The principles behind our MS-based HCP assays

Thomas Kofoed, PhD in chemestry

This page has been reviewed and verified by Thomas Kofoed, PhD in Organic Chemistry

Setting up and performing mass spectrometry (MS)-based Host Cell Protein (HCP) analysis is complex for several reasons: Firstly, sample preparation for MS, including protein digestion and peptide enrichment, is labor-intensive and prone to variability. Secondly, MS is extremely sensitive, detecting proteins as low as 1 part per million (ppm). This makes it capable of identifying trace impurities but also increases the likelihood of detecting background contaminants, complicating analysis.

Lastly, MS-based data is complex and requires specialized software and expertise in MS/MS analysis. Challenges include differentiating between genuine HCPs and background noise and quantifying proteins accurately. Alphalyse has worked on overcoming these challenges since 2014 - gathering experience from more than 600 client projects - and today has the world's most advanced and robust setup. In the following, you can learn more about our 3 types of HCP assays:

DIA-LC-MS

for reproducible, quantitative determination of all protein impurities

MRM LS-MS

for quantifying target proteins throughout the purification process and in the final DS

ELISA-MS

for documenting coverage percentage and identifying high-risk HCPs

As an orthogonal approach to traditional ELISA, the Alphalyse HCP assays provide comprehensive coverage of HCPs, addressing both regulatory requirements and critical quality attributes in biologics development. Learn more details below.

Setup for DIA-LC-MS assay - quantitation of all HCPs

This standard assay setup investigates all impurities, regardless of their source. The process begins with sample preparation, where proteins are denatured, reduced, and enzymatically digested into peptides. These peptides are then separated using liquid chromatography to ensure efficient entry into the mass spectrometer (MS).

In Data-Independent Acquisition (DIA), the MS instrument collects fragmentation data for all ions within predefined mass-to-charge (m/z) windows, independent of precursor ion selection. This approach improves reproducibility and ensures that even low-abundance peptides are captured for analysis.

Quantification is achieved by integrating the sum of all signal intensities and referencing internal standards. The entire method fully aligns with USP General Chapter 1132.1, which establishes best practices for using mass spectrometry in HCP analysis.

Eppendorf robot for digestion of biologics samples

Sample preparation

  • Automated; denaturation, alkylation, digestion
  • Spike-in of intact proteins as internal standards
Mass spectrometers

LC-MS
analysis

  • Microflow LC & Data Independent Acquisition
    (LC-MS with SWATH DIA)
HCP analysis reports

Data
analysis

  • Search in product-specific database
  • Internal SUMall™ calibration for reproducible
    quantification and quality control

Setup for MRM LC-MS assay (for GMP)

The process begins with sample preparation, where proteins are denatured, reduced, and enzymatically digested into peptides. These peptides are then separated using liquid chromatography to ensure efficient entry into the mass spectrometer (MS). The MS detects peptides based on their mass-to-charge ratio, enabling precise identification through comparison with a database of known protein sequences.

Quantification is achieved by integrating the sum of all signal intensities and referencing internal standards. The entire method fully aligns with USP General Chapter 1132.1, which establishes best practices for using mass spectrometry in HCP analysis.

Eppendorf robot for digestion of biologics samples

Sample preparation

  • Automated; denaturation, alkylation, digestion
  • Spike-in of intact proteins as internal standards
Sciex MS instrument

LC-MS
analysis

  • Qualified instrument and software
  • Standard flow LC & MRM Acquisition
    on selected proteins (on TripleQuad instrument)
HCP analysis reports

Data
analysis

  • Quantification using specific peptides
  • Internal SUMall™ calibration for reproducible quantification and quality control

Principles of ELISA-MS™

The ELISA-MS™ method combines the strengths of traditional Enzyme-Linked Immunosorbent Assay (ELISA) with Mass Spectrometry (MS) to provide a detailed characterization of the ELISA and the use of the kit for monitoring HCPs in the specific project. Below are the key principles:

1. Immunocapture using ELISA
Polyclonal antibodies capture HCPs from the mock sample directly on the ELISA plate. The immunocaptured HCPs are then concentrated to enhance the detection of even low-abundance proteins.

2. LC-MS analysis
The immunocaptured HCPs are enzymatically digested into peptides and analyzed by LC-MS. This provides a detailed profile of HCPs, including low-abundance or small-molecular-weight proteins often missed by ELISA. An aliquot of the mock sample is digested in parallel and serves as a reference for total HCP content.

3. Coverage Evaluation
The list of immunocapture HCPs is compared to the list of total HCPs identified to provide a coverage number for the specific ELISA.

The principles of the ELISA-MS assay by Alphalyse

Talk to us

Whatever protein-related challenge or question you may have, we would love to help. Our experts can help you decide on the best analytical approach for your project by email or online meeting - providing advice without obligation.

I would like an
Hidden
Hidden
Hidden
This field is for validation purposes and should be left unchanged.