Antibody Quantification Using ProteinWorks eXpress Digest Kits and Multiple Plasma Volumes

Importance of the topic

Liquid chromatography–tandem mass spectrometry (LC-MS/MS) is emerging as a powerful tool for the bioanalytical quantification of monoclonal antibodies (mAbs). The technique offers multiplexing, high specificity and broad dynamic range, but protein assays often demand complex workflows and larger sample volumes. Simplified, kit-based workflows suitable for low-volume plasma samples (<50 µL) can accelerate protein therapeutic quantification and support preclinical and clinical research studies.

Objectives and Study Overview

This study evaluated the versatility of the ProteinWorks eXpress Direct Digest Kit for accurate quantification of four monoclonal antibody drugs (infliximab, adalimumab, bevacizumab, trastuzumab) across plasma volumes ranging from 15 to 70 µL. Key goals included:

• Assessing linearity and accuracy of calibration curves for signature tryptic peptides.
• Demonstrating applicability across varying sample volumes without custom method development.
• Highlighting reproducibility and sensitivity in low-volume assays.

Methodology and Instrumentation

Monoclonal antibodies were spiked into human plasma at concentrations of 5–50 µg/mL. Aliquots of 15, 35 and 70 µL were directly digested using the ProteinWorks eXpress Direct Digest Kit following a generic protocol. Tryptic peptides were analyzed by LC-MS/MS employing multiple reaction monitoring (MRM) with 1/x weighting for calibration curves. Standard peptides representing unique antibody sequences were monitored to quantify each mAb.

• ProteinWorks kit: eXpress Direct Digest Kit (p/n 176003688).
• Instrumentation: LC system coupled to a triple quadrupole mass spectrometer (Waters Corporation) configured for MRM analysis.

Main Results and Discussion

Calibration curves over the 5–50 µg/mL range exhibited excellent linearity (R2 ≥ 0.99) across all plasma volumes. Mean accuracy of standard curve points exceeded 99% for all signature peptides. Representative infliximab peptide (DILLTQSPAILSVSPGER) maintained consistent response factors at 15, 35 and 70 µL. Chromatographic peak shapes and signal-to-noise ratios remained robust at low sample volumes, underscoring method sensitivity and reproducibility. The standardized digestion protocol eliminated the need for custom optimization per mAb and sample volume, streamlining bioanalytical workflows.

Benefits and Practical Applications

• Kit-based workflow reduces hands-on time and method development effort.
• Enables quantification of multiple mAb therapeutics in a single analytical sequence.
• Compatible with low plasma volumes typical of preclinical rodent studies.
• Delivers high linearity and accuracy for discovery and quality control applications.

Future Trends and Possibilities

Advancements in proteomics and mass spectrometry may further enhance sensitivity and throughput. Potential developments include:

• Integration with microfluidic and automated sample-preparation platforms.
• Extension to high-resolution mass spectrometry for peptide confirmation and structural analysis.
• Multiplexing additional protein biomarkers and biotherapeutics in complex matrices.
• Application of machine learning algorithms for chromatographic data processing and peak detection.

Conclusion

The ProteinWorks eXpress Direct Digest Kit provides a flexible, kit-based solution for accurate and reproducible quantification of monoclonal antibodies from plasma volumes as low as 15 µL. By unifying sample preparation and analytical conditions, the approach simplifies LC-MS/MS workflows and accelerates protein therapeutic bioanalysis.