LC-MS Quantification of Trastuzumab Using Pellet Digestion and ProteinWorks eXpress Direct Digest Kits

Importance of the Topic

The accurate quantification of therapeutic monoclonal antibodies in biological matrices is essential for drug discovery, development and bioanalytical studies. Conventional workflows often require extensive sample cleanup and affinity enrichment to overcome matrix interferences, notably from high-abundance proteins such as serum albumin. Simplifying the sample preparation while maintaining sensitivity and specificity is critical for routine analysis and early-stage research.

Goals and Overview of the Study

This study evaluates the compatibility of ProteinWorks eXpress Direct Digest Kits combined with a pellet digestion workflow to quantify trastuzumab (Herceptin) in plasma. It aims to demonstrate improved sensitivity and selectivity by introducing a simple protein precipitation (PPT) step prior to digestion, comparing several organic solvent conditions, and establishing performance in a targeted UPLC-MS/MS assay.

Methodology and Instrumentation

The workflow integrates two main steps:

• Protein Precipitation (PPT) pre-treatment: Plasma aliquots (15 µL) were subjected to different organic solvent ratios and chemistries: 1:1 isopropanol (IPA), 1:1 methanol with 1 % trichloroacetic acid (TCA), and 1:10 IPA with 1 % trifluoroacetic acid (TFA). After centrifugation, pellets were retained.
• Pellet digestion: Resuspended pellets were digested using the ProteinWorks eXpress Direct Digest Kit following the vendor protocol.

Targeted quantification was performed by UPLC-MS/MS monitoring signature tryptic peptides of trastuzumab (DTYIHWVR, IYPTNGYTR, FTISADTSK) and representative human serum albumin peptides to assess depletion.

Main Results and Discussion

• Albumin depletion: The 1:10 IPA with 1 % TFA condition achieved over 90 % removal of three monitored albumin peptides, outperforming other PPT treatments.
• Trastuzumab signal enhancement: All top three PPT conditions produced 2–8-fold increases in the peak areas of trastuzumab peptides compared to no PPT, with the IPA/TFA protocol delivering the greatest sensitivity gain.
• Calibration performance: Standard curves prepared in plasma after PPT and pellet digestion demonstrated excellent linearity (R² ≥ 0.99 with 1/x weighting) over 5.0–50.0 µg/mL and mean accuracies > 99 % for all three signature peptides.

Benefits and Practical Applications

This streamlined workflow offers:

• Reduced sample complexity by simple, low-cost PPT steps without affinity reagents.
• Enhanced digestion efficiency and MS sensitivity, lowering enzyme requirements and costs.
• Reproducible quantification of mAbs in small plasma volumes, suitable for discovery and QA/QC labs.
• Generic applicability to other protein biotherapeutics with minimal method development.

Future Trends and Potential Applications

Advancements may include:

• Automation of PPT and digestion steps for higher throughput.
• Adaptation to microflow LC-MS and high-resolution platforms for deeper proteome coverage.
• Extension to multiplexed assays of multiple antibodies or biomarkers in a single run.
• Integration with novel precipitation chemistries and microfluidic systems to further reduce sample volumes.

Conclusion

A combined PPT and pellet digestion approach using ProteinWorks eXpress Direct Digest Kits provides a robust, sensitive, and straightforward LC-MS/MS method for quantitative analysis of trastuzumab in plasma. The protocol simplifies sample preparation, maximizes depletion of interfering proteins, and achieves high analytical performance, making it well-suited for diverse biotherapeutic quantification applications.

Reference

• Mary Lame, Paula Orens, Sherri Naughton, Erin Chambers. LC-MS Quantification of Trastuzumab Using Pellet Digestion and ProteinWorks eXpress Direct Digest Kits. Waters Corporation, January 2016.