Automation of Sample Preparation for Accurate and Scalable Quantification and Characterization of Biotherapeutic Proteins Using the Agilent AssayMAP Bravo Platform

The Agilent AssayMAP Bravo platform automates a wide variety of LC/MS sample prep workflows on a single instrument that is easy to use, gives highly reproducible results, reduces hands-on time, and can simultaneously process 8–96 samples. We report results from two workflows that are routinely used to quantify and characterize biotherapeutic proteins. Peptide quantification was performed using the AssayMAP Bravo to purify 96 antibody samples in parallel with PG-W (immobilized Protein G) cartridges, digest the antibodies, and clean up the peptides with C18 cartridges. LC/MS analysis indicated that the workflow was highly reproducible with CVs < 10 %. Sequence coverage characterization was performed using the AssayMAP Bravo to purify antibody samples, digest the antibodies with three different proteases in parallel, and clean up the peptides. LC/MS analysis indicated that the combined sequence coverage from all three enzymes was 99 % and 100 % for the heavy and light chains, respectively. Finally, we report analytical figures of merit for antibody purification using PG-W and PA-W (immobilized Protein A) cartridges and the AssayMAP Affinity Purification application. Antibodies were purified with CVs less than 5 %, recovery greater than 90 %, and elution volumes of less than 10 μL.