Quantification of Monoclonal Antibody Trastuzumab in Mouse Plasma with the Agilent 1290 Infinity II Bio LC and Agilent 6495 Triple Quadrupole LC/MS Systems

Importance of the Topic

Monoclonal antibody therapeutics have become a cornerstone of modern drug development with a market projected to exceed 390 billion USD by 2030. Accurate quantification of these large biomolecules in biological matrices is essential for pharmacokinetic studies safety assessments and dose optimization. Traditional ligand binding assays provide high throughput but suffer from cross-reactivity long reagent development times and limited specificity. LC/MS workflows overcome these limitations by offering high specificity sensitivity a wide dynamic range and faster method development.

Objectives and Study Overview

This study presents a hybrid immunoaffinity LC/MS method for quantifying the monoclonal antibody trastuzumab in mouse plasma. Key goals include:

• Developing an immunoaffinity purification protocol to enrich target antibody from small plasma volumes
• Implementing trypsin digestion to generate surrogate peptides unique to trastuzumab
• Establishing an LC/MS MRM workflow on Agilent platforms for sensitive specific quantitation

Methodology and Instrumentation

Immunoaffinity Purification

• Magnetic streptavidin beads conjugated with biotinylated antihuman Fc antibody to capture trastuzumab
• Incubation with 50 μL of mouse plasma fortified at various concentrations followed by washing and elution with trifluoroacetic acid buffer

Trypsin Digestion

• Dithiothreitol reduction and iodoacetamide alkylation of captured antibody
• Digestion with sequencing‐grade trypsin at 37 °C overnight

LC/MS Analysis

• Agilent 1290 Infinity II Bio LC with Poroshell EC-C18 column (2.1 × 50 mm 2.7 μm) at 50 °C flow rate 0.4 mL/min
• Agilent 6495 Triple Quadrupole LC/MS with Jet Stream source in positive electrospray mode
• MRM transitions optimized for two unique surrogate peptides IYPTNGYTR and FTISADTSK and one universal peptide GPSVFPLAPSSK

Main Results and Discussion

The hybrid workflow achieved a lower limit of quantification of 5 ng/mL using 50 μL of mouse plasma. Calibration curves spanning 5 to 2000 ng/mL were generated with a quadratic fit and 1/x2 weighting. Key performance metrics included:

• Accuracy biases within ±10 percent across all concentration levels
• Precision with coefficients of variation below 15 percent even at the lowest concentration
• High specificity demonstrated by clear MRM chromatograms for each surrogate peptide

Benefits and Practical Applications

The hybrid immunoaffinity LC/MS method offers:

• High specificity and sensitivity without the need for a dedicated anti-idiotype reagent
• Rapid method development enabling faster bioanalysis in drug discovery
• Wide dynamic range suitable for preclinical and clinical pharmacokinetic studies
• Universal applicability to other human IgG-based monoclonal antibodies

Future Trends and Opportunities

Emerging directions include:

• Multiplexed MRM assays for simultaneous quantitation of multiple antibody therapeutics
• Automation of immunocapture and digestion steps for higher throughput
• Integration with high resolution MS for improved selectivity and expanded analyte panels
• Application to biosimilar comparability studies and clinical therapeutic monitoring

Conclusion

The Agilent hybrid immunoaffinity LC/MS workflow combining the 1290 Infinity II Bio LC and 6495 Triple Quadrupole MS platforms provides a robust sensitive and universal approach for quantifying trastuzumab in plasma. With minimal sample preparation fast turnaround and excellent assay performance this method supports accelerated drug development and can be extended to a broad range of monoclonal antibody biotherapeutics.

References

• Allied Market Research Latest Reports
• Jenkins R et al Recommendations for Validation of LC–MS/MS Bioanalytical Methods for Protein Biotherapeutics AAPS J 2015 17 1 1–16
• Boekhout AH Beijnen JH Schellens JHM Trastuzumab Oncologist 2011 16 6 800–810