Automated, Generic Reagent and Ultratargeted 2D-LC-MS/MS Enabling Quantification of Biotherapeutics and Soluble Targets down to pg/mL Range in Serum

Abstract

Mass spectrometry has recently emerged as a powerful analytical tool for the assessment of pharmacokinetics and biomarkers in drug development. Compared with ligand binding assays, a major advantage of mass spectrometry-based assays is that they are less dependent on high quality binding reagents, while a key limitation is the relatively lower sensitivity. To address the sensitivity issue, we have developed a generic reagent, ultratargeted two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) method which combines commercially available protein A affinity capture, targeted analyte isolation by 2D-LC, and targeted detection by multiple reaction monitoring (MRM). A targeted-2D-with-dilution configuration was designed to automate 2D-LC-MS/MS. This method was systematically evaluated using an anti-CD22 monoclonal antibody spiked into monkey and human serum, where lower limits of quantification (LLOQ) of 0.78 and 1.56 ng/mL were achieved, respectively. This represents an over 100-fold improvement in assay sensitivity compared to the conventional LC-MS/MS method. The performance of the method was further confirmed by analyzing another monoclonal antibody, bevacizumab, as well as a soluble antigen, circulating PD-L1. The results indicate that our method enables quantification of antibody therapeutics and antigen biomarkers in both clinical and nonclinical samples in the pg/mL to low ng/mL range. Protein A affinity capture was employed as a universal sample preparation procedure applicable to both full-length antibody therapeutics and antibody–antigen complexes. This novel method is also fully automated and proven to be highly robust for routine bioanalysis in drug development.

Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC–MS/MS Approach

“…RAS10 has been shown to possess comparable affinity to KRAS G12C with or without covalent modification by immunoprecipitation . After on-bead tryptic digestion, the concentrations of both free and GDC-6036-bound KRAS G12C were determined with 4 μg of total protein using an ultra-sensitive targeted 2D-LC–MS/MS technology, which enables up to 100-fold increase in sensitivity compared to the conventional LC–MS/MS method . KRAS G12C engagement was calculated as the ratio of GDC-6036-bound KRAS G12C/ (free KRAS G12C + GDC-6036-bound KRAS G12C).…”

“…After protein digestion, 12 μL of each sample (∼ 2 μg of total protein) was used for quantification of free or GDC-6036-bound KRAS G12C by a targeted 2D-LC−MS/MS method as described previously. 12 High-pH RPLC−MS/MS survey scan was performed to determine the retention time of the signature peptides for free and GDC-6036-bound KRAS G12C. The peptides were separated on an ACQUITY UPLC M-class 2D-LC system (Waters) equipped with a nanoEase M/Z Peptide BEH C18 Column (300 μm i.d.…”

“…13 After on-bead tryptic digestion, the concentrations of both free and GDC-6036bound KRAS G12C were determined with 4 μg of total protein using an ultra-sensitive targeted 2D-LC−MS/MS technology, which enables up to 100-fold increase in sensitivity compared to the conventional LC−MS/MS method. 12 KRAS G12C engagement was calculated as the ratio of GDC-6036-bound KRAS G12C/ (free KRAS G12C + GDC-6036-bound KRAS G12C). The selected reaction monitoring (SRM) transitions of the signature peptides (Figure 2) for free and GDC-6036bound KRAS G12C were monitored by targeted 2D-LC−MS/ MS.…”

“…Anti-RAS antibody clone RAS10 was immobilized onto protein A magnetic beads and used to capture both free and GDC-6036-bound KRAS G12C from tumor tissue lysates. After on-bead tryptic digestion, the SRM transitions of the signature peptides for free and GDC-6036-bound KRAS G12C were monitored using a targeted 2D-LC−MS/MS technique 12.…”

A Liquid Chromatography-Mass Spectrometry Method to Study the Interaction between Membrane Proteins and Low-Molecular-Weight Compound Mixtures

“…Robotic automation is needed to increase throughput. (2) Two dimensional LC-MS (2D-LC-MS), which separates ligand-protein complexes by LC such as SEC or affinity chromatography and then automatically introduces each eluate into a second LC to measure sub-molecules derived from the complexes (e.g., the dissociated ligand; [ 16 , 17 , 18 , 19 ]. The elution profile in the first dimensional LC provides information on the complex and the second dimensional LC has the advantage that the ligand is measured with high sensitivity.…”