Transitioning from concept to application: Comprehensive analytical validation of antibody-based smart sampling

Area of Science:

• Analytical Chemistry
• Biotechnology
• Biomarker Discovery

Background:

• Smart sampling integrates sample preparation into the sampling process for enhanced efficiency in LC-MS protein analysis.
• Current smart sampling applications are largely limited to proof-of-concept studies with minimal validation.
• Bioactive protein quantification in biological matrices requires robust and validated analytical methods.

Purpose of the Study:

• To fully validate the utility of antibody-based smart samplers for the quantitative analysis of bioactive proteins.
• To optimize the fabrication and performance of paper-based antibody samplers for human chorionic gonadotropin (hCG) detection.
• To assess the performance of smart samplers according to regulatory guidelines for bioanalytical methods.

Main Methods:

• Optimization of antibody sampler fabrication, reducing antibody usage by 90% while maintaining performance.
• Integration of sample preparation steps (reduction, alkylation, and cleanser extraction) into the smart sampling process.
• Validation of the optimized smart sampler for human chorionic gonadotropin (hCG) in human serum using ICH M10 guidelines.

Main Results:

• Optimized samplers demonstrated a significant reduction in antibody amount and manufacturing cost.
• Removal of the sampler post-reduction and alkylation improved signal output and reduced peptide background.
• Validation studies showed excellent linearity (r² ≥ 0.9955), accuracy (91-105%), and precision (≤ 14% CV) within regulatory limits.
• Samplers exhibited stability for at least ten weeks at room temperature.

Conclusions:

• The optimized antibody smart sampler represents a significant advancement in practical bioanalytical applications.
• Smart sampling technology offers a more efficient and cost-effective approach to protein quantification in biological samples.
• This validated method brings smart sampling closer to routine laboratory and clinical use.