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

Abstract

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.

Host cell protein profiling of commercial therapeutic protein drugs as a benchmark for monoclonal antibody-based therapeutic protein development

“…Recently, LC-MS methods have become more sensitive and can now measure tens or even hundreds of very low abundance HCP in purified drug substances. 45,46 Many of these proteins have certain biological functions or potential enzymatic activities that may be a potential immunogenicity risk. However, when MS methods have become so sensitive that HCP are identified at less than 1 ppm, it is hard to determine whether they will pose a risk to product stability or patient safety.…”

Activity-Based Protein Profiling Probe for the Detection of Enzymes Catalyzing Polysorbate Degradation

“…This DS had been previously analyzed for host cell proteins by various sensitive methods, and no lipase was detected. The HCP analysis methods used include pro-A depletion and ultralow trypsin digestion, and the details of those methods could be found in previously published works. , The DS was diluted by the ABPP binding buffer to 10 mg/mL. Recombinant human PLA2G7, recombinant human LPL, and recombinant CHO LAL were spiked into diluted DS at the indicated concentrations.…”

“…19 This sensitivity may not be sufficient because some lipases with abundances lower than 10 ppm can cause significant polysorbate degradation. 20 Therefore, different methods have been developed to increase the sensitivity of LC-MS/MS-based HCP analysis, including native digestion, 21 pro-A depletion, 22 ultralow trypsin digestion, 23 molecular weight cutoff filtering, 24 ProteoMiner enrichment, 25 and 2D LC-MS/MS. 26 These methods also have limitations.…”

The measurement and control of high-risk host cell proteins for polysorbate degradation in biologics formulation

“…In addition to advancements in sample preparation, improvements in LC–MS analysis have also been made to improve sample dynamic range and improve the sensitivity of HCP identification. For LC improvements, longer chromatographic gradients or 2D-LC have been used [ 36 , 37 ]. There are also improvements on the mass spectrometry side.…”

“…There are also improvements on the mass spectrometry side. Additional gas phase separation with ion mobility, such as high-field asymmetric waveform ion mobility spectrometry[ 32 ], and different mass spectrometry acquisition techniques, such as BoxCar and DIA [ 37 , 38 ], have been employed to improve both HCP identification and quantification. Several PSDEs, including LPLA2 (PLA2G15, phospholipase A2, group XV), LPL (lipoprotein lipase), CES (liver carboxylesterase), LIPA (lysosomal acid lipase), PPT1 (palmitoyl-protein thioesterase 1), and SIAE (sialate o-acetylesterase) have been identified by TABP and have shown to have a direct impact on PS-80 or PS-20 degradation in biotherapeutics [ 15 , 26 , 27 , 33 , 39 ].…”