Analysis of Charge Variant of Trastuzumab

Importance of the Topic

Trastuzumab is a monoclonal antibody used in targeted cancer therapy. Charge heterogeneity, arising from post-translational modifications, can impact its efficacy, stability, and immunogenicity. Accurate profiling of these charge variants is essential for ensuring product consistency and meeting regulatory standards in biopharmaceutical production.

Objectives and Overview of the Study

This application note describes a pH-gradient ion-exchange liquid chromatography (IEX-LC) method to separate and quantify the acidic, main, and basic isoforms of trastuzumab. The study evaluates method performance, resolution, and reproducibility on a Shimadzu Nexera lite inert HPLC system equipped with a Shim-pack Bio IEX SP-NP column.

Methodology and Instrumentation

The separation employs a linear pH gradient from 5 to 10 over 15 minutes using:

• Column: Shim-pack Bio IEX SP-NP (100 × 4.6 mm I.D., 5 μm)
• Mobile phase A: 20 mmol/L MES/HEPES/sodium acetate buffer (pH 5)
• Mobile phase B: 20 mmol/L MES/HEPES/sodium acetate buffer (pH 10)
• Gradient program: 0% B (0–15 min) to 100% B (15–17 min), then return to 0% B (17.1–30 min)
• Flow rate: 0.6 mL/min
• Column temperature: 40 °C
• Injection volume: 1 μL
• Detection: UV at 280 nm

All analyses were performed on a Shimadzu Nexera lite inert HPLC system using Shim-vial™ H glass vials.

Key Results and Discussion

The pH-gradient method achieved baseline resolution of multiple trastuzumab charge variants within 15 minutes. Distinct peaks representing acidic, main, and basic isoforms exhibited sharp shapes and retention time reproducibility below 0.5% RSD. The chromatogram confirmed clear separation, demonstrating the column’s high capacity and selectivity for antibody charge profiling.

Benefits and Practical Applications

This method provides:

• High-resolution separation of antibody charge variants for quality control and comparability studies
• Rapid cycle time suitable for high-throughput environments
• Simple buffer system minimizing sample preparation
• Applicability to batch release testing, stability assessments, and biosimilar development

Future Trends and Potential Applications

Future developments may include coupling pH-gradient IEX with mass spectrometry for detailed variant identification and peak assignment. Automation of gradient optimization and real-time data analysis will further enhance throughput. Integration with process analytical technology (PAT) platforms could enable in-line monitoring of charge heterogeneity during bioprocessing.

Conclusion

The pH-gradient ion-exchange chromatography method on Shim-pack Bio IEX SP-NP delivers robust, rapid, and high-resolution separation of trastuzumab charge variants. Its reproducibility and ease of use make it an ideal choice for routine biopharmaceutical quality control workflows.