Sensitive, Rapid, Robust, and Reproducible Workflow for Host Cell Protein Profiling in Biopharmaceutical Process Development

Abstract

There is a growing industry and regulatory need to detect host cell protein (HCP) impurities in the production of protein biopharmaceuticals, as certain HCPs can impact product stability, safety, and efficacy, even at low levels. In some cases, regulatory agencies require the identification and the quantification of HCPs in drug products (DPs) for risk assessment, and this is an active and growing topic of conversation in the industry and amongst regulators. In this study, we developed a sensitive, robust, and reproducible workflow for HCP detection and quantification in a significantly shorter turnaround time than that previously reported using an Evosep ONE LC system coupled to an Orbitrap Fusion Lumos mass spectrometer. Because of its fast turnaround time, this HCP workflow can be integrated into process development for the high-throughput (60 samples analyzed per day) identification of HCPs. The ability to rapidly measure HCPs and follow their clearance throughout the downstream process can be used to pinpoint sources of HCP contamination, which can be used to optimize biopharmaceutical production to minimize HCP levels. Analysis of the NIST monoclonal antibody reference material using the rapid HCP profiling workflow detected the largest number of HCPs reported to date, underscoring an improvement in performance along with an increased throughput. The HCP workflow can be readily implemented and adapted for different purposes to guide biopharmaceutical process development and enable better risk assessment of HCPs in drug substances and DPs.

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

“…We then compared our results to published HCP results for the NISTmAb that were determined using different digestion conditions, LC separation methods and MS systems (Figure 1a). 22,[26][27][28] As expected, the majority of HCP reported in other LC-MS analyses were identified in our experiment (Figure 1, Supplemental Table S1). In addition, 13 HCP identified in this study but not in the published studies were verified against much longer lists of NISTmAb HCP identified using more advanced HCP enrichment methods.…”

Advanced mass spectrometry workflows for accurate quantification of trace‐level host cell proteins in drug products: Benefits of FAIMS separation and gas‐phase fractionation DIA

“…(2021) [64] (yellow), Ma et al. (2020) [36] (purple), and in the current study (blue). All studies used a native digestion protocol adapted from Huang et al.…”

Methods for addressing host cell protein impurities in biopharmaceutical product development

“…[11] Lipoprotein lipase, in particular, can contribute to polysorbate degradation by hydrolysis. [3,62] Polysorbate degradation can lead to the formation of acetic acid, formic acid, reactive aldehyde/ketones, peroxides, and free fatty acids, which affects the stability and shelf life of the protein product. [10] The identification and assessment of these problematic impurities that cause product or surfactant degradation in the formulation can help improve the quality of a product.…”

“…[ 11 ] Lipoprotein lipase, in particular, can contribute to polysorbate degradation by hydrolysis. [ 3,62 ] Polysorbate degradation can lead to the formation of acetic acid, formic acid, reactive aldehyde/ketones, peroxides, and free fatty acids, which affects the stability and shelf life of the protein product. [ 10 ]…”