Challenges and Solutions in Large Molecule Quantitation Using Bottom-Up LC–MS/MS: Insights from Real Case Studies

Liquid chromatography–mass spectrometry (LC MS) plays a central role in pharmaceutical analysis, employing both bottom up and top down strategies to address diverse analytical needs. These complementary approaches enable comprehensive characterization of large molecules such as antibodies, antibody–drug conjugates (ADCs), fusion proteins, and therapeutic proteins. Bottom up workflows provide robustness and high throughput, making them ideal for quantitative analysis and metabolite profiling, while top-down approaches deliver critical structural insights into biotherapeutic integrity and heterogeneity. Bottom-up excels in routine quantitation and quality control, whereas top-down is indispensable for comprehensive structural characterization.
This presentation focuses on quantitative applications of large molecules using bottom-up LC–MS/MS. The workflow encompasses analyte extraction, denaturation/reduction/alkylation, enzymatic digestion, peptide selection, internal standard addition, and LC–MS/MS analysis. Each step requires careful optimization to achieve sensitivity, robustness, and high throughput. To illustrate these principles, three case studies will be presented:
Case Study One – development of a clinical assay for a therapeutic protein.
Case Study Two – establishment of a preclinical toxicology method for an ADC.
Case Study Three – implementation of an endogenous protein quantitation strategy for clinical support.