Quantification of total dinutuximab concentrations in neuroblastoma patients with liquid chromatography tandem mass spectrometry

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

Dinutuximab in adult-onset chemotherapy refractory high-risk neuroblastoma

“…This closely parallels the dinutuximab serum levels drawn in a single pediatric patient, with the highest level measured on the third day of a four-day cycle. 18 Despite availability of PCA hydromorphone bolus doses through the entire four-day course of the dinutuximab infusion, pain persisted between cycles and our patient continued to require methadone and as needed oxycodone with acetaminophen in the outpatient setting despite being opioid naive. Pain control improved during cycle 3 as her baseline opioid needs from the first two cycles provided guidance, though the patient reported that the pain was still extremely difficult to tolerate.…”

Quantification of emicizumab by mass spectrometry in plasma of people with hemophilia A: A method validation study

“…An ammonium sulfate (AS) protein precipitation method was chosen for simplicity and fast workflow. 29 From the plasma sample, 10 μl was taken and diluted with a 5‐μl IS solution and 85 μL of Tris buffer (50 mM, pH 8, 0.5% OG) in a 1‐ml LoBind 96 deep‐well plate and mixed for 1 minute at 1350 rpm. Then, 100 μL of saturated AS solution was added to each sample and mixed for 1 minute at room temperature at 1350 rpm to precipitate both therapeutic and endogenous immunoglobulins from the plasma samples.…”

Immunomodulatory Potential of anti-idiotypic Antibodies for the Treatment of Autoimmune Diseases

“…In 2010, sipuleucel-T became the first therapeutic cancer vaccine to receive FDA approval for the treatment of metastatic hormone-refractory prostate cancer [ 89 ]. In 2015, dinutuximab (marketed as unituxin) was granted orphan drug designation and approved by the FDA and EMA to be used as part of a first-line treatment on paediatric patients with high-risk neuroblastoma [ 90 , 91 ]. Subsequently, dinutuximab β (marketed as isquette), used as a second-line treatment of neuroblastoma, was also approved in 2017 [ 92 ].…”