USP 1132.1 | Host Cell Protein quantification by Mass Spectrometry
USP 1132.1 is the new United States Pharmacopeia chapter (official as of May 1, 2025) that sets best practices for measuring residual host cell proteins (HCPs) in biologics using LC-MS/MS. It expands the original chapter 1132 - focused on ELISA - to cover sample prep, quantitation methods, and validation for mass spectrometry workflows.
This page has been reviewed and verified by Ejvind Mortz, PhD in Protein Chemistry & Molecular Biology
What is USP 1132.1?
USP 1132.1 is a newly introduced chapter by the United States Pharmacopeia, official from May 1, 2025. It outlines best practices for measuring residual host cell proteins (HCPs) in biologics using liquid chromatography–mass spectrometry (LC-MS/MS). The chapter extends USP 1132 by covering MS-based workflows for HCP identification, quantification, and validation.
The United States Pharmacopeia (USP) is the most comprehensive source for standards about drug quality, purity, strength, and identity worldwide. The FDA widely uses USP standards as a reference, outlining the recommended processes, tests, and procedures for developing, manufacturing, and regulating pharmaceutical products.
When did USP 1132.1 become official?
The chapter has been available on the USP website since December 1st, 2024, and became official on May 1st, 2025. This five-month implementation window allowed biopharmaceutical companies to prepare their MS workflows and validation procedures to meet the new standards.
What's new in chapter 1132.1?
The original USP 1132 chapter on Residual Host Cell Protein Measurement in Biopharmaceuticals focuses almost exclusively on immunoassay methods, with only a limited discussion on supporting/orthogonal technologies.
USP 1132.1 provides general guidance and best practices on:
- Sample preparation – denatured digestion, depletion of product protein, and HCP enrichment
- Chromatographic separation – setting up robust LC-MS workflows
- Mass spectrometry analysis – including:
- HCP quantitation methods (relative to product protein, spiked-in proteins/peptides)
- Validation and system suitability
- Reporting strategies for comparing ELISA and LC-MS/MS results
A real-world case study is included to demonstrate normal variation in performance across instruments and analysts.
Key differences between USP 1132 vs 1132.1
The key difference between “USP 1132” and “USP 1132.1” can be seen in the table below.
| Primary focus | Immunoassay (ELISA) methods | Mass spectrometry methods |
| Analytical Approach | Total HCP content measurement | Individual HCP identification and quantification |
| MS coverage | Limited discussion of orthogonal methods | Comprehensive MS guidance including sample prep, LC separation, and quantitation |
| Validation Requirements | ELISA-specific validation | MS method validation and system suitability |
| Reporting | Total HCP levels (ppm) | Individual HCP identification with abundance levels |
Understanding the shift in analytical paradigm
The evolution from USP 1132 to 1132.1 reflects a broader industry transition from general quantification to precise characterization. Traditional ELISA techniques provide an overview of total HCP levels but lack specificity.
In contrast, mass spectrometry enables detailed identification and quantification of individual HCPs. This distinction is critical when regulators ask not just “how much” but “which” HCPs remain in the drug substance.
This deeper resolution helps identify problematic HCPs, such as proteases or lipases, which may degrade the drug product or affect stability and efficacy.
MS also helps monitor product consistency over time and across manufacturing sites. By applying orthogonal methods like LC-MS/MS, manufacturers can overcome the known limitations of polyclonal antibody-based ELISAs, which may fail to detect low-abundance or weakly immunogenic proteins.
Contents of USP General Chapter 1132.1
Chapter 1132.1 includes comprehensive sections on:
Sample preparation requirements
- Denatured digestion protocols
- Product protein depletion strategies
- HCP enrichment techniques
Chromatographic separation
Detailed guidance on LC methods optimized for HCP separation and detection
Mass spectrometry analysis
- Methods for HCP quantitation (relative to product protein, spiked-in proteins, and spiked-in peptides)
- Method validation procedures
- System suitability requirements
- Best practices for reporting ELISA vs. LC-MS/MS data
The chapter also includes a case study on low-abundance HCP detection to demonstrate normal variation in analyst and instrument performance.
