Abstract Number: LPB0116
Meeting: ISTH 2021 Congress
Theme: Hemophilia and Rare Bleeding Disorders » Novel Biotherapeutics in Hemophilia
Background: Emicizumab is approved for people with hemophilia A (PwHA) with a body-weight-based dose regimen independent of laboratory monitoring. Guidelines, however, recommend measuring the emicizumab plasma concentration when suspecting neutralizing anti-drug antibodies. In addition, monitoring can be useful in shared decision-making, adherence checks, and for research purposes.
Aims: The objective was to validate a liquid chromatography tandem mass-spectrometry (LC-MS/MS) method for the quantification of emicizumab in plasma of PwHA.
Methods: Sample preparation for LC-MS/MS analysis was performed by protein precipitation with ammonium sulphate and by trypsin digestion. A signature peptide of emicizumab was used for quantification. A stable isotopically labeled internal standard with an amino acid-sequence matching the chosen signature peptide, was added to correct for loss during sample preparation and eliminate matrix effect during analysis. The signature peptide of emicizumab was separated chromatographically from other tryptic peptides and measured with tandem mass-spectrometry. Cross validation with 48 remnant samples from PwHA was performed with a modified and calibrated (r2 Diagnostics) one stage-clotting assay (OSA). The LC-MS/MS method was validated in accordance with the European Medicines Agency (EMA) guideline on bioanalytical method validation.
Results: The calibration curve of emicizumab was linear over a concentration range from 4-512 μg/mL with an R2 of 0.999. Within-run and between-run precision and accuracy were well within acceptance criteria (Table 1). Samples were stable for three freeze and thaw cycles. Lower limit of quantification had a signal-to-noise ratio of 88 (criterion >5). The numerical values for all other parameters were well within acceptance criteria as well. Cross validation demonstrated a strong correlation between the two methods, despite their fundamental differences (Figure 1).
| Quality Control (QC) samples | Concentration (μg/mL) | Precision (%CV) | Accuracy (bias %) | Acceptance criteria | |
| Within-run | Between-run | Overall | |||
| Lower limit of quantification | 4 | 4.9 | 7.4 | 6.1 | <20 |
| Low | 10 | 4.2 | 4.5 | -4.1 | <15 |
| Medium | 200 | 2.4 | 2.8 | -3.8 | <15 |
| High | 400 | 2.1 | 3.4 | 1 | <15 |
Summary of precision and accuracy validation parameter results
Bland-Altman plot
Conclusions: We present the first, rapid, and robust LC-MS/MS-method for quantification of emicizumab in plasma of PwHA. A full validation of the analytical method, with cross validation, was performed successfully in accordance with the EMA guideline.
To cite this abstract in AMA style:
Donners A, Gerencsér L, van der Elst K, Fischer K, Urbanus R, El Amrani M. An Accurate and Simple Liquid Chromatography Tandem Mass-spectrometric Method for the Quantification of Emicizumab in People with Hemophilia A [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/an-accurate-and-simple-liquid-chromatography-tandem-mass-spectrometric-method-for-the-quantification-of-emicizumab-in-people-with-hemophilia-a/. Accessed November 29, 2021.« Back to ISTH 2021 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/an-accurate-and-simple-liquid-chromatography-tandem-mass-spectrometric-method-for-the-quantification-of-emicizumab-in-people-with-hemophilia-a/