Simple and Sensitive Method for Deep Profiling of Host Cell Proteins in Therapeutic Antibodies by Combining Ultra-Low Trypsin Concentration Digestion, Long Chromatographic Gradients, and BoxCar Mass Spectrometry Acquisition
2021
Nie, Song | Greer, Tyler | O’Brien Johnson, Reid | Zheng, Xiaojing | Torri, Albert | Li, Ning
Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for the analysis of host cell proteins (HCP) during antibody drug process development due to its sensitivity, selectivity, and adaptability. However, the enormous dynamic range between the therapeutic antibody and accompanying HCPs poses a significant challenge for LC-MS based detection of these low abundance impurities. To address this challenge, enrichment of HCPs via immunoaffinity, protein A, 2D-LC, or other strategies is typically performed. However, these enrichments are time-consuming and sometimes require a large quantity of sample. Here, we report a simple and sensitive strategy to analyze HCPs in therapeutic antibody samples without cumbersome enrichment by combining an ultra-low trypsin concentration during digestion under nondenaturing conditions, a long chromatographic gradient, and BoxCar acquisition (ULTLB) on a quadrupole-Orbitrap mass spectrometer. Application of this strategy to the NIST monoclonal antibody standard (NISTmAb) resulted in the identification of 453 mouse HCPs, which is a significant increase in the number of identified HCPs without enrichment compared to previous reports. Known amounts of HCPs were spiked into the purified antibody drug substance, demonstrating that the method sensitivity is as low as 0.5 ppm. Thus, the ULTLB method represents a sensitive and simple platform for deep profiling of HCPs in antibodies.
Afficher plus [+] Moins [-]Mots clés AGROVOC
Informations bibliographiques
Cette notice bibliographique a été fournie par National Agricultural Library
Découvrez la collection de ce fournisseur de données dans AGRIS