Bioanalysis and pharmacokinetics of monoclonal antibodies in oncology: Assay development, validation, and application

Monoclonal antibody therapies have become a key component of cancer treatment, but their distinct pharmacokinetics and high costs create a need for optimized dosing strategies. Accurate bioanalytical assays are essential for pharmacokinetic investigations that guide therapeuty optimization. Two main quantification techniques are used: ligand-binding assays (LBAs), such as ELISAs, and liquid chromatography–mass spectrometry (LC-MS). While LBAs are long-established, the advancement of LC-MS technologies offers improvements in specificity and multiplexing.

This work focused on developing, validating, and applying bioanalytical assays for mAb quantification in various biological matrices. An optimized bottom-up sample preparation protocol was designed for ipilimumab, nivolumab, and pembrolizumab, including antibody precipitation with ammonium sulfate, reduction with DTT, denaturation using methanol, followed by tryptic digestion. These steps ensured reliable peptide recovery and reproducibility. The resulting multiplexed UPLC–MS/MS method enabled simultaneous quantification of these monoklonal antibodies with high accuracy across a quantification range of 3–200 µg/mL. The technique was cross-validated against ELISA measurements, showing comparable results.

Adaptations of this LC-MS workflow successfully extended to PD-L1 inhibitors (atezolizumab, avelumab, durvalumab). Furthermore, a novel LC-QTOF-MS assay was designed for anti–SARS-CoV-2 antibodies, enabling the first quantitative mass spectrometric measurement of anti-SARS-CoV-2 antibody concentrations in infected individuals. This method introduced spike protein–based magnetic bead isolation and use of a general isotope-labeled internal standard.

Complementary work involved the development of a sensitive ELISA for ipilimumab detection in serum, plasma, breast milk, and cerebrospinal fluid. The ELISA demonstrated lower limits of quantification than LC-MS but systematically measured lower concentrations, which was attributed to the detection of free rather than total drug. Together, these assay developments demonstrated the potential for both techniques to yield accurate and clinically meaningful data depending on the research context.

The validated assays were subsequently applied in several clinical pharmacokinetic studies. Quantification of nivolumab and ipilimumab in urothelial cancer patients revealed that lower baseline clearance correlated with improved therapeutic response. Similarly, a study on atezolizumab in penile cancer patients found that low baseline clearance was associated with better treatment outcomes and longer progression-free survival. Additional analysis of breast milk from a patient treated with nivolumab showed drug accumulation across cycles, indicating that breastfeeding during therapy warrants caution due to potential infant exposure.

Overall, this thesis established robust LC-MS and ELISA methodologies for therapeutic and endogenous mAb quantification. Their application in clinical research has provided valuable insights into the pharmacokinetics and safety of mAb therapies.