Quantitation of 7 N-Nitrosamines in Monoclonal antibody (mAb) formulations using LC-MS/MS

Significance of the Topic

N nitrosamines are potent mutagens classified as Class 1 under ICH M7 and have been closely monitored in small molecule drugs. Recent findings highlight their potential presence in biopharmaceuticals such as monoclonal antibodies, arising from nitrosating reagents, process contaminants and packaging materials. A reliable analytical method is essential to ensure patient safety and regulatory compliance.

Objectives and Overview of the Study

This study aimed to develop and validate a sensitive and robust LC-MS/MS protocol for quantification of seven N nitrosamine impurities in monoclonal antibody formulations. The targeted analytes included NMBA, NDMA, NDEA, NEIPA, NDIPA, NDPA and NDBA, using isotope labelled internal standards. Method performance was assessed through calibration, detection limits and recovery tests.

Methodology and Instrumentation

Sample Preparation

• Heat formulation or spiked sample at 70°C for 30 minutes
• Centrifuge at 12000 rpm for 5 minutes at 5°C
• Collect supernatant for direct LC-MS/MS injection

LC-MS/MS Conditions

• Instrument: Nexera X3 UHPLC coupled to LCMS-8060NX triple quadrupole
• Ionization: APCI IonFocus source with patented lens system
• Column: C8 150 mm × 4.6 mm, 5 μm particle size
• Mobile phases: 0.1 percent formic acid in water (A) and methanol (B)
• Gradient: 40 percent B to 100 percent B over 12 minutes, total run time 20 minutes
• Flow rate: 0.5 mL per minute; column temperature 40°C; injection volume 30 μL
• Gas flows: nebulizing and drying gas at 3 L per minute; interface 270°C, desolvation line 220°C, heater block 220°C
• MRM transitions optimized for each analyte and internal standard

Main Results and Discussion

The method demonstrated linear responses over 50 to 2000 ppb with correlation coefficients above 0.996. The limit of quantitation was established at 50 ppb, with signal-to-noise ratios exceeding regulatory requirements. Repeatability expressed by relative standard deviation was below 20 percent for all analytes, and recoveries ranged from 70 to 130 percent in spiked samples. Analysis of six commercial mAb formulations showed most target nitrosamines below the quantifiable limit, indicating low contamination levels under current manufacturing conditions.

Benefits and Practical Applications of the Method

• High sensitivity and specificity through optimized MRM and APCI source design
• Streamlined sample preparation without complex cleanup steps
• Broad dynamic range suitable for routine quality control of biologics
• Robust instrument performance with low matrix effects and high throughput

Future Trends and Applications

Ongoing advancements in ion source technology and separation media may further improve sensitivity and reduce analysis time. Extending this approach to other biologic matrices and integrating automated sample handling can enhance routine monitoring of nitrosamine impurities. Emerging regulatory guidelines are expected to expand testing requirements to novel biotherapeutics and delivery systems.

Conclusion

A validated LC-MS/MS method was established for accurate quantification of seven N nitrosamines in monoclonal antibody formulations. The protocol meets regulatory criteria for precision, accuracy and detection limits, providing a valuable tool for ensuring the safety of biopharmaceutical products.

References

• WHO Information Note Update on Nitrosamine impurities November 2019
• EMA Questions and answers on Article 5 3 referral on nitrosamine impurities March 2023