Regulatory Consequences of New Protein-Impurity Guidelines

The biopharmaceutical industry is on the brink of a paradigm shift with the release of the United States Pharmacopeia (USP) General Chapter <1132.1> “Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry,” which was published on 1 December 2024 (1). The chapter presents best practices for standardized mass spectrometry (MS)-based host cell protein (HCP) analyses to enable comprehensive and reproducible impurity profiling in biologics, improving their quality and consistency. The chapter also marks a turning point in regulatory approaches.

HCPs are process-related impurities generated during manufacturing of monoclonal antibodies (mAbs), other recombinant proteins, vaccines, and advanced therapies; therefore, monitoring HCPs is critical for ensuring product quality, safety, and efficacy. Previously, manufacturers have focused on reducing and monitoring total HCP levels, but health authorities now emphasize the monitoring of specific HCPs that can affect product quality. Without orthogonal MS analyses, biomanufacturers risk approval delays.

ELISA Limitations

For decades, enzyme-linked immunosorbent assays (ELISAs) have been used for HCP measurement and control, usually supported by two-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (2D SDS-PAGE) and western blot methods. However, the technique has significant limitations, as outlined in USP General Chapter <1132.1>:

Those shortcomings can result in undetected or unidentified HCPs, potentially leading to product and excipient degradation by enzymatically active proteases, lipases, and esterases; harmful immune responses, such as those from anti-HCP and/or antidrug antibodies; and interference with endogenous protein activities (2). Notable examples include the following:

• The Chinese hamster ovary (CHO) HCP phospholipase-B–like 2 (PLBL2) went undetected by a platform ELISA during late-stage trials, resulting in patient immune responses and delayed phase 3 trials

• Escherichia coli ribose phosphate isomerase (RPI) went undetected by a generic E. coli protein assay and, during clinical trials, induced antidrug antibodies in 60% of patients and anti-HCP antibodies in all subjects (2, 3)

• Both monocyte chemoattractant protein (MCP-1), a CHO HCP, and flagellin, an E. coli HCP, put phase 2 trials on hold following severe adverse events after going undetected by ELISAs (2).

Inadequate HCP control has led to project delays and terminations, company bankruptcies, and adverse patient reactions. Regulatory authorities now broadly request orthogonal HCP analysis to complement immunoassays. Several clients have approached Alphalyse, a contract research organization (CRO) specializing in protein impurity analysis by MS, after their investigational new drug (IND) or clinical trial applications were delayed by the US Food and Drug Administration (FDA) or the European Medicines Agency (EMA) because of inadequate HCP monitoring.

The Need for <1132.1>

The evolution of complex biotherapeutics and advances in proteomics technology have expanded the use of MS-based analyses to overcome the limitations of HCP-ELISA and provide developers with the means to detect, identify, and eliminate HCPs of concern. A key advantage of MS-based HCP analysis over ELISA is the ability to identify and quantify residual impurities simultaneously. Whereas an HCP-ELISA provides a single total HCP value, MS enables data-driven risk assessments of individual HCPs and targeted purification strategies for specific HCPs.

In 2020, the USP convened an expert panel of industry, academic, and regulatory specialists to address variations in sample preparation and quantitation methods and to develop best practices for HCP analysis by MS. <1132.1> outlines sample preparation, chromatographic separation, MS analysis, and methods for HCP quantification (2).

Industry Impact of Standardized MS Methods

USP General Chapter <1132.1> offers a standardized approach to HCP analysis that will change industry practices. Previous USP chapters have influenced regulatory decisions, and regulatory reviewers already are encouraging MS-based analysis in INDs and biologics license applications (BLAs) (4).

Alphalyse exemplifies the benefits of standardizing MS-based HCP analysis. Since 2014, Alphalyse has developed and optimized the quantification method using spiked-in intact proteins described in <1132.1>, becoming the first laboratory in the world to perform HCP release testing by MS under good manufacturing practice (GMP) conditions (5).

By using intact proteins as internal standards for normalizing HCP quantitation, automated sample preparation, and automated data analysis, Alphalyse achieves robust, reproducible, and quantitative MS results. The intermediate precision of the assay shows a coefficient variation (CV%) of <20%, which has enabled validation of HCP assays following ICH Q2(R2) guidelines for over 10 biopharmaceutical products.

MS-based HCP methods have proven invaluable for process development, product characterization, batch comparison, verification of ELISA results, and ELISA reagent-coverage analysis across a wide range of drug types and expression systems. Regulators consistently have accepted MS-based analyses performed by Alphalyse and submitted in client applications with few or no questions. In addition, the data collected from over 600 standardized HCP analysis enable Alphalyse to advise clients on expected impurity levels and HCPs of concern in their specific drug type and expression system based on consistent, reproducible MS data from comparable projects.

The Future of HCP Analysis

The technical advancements outlined in <1132.1> represent a necessary evolution in the approach to HCP analysis, emphasizing the identification and quantitation of specific high-risk HCPs. Older methods cannot meet the complex needs of next-generation drug development, as ELISA results depend on proprietary reagents tied to specific products or production systems, limiting industry-wide standardization. The MS methods described in <1132.1>, combined with HCP reference standards, will enable comparisons of HCP levels across the industry, facilitating knowledge-based risk assessment, safer and more efficient biologics, and, ultimately, better patient outcomes.

Whether you choose to perform HCP-MS in-house or outsource to an experienced CRO, now is the time to familiarize yourself with <1132.1>. Regulatory authorities are already requesting orthogonal HCP data, shifting the focus from total HCP levels to individual HCP quantitation. mAb developers are leading the way toward individual HCP risk analysis, with those using E. coli-based expression systems following closely.

As the industry embraces chapter <1132.1>, orthogonal MS analysis will undoubtedly become the industry standard for all biologics — and a regulatory requirement within the next few years.

References

1 USP General Chapter <1132.1> Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry. United States Pharmacopeial Convention: Rockville, MD, 2024.

2 Vanderlaan M, et al. Experience with Host Cell Protein Impurities in Biopharmaceuticals. Biotechnol. Prog. 34(4) 2018: 828–837; https://doi.org/10.1002/btpr.2640.

3 Omnitrope: EPAR — Scientific Discussion. European Medicines Agency: Amsterdam, the Netherlands, 2006; https://www.ema.europa.eu/en/documents/scientific-discussion/omnitrope-epar-scientific-discussion_en.pdf.

4 Peckham N. USP Resources To Support Host Cell Protein Analysis by Mass Spectrometry. BEBPA 2024 Hybrid Host Cell Protein Conference: College Park, MD, 15 May 2024; https://www.bebpa.org/2024-hcp.

5 World’s 1st MS-Based HCP Analysis Under GMP for Release Test [press release]. Alphalyse, 3 November 2022; https://alphalyse.com/press-release-worlds-1st-ms-based-hcp-analysis-under-gmp-release-test.

Ejvind Mørtz is chief operating officer (COO) and cofounder, and Thomas Kofoed is chief executive officer (CEO) and cofounder, both at Alphalyse; [email protected]; https://www.linkedin.com/in/ejvindmortz.