High Sensitivity LC-MS/MS Quantification of Monoclonal Antibody Drugs in Rat Plasma Using a Standardized Sample Preparation Workflow

Importance of the Topic

Monoclonal antibodies (mAbs) have become critical therapeutic agents due to their high target specificity, potency and favorable safety profiles. As many originator mAbs approach or pass patent expiry, bioanalytical quantification of mAbs in preclinical species such as rat plasma is essential to support pharmacokinetic, pharmacodynamic and toxicokinetic studies. A rapid, sensitive and reproducible workflow is needed to accelerate biosimilar and next-generation antibody drug development.

Study Objectives and Overview

This study aimed to demonstrate a highly sensitive LC-MS/MS method capable of quantifying multiple mAbs at concentrations ≤5 ng/mL in rat plasma. By combining protein-level affinity purification with a standardized bottom-up digestion kit and UPLC-MS/MS analysis, the workflow seeks to minimize method development time and provide robust quantification for infliximab, adalimumab and trastuzumab.

Methodology and Instrumentation

The standardized sample preparation workflow consisted of:

• Affinity purification: 100 µL rat plasma spiked with mAbs was incubated with goat anti-human biotinylated IgG on streptavidin beads, followed by neutralization to pH 8.
• Enzymatic digestion: The ProteinWorks eXpress Digest Kit was used with a five-step protocol including reduction, alkylation and tryptic digestion to generate signature peptides.
• LC-MS/MS analysis: Peptide separation was performed on an ACQUITY UPLC Peptide BEH C18 (300 Å, 1.7 µm, 2.1 × 150 mm) column at 0.3 mL/min, using 0.1% formic acid in water (A) and acetonitrile (B). A 15 µL injection and a linear gradient achieved baseline separation of target peptides.
• Mass spectrometry: A Xevo TQ-XS tandem quadrupole instrument was operated in MRM mode. Optimized transitions, collision energies and source parameters enabled sensitive detection of signature tryptic peptides for each mAb and the 15N13C-labeled SILu™ internal standard.

Main Results and Discussion

The method achieved:

• Limits of quantification between 1–5 ng/mL and detection limits as low as 0.5 ng/mL for selected peptides.
• Linear dynamic ranges spanning three to four orders of magnitude with correlation coefficients (R2) ≥0.99.
• Accuracy of standard curve points between 90–108% across the calibration range.
• Clear, reproducible chromatographic peaks at the low ng/mL level, confirming adequate sensitivity for preclinical PK studies.
• Total sample preparation time under 8 hours, including affinity capture and digestion.

Benefits and Practical Applications

The kit-based bottom-up workflow eliminates extensive discovery-stage optimization, enabling even inexperienced users to generate precise and accurate mAb quantification data. This approach supports time-critical decision making in biosimilar development, biotherapeutic screening and routine bioanalysis for preclinical drug research.

Future Trends and Opportunities

Opportunities exist to expand this workflow to multiplexed panels of therapeutic proteins, integrate automation for higher throughput, and adapt to microflow or nanoflow LC-MS platforms. Further advancements in digestion kits and affinity reagents may extend applicability to complex biological matrices and novel antibody formats.

Conclusion

The combination of protein-level affinity purification, a standardized digest kit and UPLC-MS/MS analysis delivers a robust, highly sensitive assay for monoclonal antibody quantification in rat plasma. Achieving quantification at or below 5 ng/mL with minimal method development accelerates bioanalytical support for biosimilar and next-generation antibody programs.

Reference

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