Prospective ICH Q2(R2)-Aligned Total-Error Validation of Label-Free Untargeted Proteomics for Host Cell Protein...

Area of Science:

• Biopharmaceutical Analysis
• Analytical Chemistry
• Proteomics

Background:

• Untargeted proteomics offers quantitative host cell protein (HCP) determination in biotherapeutics.
• No current workflow is validated under ICH Q2(R2) for regulated quality control of HCPs.

Purpose of the Study:

• To perform a prospective total-error (TE) validation of label-free ddaPASEF proteomics for HCP quantification.
• To establish a validated workflow meeting ICH Q2(R2) guidelines for regulated biopharmaceutical quality control.

Main Methods:

• Prospective TE validation using label-free ddaPASEF proteomics.
• Spiking a stable isotope-labeled whole-proteome standard into NISTmAb across seven concentration levels (20-80 ng).
• Analysis via one-way random-effects ANOVA and peptide-level error evaluation.

Main Results:

• Empirical false-discovery proportions below 1% (q=0.01).
• Validated range of 20-80 ng with 95% tolerance intervals within ±30%.
• Robustness demonstrated across independent software and acquisition platforms.

Conclusions:

• First prospective ICH Q2(R2)-aligned validation of untargeted proteomics for HCP quantification.
• Established a transferable statistical framework for high-dimensional analytical methods.
• The validated method provides accurate and reliable HCP quantification for biotherapeutics.