Extensive Biotransformation Profiling of AZD8205, an Anti-B7-H4 Antibody-Drug Conjugate, Elucidates Pathways Underlying Its Stability In Vivo.

What happens to macromolecules in vivo? What drives the structure-activity relationship and in vivo stability for antibody-drug conjugates (ADCs)? These interrelated questions are increasingly relevant due to the re-emerging importance of ADCs as an impactful therapeutic modality and the gaps that exist in our understanding of ADC structural determinants that underlie ADC in vivo stability. Complex macromolecules, such as ADCs, may undergo changes in vivo due to their intricate structure as biotransformations may occur on the linker, the payload, and/or at the modified conjugation site. Furthermore, the dissection of ADC metabolism presents a substantial analytical challenge due to the difficulty in the identification or quantification of minor changes on a large macromolecule. We employed immunocapture-LCMS methods to evaluate in vivo changes in the drug-antibody ratio (DAR) profile in four different lead ADCs. This comprehensive characterization revealed that a critical structural determinant contributing to the ADC design was the linker, and competition of the thio-succinimide hydrolysis reaction over retro-Michael deconjugation can result in superb conjugation stability in vivo. These data, in conjunction with additional factors, informed the selection of AZD8205, puxitatug samrotecan, a B7-H4-directed cysteine-conjugated ADC bearing a novel topoisomerase I inhibitor payload, with durable DAR, currently being studied in the clinic for the potential treatment of solid malignancies (NCT05123482). These results highlight the relevance of studying macromolecule biotransformation and elucidating the ADC structure-in vivo stability relationship. The comprehensive nature of this work increases our confidence in the understanding of these processes. We hope this analytical approach can inform future development of bioconjugate drug candidates.

Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody-Drug Conjugate.

Deamidation, a common post-translational modification, may impact multiple physiochemical properties of a therapeutic protein. MEDI7247, a pyrrolobenzodiazepine (PBD) antibody-drug conjugate (ADC), contains a unique deamidation site, N102, located within the complementarity-determining region (CDR), impacting the affinity of MEDI7247 to its target. Therefore, it was necessary to monitor MEDI7247 deamidation status in vivo. Due to the low dose, a sensitive absolute quantification method using immunocapture coupled with liquid chromatography-tandem mass spectrometry (LBA-LC-MS/MS) was developed and qualified. We characterized the isomerization via Electron-Activated Dissociation (EAD), revealing that deamidation resulted in iso-aspartic acid. The absolute quantification of deamidation requires careful assay optimization in order not to perturb the balance of the deamidated and nondeamidated forms. Moreover, the selection of capture reagents essential for the correct quantitative assessment of deamidation was evaluated. The final assay was qualified with 50 ng/mL LLOQ for ADC for total and nondeamidated antibody quantification, with qualitative monitoring of the deamidated antibody. The impact of deamidation on the pharmacokinetic characteristics of MEDI7247 from clinical trial NCT03106428 was analyzed, revealing a gradual reduction in the nondeamidated form of MEDI7247 in vivo. Careful quantitative biotransformation analyses of complex biotherapeutic conjugates help us understand changes in product PTMs after administration, thus providing a more complete view of in vivo pharmacology.

Multiplex Hybrid Antigen-Capture LC-MRM Quantification in Sera and Nasal Lining Fluid of AZD7442, a SARS-CoV-2-Targeting Antibody Combination.

AZD7442 (tixagevimab [AZD8895]/cilgavimab [AZD1061]) is a monoclonal antibody (mAb) combination in development for the prevention and treatment of coronavirus disease 2019. Traditionally, bioanalysis of mAbs is performed using ligand binding assays (LBAs), which offer sensitivity, robustness, and ease of implementation. However, LBAs frequently require generation of critical reagents that typically take several months. Instead, we developed a highly sensitive (5 ng/mL limit of quantification) method using a hybrid LBA-liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) approach for quantification of the two codosed antibodies in serum and nasal lining fluid (NLF), a rare matrix. The method was optimized by careful selection of multiple reaction monitoring, capture reagents, magnetic beads, chromatographic conditions, evaluations of selectivity, and matrix effect. The final assay used viral spike protein receptor-binding domain as capture reagent and signature proteotypic peptides from the complementarity-determining region of each mAb for detection. In contrast to other methods of similar/superior sensitivity, our approach did not require multidimensional separations and can be operated in an analytical flow regime, ensuring high throughput and robustness required for clinical analysis at scale. The sensitivity of this method significantly exceeds typical sensitivity of ∼100 ng/mL for analytical flow 1D LBA-LC-MS/MS methods for large macromolecules, such as antibodies. Furthermore, infection and vaccination status did not impact method performance, ensuring method robustness and applicability to a broad patient population. This report demonstrated the general applicability of the hybrid LBA-LC-MS/MS approach to platform quantification of antibodies with high sensitivity and reproducibility, with specialized extension to matrices of increasing interest, such as NLF.

