeutic index. Assessing the biotransformation of ADCs in vitro and in vivo is important in understanding their stability and pharmacokinetic properties. We have previously reported biotransformation pathways for the anti-B7H4 topoisomerase I inhibitor ADC, AZD8205, that underpin its structural stability in vivo using intact protein mass LC-HRMS approach. Herein we employed LC-MRM method with both CID and EAD fragmentation that confirmed our earlier findings. Furthermore, we were able to obtain additional detailed structural information of these biotransformation products expanding on earlier intact mass method analyses. We also highlight the high sensitivity of LC-MRM for successfully identifying minor biotransformation products at low concentrations, that were not distinguishable using the intact mass LC-HRMS workflow. Especially, the EAD fragmentation aided in the confirmation of biotransformation species that contain newly formed disulfide bonds, due to the preferential fragmentation of disulfide bonds using this method. We observed biotransformation reactions that vary between linker-payload (PL) conjugation sites on the antibody, including the trend towards constitutional isomerism in thio-succinimide linker hydrolysis, and linker-payload deconjugation. The reported orthogonal analytical approach highly complements and fortifies the earlier findings from intact protein mass LC-HRMS explaining AZD8205 stability in vivo. This study sheds further light upon detailed structural confirmation of ADC biotransformation and validates our earlier findings that explain AZD8205 stability in vivo.

Oral peptide therapeutics typically suffer from short half-lives as they are readily degraded by digestive enzymes. Systematic peptide engineering along with formulation optimization led to the development of a clinical candidate MEDI7219, an orally-bioavailable GLP-1 peptide, that is much more stable than wild type GLP-1 or semaglutide. In this study, we elucidated peptide biotransformation products using in vitro pancreatin assay that employed both collision-induced dissociation (CID) and electron-activated dissociation (EAD) LC-MS/MS methods. Using this approach, we have confidently identified a total of 13 metabolites. Relative quantification of these metabolites over time showed sequential cleavage pattern as peptides were further digested to smaller fragments. These 13 metabolites mapped to 8 cleavage sites on MEDI7219 structure. Most of these cleavage sites can be explained by the specificity of digestive enzymes, e.g. ,trypsin, pepsin and elastase. α-methyl-L-phenylalanine appeared to be well protected from chymotrypsin and pepsin digestion since no cleavage peptides ending with α-methyl-L-phenylalanine were observed. These study results expand upon previously published stability data and provide new insights on potential GLP1 proteolytic liabilities for future engineering. Furthermore, this study exemplifies the application of pancreatin in vitro system methodology as a valuable tool for understanding metabolism of oral peptide therapeutics in vitro. Additionally, orthogonal MS fragmentation modes offered improved confidence in identification for peptide unknown metabolites. ### Competing Interest Statement K.L., Y.H., T.W., R.M., and A.I.R. are or were employees of AstraZeneca at the time this work was conducted and may hold stock ownership and/or stock options or interests in the company. This study was funded by AstraZeneca.