Host cell protein quantitation by LC-MS. Experimental demonstration, qualification, and comparison of methods in USP 1132.1
Victor G Chrone, Anthony J. Blaszczyk, Derrick H. Zhang, Solveig B. Nielsen, Janne Crawford, Veit Schwämmle, Peter Højrup, Thomas Kofoed, Niomi Peckham, Ejvind Mørtz
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.