89 LC-MS/MS-method validation quantifying emicizumab Cross-validation parameters Plasma samples from patients with haemophilia A were measured with mcOSA and LC-MS/MS. The following EMA criterion for cross-validation was applied “the difference between the two values obtained should be within 20% of the mean for at least 67% of the repeats” [30]. Samples with >20% difference were re-analyzed with LC-MS/MS method. Cross-validation results were analyzed with weighted Deming regression and Bland-Altman analysis. The regression was performed with jackknife based calculation of 95% confidence intervals (CI) according to Linnet’s method and a Pearson’s correlation coefficient, with a validated web-based tool [32]. The Bland-Altman analysis of absolute and relative differences included mean bias (in µg/mL or %, respectively) with standard deviation (SD) and 95% Limits of Agreement (LoA). Relative differences (%) were calculated as: The influence of covariates on absolute differences was assessed with an unpaired Student’s t-test (dichotomous). Anti-FVIII antibodies and FVIII in samples were scored based on lab results and reviewing the Electronic Patient Records. Titers of anti-FVIII antibodies were determined when indicated by the local protocol with the Bethesda assay (Nijmegen modified chromogenic assay with bovine reagents) for which the clinical cut-off ≥0.6 Bethesda Units (BU) per milliliter was used [16]. No FVIII activity was measured during emicizumab therapy in our clinic. Statistics were performed in GraphPad Prism (GraphPad Software LLC, Version 8.3.0, San Diego, CA, USA). DISCUSSION Analytical validation study Linearity of the LC-MS/MS method was established from 4 to 512µg/mL with an R2 of 0.999. The RSD of the within-run precision ranged from 2.1% to 4.9% and the RSD of the between-run precision ranged from 2.8% to 7.4%. The accuracy (%bias) ranged from -4.1% to 6.1%. All other validation parameters were also well within the acceptance criteria of the EMA guideline (Table 2). The validated LLOQ was 4 µg/mL and had a signalto-noise ratio of 88, which indicated an even lower LLOQ can be achieved. Emicizumab in QC samples was stable during three freeze-and-thaw cycles. The QC low and high samples remained stable after sample preparation for one week at 10 °C. 5
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