NISTmAb characterization with a high-performance RP chromatography column

Importance of the Topic

Monoclonal antibodies represent a critical class of therapeutics with increasing complexity and stringent quality requirements. High-performance reversed-phase chromatography enables detailed characterization of intact antibodies and their variants, supporting reliable product development and regulatory compliance.

Objectives and Study Overview

This study assessed the robustness, reproducibility, carry-over, recovery, accuracy, and precision of NISTmAb quantification using the Thermo Scientific Vanquish Horizon UHPLC system and MAbPac RP column. Results were benchmarked against a traditional wide-pore silica C4 column.

Instrumentation Used

• Vanquish Horizon UHPLC system including System Base, Binary Pump H, Column Compartment H, Split Sampler HT (25 μL loop) and Diode Array Detector HL
• Thermo Scientific Chromeleon CDS 7.2 SR5 software

Methodology and Chromatographic Conditions

Separation was performed on a polymer-based MAbPac RP column (2.1 × 50 mm, 4 μm, 1500 Å) and compared to a silica-based C4 column (2.1 × 50 mm, 5 μm, 300 Å). Mobile phases were water/TFA (99.9:0.1 v/v) and acetonitrile/water/TFA (90:9.9:0.1 v/v/v). A 0.6 mL/min flow rate, 80 °C column temperature and 1 μL injection volume were used. Detection was at 280 nm with a gradient from 20% to 55% organic over 10 minutes.

Main Results and Discussion

• Resolution and Peak Performance: The MAbPac RP column delivered sharper peaks (2.2 s width at half height versus 4.4 s) and higher plate counts, resolving eight variants including a lysine form, compared to a single peak on the silica column.
• Column Lifetime: Over 1100 consecutive injections at 80 °C showed stable retention times (0.12% RSD), peak widths (2.17% RSD) and asymmetry (0.96% RSD), with minimal backpressure increase.
• Lot-to-Lot Reproducibility: Four column lots exhibited consistent retention times (0.33% RSD for the main peak) and variant peaks (0.30% RSD), confirming manufacturing precision.
• Quantification Performance: A linear range from 0.1 to 20 μg (R2 > 0.999) was demonstrated. Quality control samples at 0.15, 0.7 and 7 μg showed accuracies between 93% and 104% and RSDs below 3.1%.
• Recovery and Carry-over: The polymer column achieved 96.4% recovery at 1 μg load, outperforming 87% on the silica column. Column-related carry-over remained below 0.25% across a 0.02–20 μg range.

Benefits and Practical Applications

The combination of Vanquish Horizon UHPLC and MAbPac RP column offers high resolution, excellent reproducibility and minimal carry-over, making it suitable for routine mAb characterization, QC testing and method development in biopharmaceutical laboratories.

Future Trends and Applications

Advancements may include integration with mass spectrometry for detailed variant mapping, further improvements in polymer column chemistries for enhanced stability and expansion of high-throughput workflows in bioprocessing environments.

Conclusion

The MAbPac RP column on the Vanquish Horizon UHPLC platform provides a robust, precise and high-resolution solution for monoclonal antibody analysis, supporting regulatory compliance and streamlined method transfer.

References

• High Performance RP Chromatography Column for mAb Analysis, Thermo Fisher Scientific
• Using the NISTmAb Reference Standard for Charge Variant Analysis, Thermo Fisher Scientific, 2017
• Report of Investigation: NIST Reference Material 8671, NIST, 2016