Host Cell Protein (HCP) analysis by mass spectrometry (LC-MS/MS) has obviously been growing in importance in the last decade. However, LC-MS/MS analysis is generally used for characterization, so the practitioners have been on their own to develop methods that work for their particular applications. HCP analysis has unique challenges that make it different from the MS-based general proteomics or characterization workflows that dominate the biotech and biopharma landscape. In HCP analysis, the analyst is trying to detect parts-per-million level peptides in a vast ocean of drug product (DP) peptides. Mass spectrometers do not have dynamic (let alone linear) ranges of six orders of magnitude, so LC columns are often overloaded, compromising chromatographic performance. At these overloaded conditions, DP peptides can cause interference and ionization suppression of the HCP peptides’ signals. Additionally, because HCP peptides are at low levels, their signals are closer to the noise level, and the MS/MS spectra are lower quality. Compounding this, mass spectrometers and proteomics search engines used in data analysis are not designed specifically for HCP analyses – analysts must use instruments and software “off label,” and one must often accept or work around the inherent limitations in the equipment and software. These challenges have led to a diversity of options for sample preparation, LC conditions, MS data acquisition methods, data analysis, HCP quantitation, reporting, and comparison of MS and ELISA results.

In 2020, the United States Pharmacopeia (USP) convened an Expert Panel (EP) to draft a guidance chapter for the use of mass spectrometry in HCP analysis. Chapter 1132.1, Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry, has gone through one round of public comments and is currently in the revision stage. The objective of this chapter is to provide a summary of current best-practices and encourage more consistency in the field. While the chapter is intended for those already knowledgeable in proteomics analyses, it should also be accessible to a broad audience in the HCP field and should be able to answer many questions that non-experts have about LC-MS/MS analysis.

At BEBPA’s 2024 Host Cell Protein Conference, Niomi Peckham from USP will provide an overview and update on 1132.1, as well as USP’s other efforts in bringing standardization to the analysis of HCPs by LC-MS/MS. Additionally, there will be an Interest Group led by USP EP members that will focus specifically on 1132.1. This Interest Group will consist of several presentations and extended Q&A and panel discussions. (Note: Interest Group presentations and discussions are only available to in-person attendees.) BEBPA HCP conferences are known for their cordial and vibrant discussions about technical problems in the field. Come learn from the experts and get the inside scoop on how analysts are adopting better best-practices.