How to comply with USP 1132.1
To meet USP 1132.1 requirements, biopharmaceutical companies need to establish validated LC-MS/MS workflows. The regulatory landscape is changing - health authorities increasingly demand orthogonal data such as mass spectrometry analysis to support ELISA, pushing for more thorough and reliable documentation.
At the BEBPA 2024 HCP conference, Niomi Peckham from the USP discussed implementation strategies. Key compliance steps include:
- Establishing sample preparation protocols that ensure complete protein digestion
- Implementing chromatographic methods with sufficient peak capacity
- Validating quantitation approaches using appropriate standards
- Developing system suitability criteria for routine analysis
- Creating comprehensive reporting templates that align ELISA and MS data
Learn more about how to investigate HCPs in biologics using mass spectrometry >>
Impact on the biopharmaceutical industry
The update of the USP 1132 chapter marks a significant shift in how HCP analysis is perceived and executed. While ELISA remains the most widespread method for HCP analysis, it will increasingly be supported by characterization and coverage analysis by MS. Some companies have already been required by the FDA and EMA to support their ELISA HCP data with MS data.
This shift represents a move away from examining total HCP content to evaluating specific HCPs of concern. The purpose of monitoring HCP levels in drug substances is to avoid the harmful effects of HCPs with enzymatic or immunogenic activity. While most protein impurities do not impact patient safety or drug stability, a few can cause problems even at low levels, requiring careful documentation and control.
In this video, CEO Thomas Kofoed asked CMC Executive Bryant McLaughlin how he anticipates the new USP chapter will affect the industry.
What does USP 1132.1 mean for the development of medicine and regulatory approval?
Shaping the future of biopharmaceutical development
The implementation of USP 1132.1 has significant implications across the biopharmaceutical lifecycle - from early development to regulatory submission. By emphasizing mass spectrometry-based HCP analysis, the chapter addresses the limitations of ELISA-based quantification, especially when precise identification of low-abundance or immunogenic proteins is required.
1. Earlier and smarter process development
Biopharmaceutical developers can now apply MS early in the process to identify which HCPs persist throughout purification. This supports smarter process optimization - allowing developers to improve purification strategies before clinical trials begin, reducing the risk of late-stage failures.
2. Faster, data-driven regulatory approval
Regulators such as the FDA and EMA increasingly expect orthogonal HCP data to complement ELISA results. Mass spectrometry not only meets these expectations but can accelerate regulatory acceptance. Alphalyse clients have successfully used LC-MS/MS data as standalone documentation in BLA and IND submissions, in some cases avoiding the need for process-specific ELISAs altogether.
3. Greater assurance for patient safety
Identifying individual HCPs helps assess their risk potential - whether enzymatic degradation, immunogenicity, or interaction with excipients. This level of precision ensures that even trace-level contaminants are well understood, monitored, and documented, contributing to patient safety and long-term product consistency.
4. Improved flexibility for complex therapies
For advanced modalities - such as viral vectors, gene therapies, and complex biologics - ELISAs may be impractical due to poor immunogenic response or low coverage. In such cases, MS becomes the only viable analytical strategy, providing both quantitative and qualitative data under GMP conditions.
The update of the USP 1132 chapter paves the way for significant changes in the pharmaceutical industry
The new chapter 1132.1 marks a shift in how HCP analysis is perceived and executed.
ELISA remains the most widespread method for HCP analysis, but it will increasingly be supported by characterization and coverage analysis by MS. Some of our clients have already been required by the FDA and EMA to support their ELISA HCP data with MS data.
What prompted the development of USP 1132-1?
Ejvind Mortz, COO at Alphalyse, has examined the content of the upcoming US Pharmacopeia General Chapter 1132.1, which is set to transform protein impurity analysis in the biopharma industry in 2025.
- What prompted the USP to develop this addition to chapter 1132?
- Which analytical methods are covered by 1132.1?
FAQ
On demand webinar
Initiatives for enhancing the quality and consistency of MS-based HCP analysis
Derrick Zhang, US Pharmacopeia, USA
This presentation focuses on the USP's initiatives to enhance the quality and consistency of MS-based HCP analysis.
- The introduction of a new general chapter, <1132.1> 'Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry.'
- Documentary and physical standards for MS HCP analysis
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