Abstract
Neuroblastoma is one of the most commonly found solid tumors in children. The monoclonal antibody dinutuximab (DNX) targets the sialic acid-containing glycosphingolipid GD2 expressed on almost all neuroblastoma tumor cells and induces cell lysis. However, the expression of GD2 is not limited to tumor cells only, but is also present on central nerve tissue and peripheral nerve cells explaining dinutuximab toxicity. The most common adverse reactions are pain and discomfort, which may lead to discontinuation of the treatment. Furthermore, there is little to no data available on exposure and effect relationships of dinutuximab. We, therefore, developed an easy method in order to quantify dinutuximab levels in human plasma. Ammonium sulfate (AS) was used to precipitate all immunoglobulins (IgGs) in human plasma. After centrifugation, supernatant containing albumin was decanted and the precipitated IgG fraction was re-dissolved in a buffer containing 0.5% sodium dodecyl sulfate (SDS). Samples were then reduced, alkylated, and digested with trypsin. Finally, a signature peptide in complementarity determining region 1 of DNX heavy chain was quantified on LC-MS/MS using a stable isotopically labeled peptide as internal standard. AS purification efficiently removed 97.5% of the albumin fraction in the supernatant layer. The validation performed on DNX showed that within-run and between-run coefficients of variation (CV) for lower limit of quantification (LLOQ) were 5.5 and 1.4%, respectively. The overall CVs for quality control (QC) low, QC med, and QC high levels were < 5%. Linearity in the range 1–32 mg/L was excellent (r2 > 0.999). Selectivity, stability, and matrix effect were in concordance with EMA guidelines. In conclusion, a method to quantify DNX in human plasma was successfully developed. In addition, the high and robust process efficiency enabled the utilization of a stable isotopically labeled (SIL) peptide instead of SIL DNX, which was commercially unavailable.Graphical abstract
Highlights
IntroductionAdministration (FDA) in 2015 and is used in combination with granulocyte-macrophage colony-stimulating factor, interleukin-2, and isotretinoin [10, 11]
Neuroblastoma (NB) is the third most common childhood cancer with a prevalence of 10.2 cases per million childrenAdministration (FDA) in 2015 and is used in combination with granulocyte-macrophage colony-stimulating factor, interleukin-2, and isotretinoin [10, 11]
sodium dodecyl sulfate (SDS) was efficiently removed by protein precipitation with methanol, as SDS remains in solution in the aqueous layer
Summary
Administration (FDA) in 2015 and is used in combination with granulocyte-macrophage colony-stimulating factor, interleukin-2, and isotretinoin [10, 11]. This therapeutic antibody targets the sialic acid-containing glycosphingolipid GD2 which is overexpressed on almost all NB tumor cells and induces cell lysis trough complement-dependent cytotoxicity and cell necrosis and apoptosis trough antibodydependent cell-mediated cytotoxicity [8, 10, 12, 13]. We have developed an easy method to quantify total DNX in plasma using a novel sample preparation in combination with tandem mass spectrometry analysis
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