ProteinWorks - TAKE THE COMPLEXITY OUT OF LARGE MOLECULE QUANTIFICATION

Significance of the Topic

In modern bioanalytical laboratories, accurate quantification of large molecules such as therapeutic monoclonal antibodies is essential for pharmacokinetics, pharmacodynamics, and drug development. Variability in sample preparation workflows can compromise reproducibility and sensitivity, making standardized methods critical for reliable decision-making.

Objectives and Study Overview

This work introduces the ProteinWorks Sample Preparation Kits, which integrate pre-measured, lot-traceable reagents with robust LC-MS protocols. The goal is to assess kit performance in terms of accuracy, precision, reproducibility, and detection limits for surrogate peptide quantification of multiple monoclonal antibodies in plasma and serum.

Methodology and Instrumentation

ProteinWorks employs a surrogate peptide approach with two main kit formats:

• eXpress Digest Kits for immunopurified samples
• Direct Digest Kits for whole plasma or serum

Key workflow steps include protein denaturation, enzymatic digestion, µElution SPE cleanup, and LC-MS analysis. Instrumentation and consumables used:

• UPLC system with ACQUITY UPLC Peptide BEH C18 column (2.1 x 150 mm, 1.7 µm, 300 Å)
• Triple quadrupole mass spectrometer operating in MRM mode
• µElution SPE 96-well plates for peptide cleanup
• Positive Pressure 96-processor and modular heat block

Main Results and Discussion

Reproducibility studies across five kit lots demonstrated single-digit intra-kit and inter-kit CVs for signature peptides of bevacizumab (Avastin). Inter-day and inter-analyst evaluations yielded consistent calculated concentrations for bevacizumab in plasma.
Sensitivity tests showed reliable detection of infliximab (Remicade) down to 10 ng/mL in rat plasma. Chromatographic profiles exhibited clear MRM peaks for the unique peptide SINSATHYAESVK with negligible signal in blank matrices.
Linear dynamic range experiments for four monoclonal antibodies (infliximab, trastuzumab, bevacizumab, adalimumab) using plasma volumes of 15, 35, and 70 µL achieved correlation coefficients (r2) above 0.994 and mean accuracies between 98.8% and 100.2%.

Benefits and Practical Applications

The ProteinWorks kits offer:

• Standardized reagents and protocols to reduce analyst-to-analyst variability
• High sensitivity and reproducibility suitable for PK/PD studies
• Flexibility to accommodate various sample volumes and assay workflows
• Rapid deployment and transferability between laboratories and CROs

Future Trends and Opportunities

Emerging directions include expanding the toolkit to additional biologic modalities, integrating automated sample preparation platforms for higher throughput, and leveraging advanced data analytics for deeper insight into protein quantification. Development of companion software for real-time QC monitoring and method optimization may further enhance workflow efficiency.

Conclusion

ProteinWorks Sample Preparation Kits provide a unified, kit-based solution for large molecule quantification by LC-MS, delivering high reproducibility, sensitivity, and flexibility. Standardized workflows and pre-validated reagents support reliable data generation across diverse research and development settings.

References

• Waters technology brief 720005570EN
• Waters application note 720005535EN
• Waters technology brief 720005575EN