LCMS Bioanalysis of Antibody Drugs Using Fab-Selective Proteolysis nSMOL - Trastuzumab analysis -

Significance of the Topic

The precise quantification of monoclonal antibody drugs in biological matrices is essential for pharmacokinetic profiling, drug efficacy and safety assessment. Traditional immunoassays face limitations from cross-reactivity and matrix interference. The nSMOL™ technique offers targeted proteolysis of the antibody Fab region, enabling mass spectrometric bioanalysis with higher specificity and reproducibility.

Study Overview

This application note presents a method for quantitative analysis of trastuzumab in human plasma using Shimadzu nSMOL Antibody BA Kit. The aim is to demonstrate selective digestion, robust sample preparation and reliable LC–MS/MS quantification compatible from preclinical to clinical testing.

Methodology and Instrumentation

Sample preparation uses the nSMOL protocol for Fab‐selective proteolysis, applicable to diverse antibody drugs. Digestion yields signature peptides from complementarity‐determining regions (CDR) with no interference from endogenous IgGs.

• Liquid Chromatography: Shimadzu Nexera X2, Shim-pack GISS C18 (50×2.1 mm), 0.1 % formic acid mobile phases, gradient 1–95 % B, 0.4 mL/min, 50 °C, 10 µL injection.
• Mass Spectrometry: LCMS-8050/8060 with positive ESI, DL 250 °C, heat block 400 °C, interface 300 °C, nebulizer gas 3 L/min, drying/heating gas 10 L/min.
• Signature Peptides: P14R and IYPTNGYTR, quantitation and structural confirmation via MRM transitions.

Main Results and Discussion

The assay achieved a quantitation range of 0.0610–250 µg/mL with average accuracy of 100.7 %. The lower limit of quantitation was 0.06 µg/mL. Validation tests showed:

• Precision and accuracy over 15 replicates: 88.1–106 % accuracy and CV ≤ 8.2 %.
• Freeze–thaw stability at –20 °C: 98.1–99.7 % accuracy.
• Long-term stability at –20 °C: 101–104 % accuracy.
• Processed sample stability over 48 h at 5 °C: 91.2–106 % accuracy.

Use of structure‐confirming transitions enhances specificity and data quality compared to single MRM quantitation.

Benefits and Practical Applications

The nSMOL method streamlines sample processing and reduces matrix noise, shortening analysis time while maintaining high throughput. It supports consistent assay performance from early discovery through clinical phases without the need for assay redevelopment for each antibody.

Future Trends and Potential Uses

Expansion of nSMOL to multiplexed antibody panels and combination therapies is anticipated. Integration with high‐resolution mass spectrometry could further improve sensitivity and broaden the range of detectable biotherapeutics. Automation of sample preparation may enhance throughput in clinical settings.

Conclusion

The Shimadzu nSMOL Antibody BA Kit delivers a validated, robust LC–MS/MS workflow for selective proteolysis and quantitation of trastuzumab in plasma. The method meets regulatory guidelines for low-molecular-weight compounds while offering the specificity of structure‐based mass spectrometry.

References

• Iwamoto N et al. Analyst 2014, DOI:10.1039/c3an02104a
• Iwamoto N et al. Anal Methods 2015, DOI:10.1039/c5ay01588j