Analysis of Tryptic Digests of a Monoclonal Antibody and an Antibody-Drug Conjugate with the Agilent 1290 Infinity II LC

Significance of the topic

Monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) represent a growing class of targeted therapies for cancer and autoimmune disorders. Detailed peptide mapping of these biopharmaceuticals is essential to ensure product integrity, identify post-translational modifications, and detect low-abundance variants critical for safety and efficacy.

Objectives and study overview

This application note evaluates the performance of an Agilent 1290 Infinity II liquid chromatography system coupled with an AdvanceBio Peptide Map column for the analysis of tryptic digests of trastuzumab (Herceptin) and ado-trastuzumab emtansine (Kadcyla). Key goals include optimizing mobile phase mixing to reduce baseline noise, adjusting gradient slopes to maximize peak capacity, and comparing the separation profiles of mAb and ADC samples.

Methodology and instrumentation

• Instrumentation: Agilent 1290 Infinity II LC with High Speed Pump, Multisampler, Multicolumn Thermostat, Diode Array Detector (10 mm flow cell).
• Column: AdvanceBio Peptide Map, 2.1×250 mm, 2.7 µm superficially porous C18, 120 Å.
• Mobile phase A: 0.05% TFA in water/acetonitrile (99:1 v/v); B: 0.045% TFA in acetonitrile.
• Flow rate: 0.35 mL/min; Column temperature: 60 °C; Injection volume: 5 µL with needle wash in 0.05% TFA/water/acetonitrile (20:80 v/v).
• Detection: UV at 214 nm for peptide profiling; 252 nm for visualizing ADC-linked peptides.
• Mobile phase mixers tested: V35 (35 µL), V100 (100 µL), V380 (380 µL) Jet Weaver variants to assess TFA-related baseline noise.
• Sample preparation: 100 µg of mAb or ADC in 0.05% Rapigest/100 mM Tris-HCl pH 8; reduction (5 mM DTT, 60 °C, 30 min), alkylation (10 mM iodoacetamide, 37 °C, 1 h), trypsin digestion (1:25 w/w, 37 °C, 16 h).

Main results and discussion

• Jet Weaver performance: The V380 High-Performance mixer virtually eliminated baseline noise from TFA, enabling reliable detection of low-level peptides, whereas V35 displayed significant fluctuations.
• Delay compensation: Adding a short delay to the gradient profile aligned retention times and selectivity across all mixer configurations.
• Peak capacity vs gradient time: Controlled slopes produced peak capacities of ~300 (25 min), ~450 (45 min), and ~900 (205 min). Beyond ~120 min gradients, gains in peak capacity diminished.
• Herceptin vs Kadcyla profiles: UV 214 nm chromatograms were largely similar, with late-eluting clusters (25–35 min) corresponding to peptide–emtansine conjugates. These were more pronounced at 252 nm due to emtansine absorbance.
• Method precision: Five replicate injections of the Kadcyla digest showed excellent retention time and peak area reproducibility, confirming method robustness.

Benefits and practical applications of the method

This optimized LC method delivers high-resolution peptide mapping for mAbs and ADCs with stable baselines, enhanced sensitivity to low-abundance species, and strong reproducibility. It supports critical quality control and characterization workflows in biopharmaceutical development.

Future trends and possibilities for application

Further depth of analysis can be achieved by integrating two-dimensional LC×LC separations and coupling to high-resolution mass spectrometry for detailed structural elucidation. Advances in automation, faster gradients, and data analysis will further accelerate routine characterization in high-throughput environments.

Conclusion

The combination of the Agilent 1290 Infinity II LC system, AdvanceBio Peptide Map column, and V380 Jet Weaver mixer offers a powerful platform for comprehensive peptide mapping of mAbs and ADCs. It achieves high separation efficiency, low baseline noise, and excellent reproducibility, making it well suited for biopharmaceutical characterization.

References

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