A multiplex UPLC-MS/MS method for the quantification of three PD-L1 checkpoint inhibitors, atezolizumab, avelumab,
Pauline L M Buitelaar1, Karen A M de Jong1, Leon Aardenburg1
1Department of Pharmacy & Pharmacology, Antoni van Leeuwenhoek Hospital, The Netherlands Cancer Institute, Amsterdam, the Netherlands.
Background And Aim:
To support pharmacokinetic studies, a multiplex UPLC-MS/MS assay was developed and validated to quantify PD-L1 checkpoint inhibitors atezolizumab, avelumab, and durvalumab in serum.
Methods:
A bottom-up sample pre-treatment procedure was developed to determine atezolizumab, avelumab, and durvalumab in serum. This procedure consisted of (1) precipitation of the monoclonal antibody with ammonium sulfate, (2) reduction with dithiothreitol, (3) denaturation with methanol, and (4) tryptic digestion of the protein. The unique signature peptides resulting after sample pre-treatment of the antibodies were measured using UPLC-MS/MS with a total run time of 11 minutes. The clinical application was evaluated by analyzing 114 atezolizumab patient samples.
Results:
The developed method was found to be accurate and precise for all three analytes over a concentration range of 3.00-150 µg/mL. No endogenous interference was present in serum samples. Cross-interference experiments showed no cross-analyte interference and acceptable cross-internal standard interference. In addition, no substantial carry-over was observed. The stable isotopically labeled signature peptides were most effective in compensating for matrix effects. Recovery based on back-calculated concentrations of calibration standards and quality control samples was found to be high. The analytes were stable for at least three freeze-thaw cycles, for 42 hours at processing conditions, for at least two days at 2-8°C in the final extract, for five days before re-injection analysis at 4°C, and long-term for at least 11 months at -70°C. The assay was tested for its applicability in clinical practice. For this purpose, 114 atezolizumab patient samples were measured.
Conclusion:
A multiplex UPLC-MS/MS assay was developed and validated to quantify atezolizumab, avelumab, and durvalumab in human serum. The applicability of this method was demonstrated by the analysis of clinical atezolizumab samples. The method is suitable to support clinical pharmacokinetic studies involving atezolizumab, avelumab, or durvalumab.
Insights
A new UPLC-MS/MS assay quantifies three PD-L1 inhibitors (atezolizumab, avelumab, durvalumab) in serum. This validated method supports pharmacokinetic studies for these important cancer immunotherapies.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Pharmacology
Background:
- Quantifying programmed death-ligand 1 (PD-L1) checkpoint inhibitors is crucial for pharmacokinetic studies.
- Atezolizumab, avelumab, and durvalumab are key PD-L1 inhibitors used in cancer therapy.
Purpose of the Study:
- To develop and validate a multiplex UPLC-MS/MS assay for quantifying atezolizumab, avelumab, and durvalumab in human serum.
- To support clinical pharmacokinetic studies of these PD-L1 inhibitors.
Main Methods:
- A bottom-up sample pre-treatment involving precipitation, reduction, denaturation, and tryptic digestion was employed.
- Unique signature peptides of the antibodies were measured using UPLC-MS/MS.
- Assay validation included accuracy, precision, interference, and stability assessments.
Main Results:
- The assay accurately and precisely quantified the three analytes across a concentration range of 3.00-150 µg/mL.
- No significant endogenous or cross-analyte interference was observed.
- Analytes demonstrated stability under various conditions, including long-term storage at -70°C.
Conclusions:
- A validated multiplex UPLC-MS/MS assay for quantifying atezolizumab, avelumab, and durvalumab in serum was successfully developed.
- The assay's applicability was confirmed through the analysis of clinical samples.
- This method is suitable for supporting pharmacokinetic studies of these PD-L1 inhibitors.
