Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

Area of Science:

• Biotechnology
• Analytical Chemistry
• Proteomics

Background:

• Host cell proteins (HCPs) are critical impurities in therapeutic protein products, posing potential risks even at low concentrations.
• Accurate identification and quantification of HCPs are essential for drug safety and regulatory compliance.
• Existing methods for HCP detection often require sensitive enrichment strategies to overcome low abundance challenges.

Purpose of the Study:

• To develop and validate a user-friendly protocol for the enrichment of low-abundance host cell proteins (HCPs) from therapeutic protein samples.
• To simultaneously remove monoclonal antibodies (mAbs) to improve HCP detection sensitivity.
• To establish a highly sensitive method for HCP detection using liquid chromatography-mass spectrometry (LC-MS).

Main Methods:

• Utilized commercially available proteome enrichment beads with a diverse library of hexapeptide ligands for HCP enrichment.
• Implemented a limited digestion protocol followed by peptide detection using nano liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS).
• Developed a protocol integrating HCP enrichment and mAb removal prior to LC-MS analysis.

Main Results:

• Achieved over 7000-fold enrichment of host cell proteins (HCPs).
• Established a detection limit as low as 0.002 ppm for HCPs.
• Successfully detected 850 HCPs with high confidence in a NIST mAb reference standard.
• Demonstrated a user-friendly protocol with a video demonstration for implementation.

Conclusions:

• The developed protocol offers a highly sensitive and effective method for enriching and detecting low-abundance HCPs.
• This approach significantly enhances the accuracy of risk assessment for therapeutic protein drug products.
• The user-friendly nature and high sensitivity of the protocol facilitate its adoption in biopharmaceutical quality control.