86 Chapter 5 MATERIAL AND METHODS Liquid chromatography-tandem mass spectrometry method The development of the LC-MS/MS method was reported previously [28]. Here, the protocol and validation procedures are described in detail. Chemicals and reagents The vials containing emicizumab (batch # B2002) at a concentration of 150 µg/µL were obtained from F. Hoffmann-La Roche Ltd. (Basel, Switzerland). A stable isotope-labeled (SIL) internal standard (IS) was used to correct for variations during sample preparation and to eliminate the matrix effect. The amino acid sequence of this SIL-IS, matching the signature peptide, was SGGSIYNEEFQD(R*), where (R*) = Arg (13C 6, 15N 4). The SIL-IS was synthesized by and obtained from Pepscan (Lelystad, the Netherlands). The tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin was supplied by Thermo Scientific (Breda, the Netherlands) as a lyophilized powder and was dissolved in acetic acid (50 mM) to a concentration of 10 mg/mL; aliquots were stored in Eppendorf LoBind® microcentrifuge tubes at -80 °C. The methanol mobile phase solvent (LC-MS grade) and all remaining reagents were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Standard working solution, calibration standard, internal standard and quality controls The working emicizumab standard solution was prepared by pipetting 10 μL stock solution of Hemlibra® (150 mg/mL) and 140 μL pooled human plasma in a LoBind® tube (10 mg/mL). Calibration standard solutions with concentrations of 512, 256, 128, 64, 32, 16, 8, and 4 µg/mL were prepared freshly from the working standard solution by serial dilution in pooled human plasma and aliquots were stored at -80 °C. The working IS solution (50 µg/mL) was prepared in tris(hydroxymethyl)aminomethane (Tris) buffer pH 8.5, 100 mM, containing 0.5% octyl glucoside (OG). The following quality control (QC) samples were prepared in pooled human plasma: lower limit of quantification (LLOQ; 4 µg/mL), low (10 µg/mL), medium (200 µg/mL), and high concentration (400 µg/mL). Aliquots of QC samples were stored at -80 °C. Sample preparation for LC-MS/MS An ammonium sulphate (AS) protein precipitation method was chosen for simplicity and fast workflow [29]. From the plasma sample, 10 μL was taken and diluted with 5 μL IS solution and 85 μL Tris buffer (50 mM, pH 8, 0.5% OG) in a 1 mL LoBind® 96 Deep-well plate and mixed for 1 min at 1350 rpm. Then, 100 μL saturated AS solution was added to each sample and mixed for 1 min at room temperature at 1350 rpm to precipitate both therapeutic and endogenous immunoglobulins from the plasma samples. The 96-well plate was centrifuged at 4000 G for 5 min to collect the IgG pellet at the bottom. The supernatant containing albumin was decanted, and the pellet was re-dissolved in 50 μL Tris buffer (100 mM, pH 8.5, 6 M guanidine chloride, 20 mM 1,4-dithiothreitol [DTT]).
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