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

Abstract

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.

In-Membrane Enrichment and Peptic Digestion to Facilitate Analysis of Monoclonal Antibody Glycosylation

“…Drying requires 15 min, and the LC–MS separation and analysis take 48 min, including column washing and equilibration. In total, the time cost is under 2 h. In conventional approaches, the PNGase or trypsin enzymatic reaction alone can be at least 2 h. The use of analytical high-performance LC and multiple reaction monitoring mass spectrometry might shorten the time for analysis of each sample to ∼5 min. − We also propose integrating affinity or protease membranes into spin columns or 96-well plates to reduce the time for enrichment and washing and potential coupling with liquid handling robots for automation.…”

Accelerating therapeutics development during a pandemic: population pharmacokinetics of the long-acting antibody combination AZD7442 (tixagevimab/cilgavimab) in the prophylaxis and treatment of COVID-19

“…PK sampling schemes are summarized in Table S6 . Tixagevimab and cilgavimab serum concentrations were measured by PPD Laboratories (Richmond, VA, USA) using validated hybrid ligand binding liquid chromatography with a tandem mass spectrometry assay, which is capable of distinguishing tixagevimab and cilgavimab in human sera based on distinct complementarity-determining region peptide sequences ( 35 ). The assay’s lower limit of quantification (LLOQ) was 0.3 µg/mL.…”

Serum AZD7442 (tixagevimab–cilgavimab) concentrations and in vitroIC50 values predict SARS‐CoV‐2 neutralising antibody titres

“…Bioanalytical analyses of tixagevimab and cilgavimab in serum were performed by PPD Laboratories (Richmond, VA, USA), using immunocapture to the SARS‐CoV‐2 receptor‐binding domain followed by denaturation and detection of AZD7442‐specific peptides by liquid chromatography‐mass spectrometry as described by Mu et al . (2022), 42 which was validated in accordance with relevant regulatory guidelines.…”