Comprehensive performance evaluation of ligand-binding assay-LC-MS/MS method for co-dosed monoclonal anti-SARS-CoV-2 antibodies (AZD7442)

Abstract

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 μg/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 (∼30,000 samples and ∼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.

Keywords: SARS-CoV-2; hybrid IA–LC–MS/MS; hybrid LBA–LC–MS/MS; incurred sample reanalysis; monoclonal antibody; pharmacokinetics; tixagevimab and cilgavimab; validation.

Figure 1.. Accuracy and precision for AZD1061 and AZD8895.

QCs were prepared at LLOQ, low-, mid- and high-QC levels; n = 6 replicates for each QC level per run (n = 3 validation runs). (A) Individual accuracy values for all QC replicates are depicted in raincloud plots consisting of jittered univariate dot plots, Tukey box-and-whisker plots and density (half-eye) plots. (B) Assay accuracy. Intra- and inter-run accuracy values: colored diamonds and solid black line, respectively. (C) Assay precision. Intra- and inter-run precision values: white diamonds and solid bars, respectively. LLOQ: Lower limit of quantification; QC: Quality control; DT: Difference from theoretical.

Figure 2.. Stability and dilutional study results.

Stability data at low (0.600 μg/ml), mid (22.5 μg/ml) and over-the-curve (900 μg/ml) concentrations. Shaded grey bands denote the acceptance ranges for these studies (±20% from theoretical). (A–C) Stability stress conditions for samples that were thawed and stored on ice for 24 h, exposed to five freeze–thaw cycles from -25°C or -80°C, stored for 6 days at either -25°C or -80°C or refrigerated at 2–8°C for prolonged periods (postprep). (B) Data points for insufficient volume at the mid-QC level, 14 μg/ml diluted twofold. (C) Data points for dilutional linearity at the over-the-curve quality control level, 900 ng/ml diluted 40-fold. Post-prep: Post-preparation; QC: Quality Control.

Figure 3.. Details of analytical runs and incurred sample reanalysis trends across the three clinical trials.

(A) Distribution of analytical runs across all three clinical trials. Stacked bar plots showing run performance (pass/fail) by clinical trial. (B) Distribution of ISR samples across all three clinical trials. Stacked bar plots showing ISR performance (pass/fail) by clinical trial. ISR: Incurred sample reanalysis.

Figure 4.. Quality control performance data trends.

QC performance datasets for (A) AZD1061 and (B) AZD8895 include low, mid and high QCs for 676 analytical runs: 0.600, 14.00 and 22.50 µg/ml, respectively. For each analytical run, both replicates at each QC level are plotted. Horizontal dashed lines represent acceptance limits for these QCs (±20% difference from theoretical). Symbols with solid, black fill denote data points that fall outside acceptance limits. QC: Quality control.

Figure 5.. Sequence-dependent (time-dependent) incurred sample reanalysis trend analysis.

Relative percent difference as a function of ISR sample index. As of October 2022, ISR pass rates for (A) AZD1061 and (B) AZD8895 were 98.17 and 98.27%, respectively. Vertical dashed lines appear at 3-month intervals. Black diamonds indicate ISR samples that fell outside acceptance limits. ISR: Incurred sample reanalysis.

Figure 6.. Concentration-dependent incurred sample reanalysis trend analysis.

(A & B) Modified Bland–Altman plots of relative percent difference as a function of mean reportable concentration. Symbols with solid black fill denote data points that fall outside the acceptance limits. (C & D) Frequency distributions of mean reportable ISR sample concentrations: bin width fixed at 2 with first bin centered on 1.0 μg/ml. For AZD1061 and AZD8895, bin count dropped below 15 after 23.0 and 25.0 μg/ml, respectively (right y-axis). Binwise ISR pass rates for AZD1061 and AZD8895 are presented as point-to-point line graphs (right y-axis). (E & F) Histograms of ISR mean concentration were reconstructed as uniform frequency distributions with variable bin widths and equal, or roughly equal, bin counts (right y-axis). Binwise ISR pass rates for these reconstructed distributions are presented as point-to-point line graphs (left y-axis). (E) The histogram depicting AZD1061 ISR frequencies comprises 2997 observations that are split between 37 equally sized bins (81 observations each). (F) The histogram depicting AZD8895 ISR frequencies comprises 2938 observations that are split between 33 bins. In this histogram, the first 32 bins contain 89 observations each, while the last bin contains 90 observations. ISR: Incurred sample reanalysis.