BACKGROUND:Quantitating antibody drug conjugates (ADCs) with traditional ligand binding assays (LBAs) requires cytotoxic payload targeting reagents, which may be needed for ADC capture/detection. LBAs are drug antibody ratio insensitive (DAR insensitive) and prone to under- or over-estimating DAR species. METHODS:This hybrid LBA LC MS/MS assay measures concentrations for a novel ADC drug (BMS-X) with an engineered, site‑specific, non-cleavable linker, and a unique payload. Recombinant target protein-extracellular domain fused with mouse IgG-Fc was used for ADC pull down in human serum. Pepsin digestion released unique toxin linker peptide complexes for two conjugation sites. RESULTS AND CONCLUSIONS:This novel, validated DAR-sensitive hybrid-LCMS PK assay measured payload-conjugated BMS-X levels in human serum, quantitated surrogate analytes for both unique conjugation sites, and was used to support a first-in-human clinical trial.

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.

PYX-201 is an anti-extra domain B splice variant of fibronectin (EDB + FN) antibody drug conjugate (ADC) composed of a fully human IgG1 antibody, a cleavable mcValCitPABC linker, and four Auristatin 0101 (Aur0101, PF-06380101) payload molecules. To better understand the pharmacokinetic (PK) profile of PYX-201 after it is administered to cancer patients, the development of a reliable bioanalytical assay to accurately and precisely quantitate PYX-201 in human plasma is required. In this manuscript, we present a hybrid immunoaffinity LC-MS/MS assay used to successfully analyze PYX-201 in human plasma. PYX-201 was enriched by MABSelect beads coated with protein A in human plasma samples. The bound proteins were subjected to "on-bead" proteolysis with papain to release the payload Aur0101. The stable isotope labelled internal standard (SIL-IS) Aur0101-d8 was added and the released Aur0101 was quantified as a surrogate for the total ADC concentration. The separation was performed on a UPLC C18 column coupled with tandem mass spectrometry. The LC-MS/MS assay was validated over the range 0.0250 to 25.0 mu g/mL with excellent accuracy and precision. The overall accuracy (%RE) was between-3.8% and-0.1% and the inter-assay precision (%CV) was <5.8%. PYX-201 was found to be stable in human plasma for at least 24 h on ice, 15 days after being stored at -80 C, as well as after five freeze/thaw cycles of being frozen at-25 degrees C or -80 degrees C and thawed on ice. The assay this paper reports on, has been successfully applied to human sample analysis to support clinical studies.

A hybrid immunoaffinity LC-MS/MS assay was developed and validated for the quantitation of total antibody (TAb) from an antibody drug conjugate (ADC) PYX-201 in human plasma. PYX-201 was proteolyzed using trypsin, and a characteristic peptide fragment PYX-201 P1 with ten amino acids IPPTFGQGTK from the complementarity-determining regions (CDRs) was used as a surrogate for the quantitation of the TAb from PYX201. Stable isotope labelled (SIL) peptide I(13C6, 15N)PPTFG(13C9, 15N)QGTK was used as the internal standard (IS). We performed chromatographic analysis using a Waters Acquity BEH Phenyl column (2.1 mm x 50 mm, 1.7 & mu;m). Quantification of PYX-201 TAb was carried out on a Sciex triple quadrupole mass spectrometer API 6500 using multiple reaction monitoring (MRM) mode with positive electrospray ionization. To validate PYX-201 TAb, a concentration range of 0.0500 & mu;g/mL to 20.0 & mu;g/mL was used, yielding a correlation coefficient (r) of & GE; 0.9947. For intra-assay measurements, the percent relative error (%RE) ranged from -23.2% to 1.0%, with a coefficient of variation (%CV) of & LE; 14.2%. In terms of inter-assay measurements, the %RE was between -10.5% and -5.7%, with a %CV of & LE; 12.7%. The average recovery of the analyte was determined to be 81.4%, while the average recovery of the internal standard (IS) was 97.2%. Furthermore, PYX-201 TAb demonstrated stability in human plasma and human whole blood under various tested conditions. This assay has been successfully applied to human sample analysis to support a clinical study.

PYX-201 is an investigational antibody drug conjugate (ADC) with an engineered, fully human IgG1 antibody, a cleavable chemical linker, and a toxin (Aur0101) with an average drug-antibody ratio (DAR) of similar to 4. A sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated to determine the presence in human plasma, of free payload Aur0101 from PYX-201 to assess drug safety and efficacy. Aur0101 and its deuterated internal standard (IS), Aur0101_d8, were extracted from 25 mu L of human plasma using a solid liquid extraction (SLE) method. Chromatographic analysis was carried out on a Waters Acquity UPLC BEH C18 (2.1 mm x 50 mm, 1.7 mu m, 130 A) column. Quantitation of free Aur0101 was conducted on a Sciex triple quadrupole mass spectrometer API 6500 + using multiple reaction monitoring (MRM) mode via positive electrospray ionization. The calibration curve was linear over the concentration range of 25.0 to 12,500 pg/mL with correlation coefficient, r(2) = 0.9988. The intra-assay %RE was between 4.3% to 14.3% with % CV was = 6.2%. The inter-assay %RE was between 0.2% to 9.5% with % CV was <= 6.1%. The average analyte recovery was 89.7% and the average IS recovery was 88.7%. Aur0101 was found to be stable in human plasma and human whole blood under various tested conditions with and without the presence of PYX-201. To our knowledge, this is the first published fully validated assay for free, unconjugated Aur0101 in any matrix, from any species. This assay has been successfully applied to clinical sample analysis to support clinical studies.