Bioanalytical Methods and Strategic Perspectives Addressing the Rising Complexity of Novel Bioconjugates and Delivery Routes for Biotherapeutics.

In recent years, an increase in the discovery and development of biotherapeutics employing new modalities, such as bioconjugates or novel routes of delivery, has created bioanalytical challenges. The inherent complexity of conjugated molecular structures means that quantification of the bioconjugate and its multiple components is critical for preclinical/clinical studies to inform drug discovery and development. Moreover, bioconjugates involve additional multifactorial complexity because of the potential for in vivo catabolism and biotransformation, which may require thorough investigations in multiple biological matrices. Furthermore, excipients that enhance absorption are frequently evaluated and employed for the development of oral and inhaled biotherapeutics. Risk-benefit assessments are required for novel or existing excipients that utilize dosages above previously approved levels. Bioanalytical methods that can measure both excipients and potential drug metabolites in biological matrices are highly relevant to these emerging bioanalysis challenges. We discuss the bioanalytical strategies for analyzing bioconjugates such as antibody-drug conjugates and antibody-oligonucleotide conjugates and review recent advances in bioanalytical methods for the quantification and characterization of novel bioconjugates. We also discuss bioanalytical considerations for both biotherapeutics and excipients through novel administration routes and review analyses in various biological matrices, from the extensively studied serum or plasma to tissue biopsy in the context of preclinical and clinical studies from both technical and regulatory perspectives.

Differences in levels of phosphatidylinositols in healthy and stable Coronary Artery Disease subjects revealed by HILIC-MRM method with SERRF normalization.

Quantification of endogenous biomarkers in clinical studies requires careful evaluation of a number of assay performance parameters. Comparisons of absolute values from several clinical studies can enable retrospective analyses further elucidating the biology of a given biomarker across various study populations. We characterized the performance of a highly multiplex bioanalytical method for quantification of phosphatidylinositols (PI). Hydrophilic interaction chromatography (HILIC) and multiple reaction monitoring (MRM) were employed for targeted multiplex quantification. Odd-chain PI species that are not normally present in human plasma were utilized as surrogate analytes (SA) to assess various assay performance parameters and establish a definitive dynamic linear range for PI lipids. To correct for batch effects, Systematic Error Removal using Random Forest (SERRF) normalization algorithm was employed and used to bridge raw values between two clinical studies, enabling quantitative comparison of their absolute values. A high throughput method was developed, qualified, transferred to an automation platform and applied to sample testing in two clinical trials in healthy volunteers (NCT03001297) and stable Coronary Artery Disease (CAD, NCT03351738) subjects. The method demonstrated acceptable precision and accuracy (±30%) over linear range of 1-1000 nM for SA and 8-fold dilutional linearity for endogenous PI. We determined that mean-adjusted average QC performed best for normalization using SERRF. The comparison of two studies revealed that healthy subject levels of PI are consistently higher across PI species compared to CAD subjects identifying a potential lipid biomarker to be explored in future studies.

Characterization of Antibody-Drug Conjugate Pharmacokinetics and in Vivo Biotransformation Using Quantitative Intact LC-HRMS and Surrogate Analyte LC-MRM.

Antibody-drug conjugates (ADCs) pose challenges to bioanalysis because of their inherently intricate structures and potential for very complex catabolism. Common bioanalysis strategy is to measure the concentration of ADCs and Total Antibody (Ab) as well as deconjugated warhead in circulation. The ADCs and the Total Ab can be quantified with ligand binding assays (LBA) or with hybrid immunocapture-liquid chromatography coupled with multiple reaction monitoring mass spectrometry (LBA-LC-MRM). With the LBA-LC-MRM approach, a surrogate analyte, often the signature peptide, and released warhead can be used for the quantification of the Total Ab and ADCs, respectively. Recent advances in analytical instrumentation, especially the development of high resolution mass spectrometers (HRMS), have enabled characterization and quantification of intact macromolecules such as ADCs. The LBA-LC-HRMS approach employs immunocapture, followed by chromatographic separation at the macromolecule level and detection of the intact analyte. We developed an intact quantification method with 1-10 µg/mL linear dynamic range using 25 µL of plasma sample volume. This method was qualified for the measurement of naked monoclonal antibody (mAb), a site-specific cysteine-conjugated ADC with drug to antibody ratio ∼2 (DAR2) and a site-nonspecific cysteine-conjugated ADC (DAR8) in rat plasma. Samples from a rat pharmacokinetic (PK) study were analyzed with both methods. For the naked mAb, the results from both assays matched well. For ADCs, new species were observed from the LBA-HRMS method. The results demonstrated that potential biotransformation of the ADC was unveiled using the intact quantification approach while not being observed with traditional LBA-LC-MRM approach. Our work demonstrated an application of novel intact quantification by supporting animal PK studies. Moreover, our results suggest that the intact quantification method can provide novel perspectives on ADC in vivo characterization and quantification, which can benefit future drug candidate optimization as well as the immunogenicity impact evaluation and safety assessment.