Purpose: Therapeutic antibodies have increasingly been utilized for cancer treatment and other therapeutic interventions. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) is increasingly being used as an alternative platform to ligand binding assays (LBA) as an accurate and reliable technique to measure these proteins in biological fluids. Mass spectrometry-based quantitation of the therapeutic antibodies is generally performed using the surrogate peptide approach, however intact mass analysis is also of interest within the industry. The surrogate peptide approach is known to offer good sensitivity and is amenable to conventional MRM quantitation whereas the intact mass analysis enables the quantitation of protein at an intact level which provides structural information that LBA infers from using specific capture and detection reagents. Within the intact mass analysis approach, variants of data processing are also reported to play a key role in the assay performance. The purpose of this work is to use Pembrolizumab (Keytruda®) as a representative biologic to perform a comparative study to assess quantitative results generated using the surrogate peptide and the intact mass analysis approach. We used Trastuzumab (Herceptin®) as an internal standard in this study.
Methods: The methodology for this study includes the sample preparation and data acquisition for the surrogate peptide and the intact protein analysis approaches in human serum. We applied similar technique for capturing the antibodies for these two approaches, however, the methods differ in the downstream sample preparation and data acquisition processes. The sample was subjected to immunoaffinity enrichment by a generic immunoprecipitation technique using the Protein G Mag Sepharose® Xtra magnetic beads.
For the surrogate peptide approach, the signature peptides for the Pembrolizumab and Herceptin were confirmed post tryptic digestion using Thermo Q-Exactive Plus hybrid quadrupole-Orbitrap which are in agreement with the insilico digestion and Basic Local Alignment Search Tool (BLAST). The immunoaffinity enriched sample was digested using SMART Digest soluble trypsin. The unique signature peptides which were used to represent Pembrolizumab and Trastuzumab were separated on a UHPLC reverse phase column using AB Sciex API 6500 QqQ via multiple reaction monitoring (MRM) mode of acquisition.
For intact protein analysis, the immunoaffinity enriched sample was subjected to reverse phase chromatographic separation and mass spectrometric analysis using Thermo Q-Exactive Plus BioPharma hybrid quadrupole-Orbitrap operated in protein mode.
Results: For the surrogate peptide approach, the unique signature peptides selected for quantitation of Pembrolizumab and Trastuzumab were DLPLTFGGGTK and LLIYSASFLYSGVPSR respectively. Our preliminary results indicated a linear range from 1.00 to 100 µg/mL in human serum. The accuracy and precision were acceptable within the range. The comparison of the results obtained from the surrogate peptide-based quantitation with the intact mass analysis was performed. Additionally, assay performance using the extracted ion chromatogram (XIC) and deconvolution based data processing of the intact analysis was also compared in this work.
Conclusion: Each approach has its own pros and cons. Surrogate peptide-based quantitation remains the more popular choice for protein therapeutic bioanalysis due to the ability to achieve higher sensitivity and importantly to utilize software and systems validated within the regulated environment. However, it fails to determine if the measured signal is from a homogenous parent population or potentially a mixture of catabolites. This concern can be addressed by intact mass analysis since it conserves the integrity of the protein, however, intact analysis requires complex data processing including considerations such as how to manage deconvolution and/or charge state summing as well as additional complexity due to post-translational modifications. Fundamentally, both approaches bring value and will continue to evolve with the goal of enabling increasingly high-quality decision making within the drug development process.