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.

Complex biotherapeutic modalities, such as antibody-drug conjugates (ADC), present significant challenges for the comprehensive bioanalytical characterization of their pharmacokinetics (PK) and catabolism in both preclinical and clinical settings. Thus, the bioanalytical strategy for ADCs must be designed to address the specific structural elements of the protein scaffold, linker, and warhead. A typical bioanalytical strategy for ADCs involves quantification of the Total ADC, Total IgG, and Free Warhead concentrations. Herein, we present bioanalytical characterization of the PK and catabolism of a novel ADC. MEDI3726 targets prostate-specific membrane antigen (PMSA) and is comprised of a humanized IgG1 antibody site-specifically conjugated to tesirine (SG3249). The MEDI3726 protein scaffold lacks interchain disulfide bonds and has an average drug to antibody ratio (DAR) of 2. Based on the structural characteristics of MEDI3726, an array of 4 bioanalytical assays detecting 6 different surrogate analyte classes representing at least 14 unique species was developed, validated, and employed in support of a first-in-human clinical trial (NCT02991911). MEDI3726 requires the combination of heavy-light chain structure and conjugated warhead to selectively deliver the warhead to the target cells. Therefore, both heavy-light chain dissociation and the deconjugation of the warhead will affect the activity of MEDI3726. The concentration-time profiles of subjects dosed with MEDI3726 revealed catabolism of the protein scaffold manifested by the more rapid clearance of the Active ADC, while exhibiting minimal deconjugation of the pyrrolobenzodiazepine (PBD) warhead (SG3199).

Bioanalysis of complex biotherapeutics, such as antibody-drug conjugates (ADCs), is challenging and requires multiple assays to describe their pharmacokinetic (PK) profiles. To enable exposure-safety and exposure-efficacy analyses, as well as to understand the metabolism of ADC therapeutics, three bioanalytical methods are typically employed: Total Antibody, Antibody Conjugated Toxin or Total ADC and Unconjugated Toxin. MEDI4276 is an ADC comprised of biparatopic humanized antibody attached via a protease-cleavable peptide-based maleimidocaproyl linker to a tubulysin toxin (AZ13599185) with an approximate average drug-antibody ratio of 4. The conjugated payload of MEDI4276 can undergo ester hydrolysis to produce the conjugated payload AZ13687308, leading to the formation of MEDI1498 (de-acetylated MEDI4276). In this report, we describe the development, validation and application of three novel multiplex bioanalytical methods. The first ligand-binding liquid chromatography coupled with tandem mass spectrometry (LBA-LC-MS/MS) method was developed and validated for simultaneous measurement of total antibody and total ADC (antibody-conjugated AZ13599185) from MEDI4276. The second LBA-LC-MS/MS assay quantified total ADC (antibody-conjugated AZ13687308) from MEDI1498. The third multiplex LC-MS/MS assay was used for simultaneous quantification of unconjugated AZ13599185 and AZ13687308. Additional stability experiments confirmed that quantification of the released warhead in the presence of high concentrations of MEDI4276 was acceptable. Subsequently, the assays were employed in support of a first-in-human clinical trial (NCT02576548).

Dapagliflozin (Farxiga), alone, or in the fixed dose combination with metformin (Xigduo), is an orally active, highly selective, reversible inhibitor of sodium-glucose cotransporter type 2 (SGLT2) that is marketed in United States, Europe, and many other countries for the treatment of type 2 diabetes mellitus. Here we report a liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical assay of dapagliflozin in human plasma. A lower limit of quantitation (LLOQ) at 0.2 ng/mL with 50 μL of plasma was obtained, which reflects a 5-fold improvement of the overall assay sensitivity in comparison to the previous most sensitive assay using the same mass spectrometry instrumentation. In this new assay, acetate adduct ions in negative electrospray ionization mode were used as the precursor ions for selective reaction monitoring (SRM) detection. Sample preparation procedures and LC conditions were further developed to enhance the column life span and achieve the separation of dapagliflozin from potential interferences, especially its epimers. The assay also quantifies dapagliflozin's major systemic circulating glucuronide metabolite, BMS-801576, concentrations in human plasma. The assay was successfully transferred to contract research organizations (CROs), validated, and implemented for the sample analysis of pediatric and other critical clinical studies. This assay can be widely used for bioanalytical support of future clinical studies for the newly approved drug Farxiga or any combination therapy containing dapagliflozin.