Anouk Donners

87 LC-MS/MS-method validation quantifying emicizumab Then, the 96-well plate was placed in a ThermoMixer at 60 °C, at 1000 rpm for 30 min to denature the proteins and enable the DTT to reduce the disulfide bonds. The thiol groups were alkylated by adding 20 μL iodoacetamide (IAA) solution (100 mM) and placed on the ThermoMixer at 37 °C for 30 min of mixing in the dark. Subsequently, 150 μL ultrapure water was added and mixed for 1 min to dilute guanidine and IAA. After mixing, 400 μL methanol was added to precipitate the IgG fragments, and the 96-well plate was centrifuged at 4000 G for 5 min. The supernatant, containing guanidine and IAA, was decanted. Subsequently, 90 μL Tris buffer (pH 8.5, 50 mM) with 0.5% OG was added to the pellet, followed by addition of 10 μL TPCK-trypsin solution (2 mg/mL). Samples were placed on the ThermoMixer for overnight digestion at 37 °C at 1000 rpm. Trypsin activity was stopped by adding 20 μL 10% formic acid in acetonitrile (pH 3) and centrifugation at 4000 G for 5 min. Finally, a 5 μL sample was injected into the LC-MS/MS system. Instrumentation and chromatographic conditions As described previously [28]. Signature peptide selection The amino acid sequence of emicizumab was obtained from the International Immunogenetics Information System ( From in silico (tryptic) digestion of emicizumab, potential signature peptides within the variable chains with amino acids 6<n<20, were identified with the Skyline® software (University of Washington, Seattle, WA, USA). These peptides were screened for absence from the human genome using the basic local alignment search tool (Blast) ( Finally, the retention time and the signal intensity of peptides were assessed with Skyline®. Three stable, unique signature peptides on the heavy chain were identified: the SGG peptide had the smallest isobaric interferences, a high signal-to-noise ratio and was selected as the quantifier; the remaining QAP and ASG peptides were adequate to function as qualifiers (Table 1). Table 1. Optimized SRM transition information for signature tryptic peptides and SIL-IL of emicizumab. Signature peptide sequence Analyte Function Precursor (m/z) Product (m/z) Product ion Charge CE (V) SGGSIYNEEFQDR EMI Quantifier 751.331 1100.46 y8 1+ 23.8 QAPGQGLEWMGDINTR EMI Qualifier 886.923 787.375 y14 2+ 26.4 ASGYTFTDNNMDWVR EMI Qualifier 888.886 1150.50 y9 1+ 28.5 SGGSIYNEEFQDR*[13C 6, 15N 4] IS SIL-IS 756.335 1110.47 y8 1+ 23.8 Abbreviations: CE: optimized collision energy; EMI: emicizumab; SIL-IS: stable isotope-labeled internal standard; SRM: selected reaction monitoring. Analytical validation study The analytical validation was performed in accordance with the EMA guideline on bioanalytical method validation [30]. The selectivity and matrix effect were investigated 5