Host cell protein quantitation by LC-MS. Experimental demonstration, qualification, and comparison of methods in USP 1132.1

Abstract

The development of biologics necessitates reliable assays to characterize and control Host Cell Protein (HCP) impurities. Liquid Chromatography-Mass Spectrometry (LC-MS)-based HCP assays have emerged as a powerful orthogonal method to HCP ELISA, providing detailed information on individual HCPs. In response to a growing need, the U.S Pharmacopeia (USP) introduced General Chapter < 1132.1 > , which provides the best practices and outlines three quantitative LC-MS methods. This study explores the practical application and validation readiness of the following strategies: A - Relative to Product Protein, B - Relative to Spiked-in Protein, and C - Relative to Spiked-in Peptide. Two common HCPs-Clusterin and Lipoprotein Lipase-were quantified using LC-MS in a purified mAb drug substance spiked with a CHO cell culture harvest to simulate in-process HCP levels. All three methods were demonstrated in the same samples and dilutions, enabling direct comparison of the three methods from a single dataset. Method performance was assessed according to ICH Q2(R2) guidelines for analytical method validation, focusing on linearity, accuracy, precision, and specificity. Results include a comparative assessment and discussion of the advantages and disadvantages, and application of each aforementioned HCP quantification method. This study provides practical insights into the implementation of USP < 1132.1 > supporting the growing role of LC-MS in HCP analysis for biologics development.