A Novel Strategy to Rapidly Profile and Quantify High-Risk Host Cell Proteins Using Integrated DDA-PRM-dMRM Mode

Area of Science:

• Biopharmaceutical analysis
• Proteomics
• Analytical chemistry

Background:

• Host cell proteins (HCPs) are critical process-related impurities in biotherapeutics, posing risks to drug stability and safety.
• The wide dynamic range between therapeutic proteins and HCPs complicates the detection of high-risk species.
• Accurate quantification of HCPs is essential for ensuring the safety and efficacy of biopharmaceutical products.

Purpose of the Study:

• To develop and validate a novel strategy for comprehensive profiling and accurate quantification of high-risk HCPs in biotherapeutics.
• To establish a Chinese hamster ovary (CHO) cell spectral library for HCP identification.
• To enable simultaneous quantification of known high-risk HCPs and profiling of unknown HCPs.

Main Methods:

• Integration of data-dependent acquisition (DDA), parallel reaction monitoring (PRM), and multiple reaction monitoring (MRM) techniques.
• Construction of a CHO cell spectral library using DDA.
• Development and validation of a dynamic MRM (dMRM) method for simultaneous quantification.

Main Results:

• A CHO cell spectral library covering potential HCPs was constructed.
• 28 high-risk HCPs were validated using PRM and MRM.
• The dMRM method was established for simultaneous quantification of 28 high-risk HCPs.
• The strategy was applied to analyze five purified monoclonal antibody process samples, enabling profiling of unknown and quantification of known HCPs.

Conclusions:

• The developed strategy enables thorough analysis of both known and unknown HCPs in biopharmaceutical processes.
• This approach optimizes biopharmaceutical process development by improving HCP detection and quantification.
• The findings contribute to enhanced safety and stability of biotherapeutic products.