A Single Analytical Platform for Glycan Analysis, Charge Heterogeneity, and Purity Determination of the NISTmAb

Importance of the Topic

Monoclonal antibodies (mAbs) are essential biologic therapeutics but their safety, stability, and efficacy can be compromised by structural heterogeneity and impurities. Comprehensive characterization of glycan microheterogeneity, charge variants, and product purity is critical in manufacturing, formulation, and quality control.

Goals and Overview of the Study

This whitepaper demonstrates the use of a single analytical platform—the SCIEX PA 800 Plus Pharmaceutical Analysis System—to assess N-glycan profiles, charge heterogeneity, and purity of the NISTmAb reference material (RM 8671). The aim was to streamline workflows, reduce sample preparation time, and achieve rapid, high-resolution results comparable to traditional multi-instrument approaches.

Used Instrumentation

• SCIEX PA 800 Plus Pharmaceutical Analysis System
• SCIEX EZ-CE cartridge
• SCIEX Fast Glycan Technology

Methodology

- Glycan analysis: N-linked glycans were released from NISTmAb using on-bead PNGase F digestion at 60 °C, labeled with aminopyrene trisulfonate (APTS), purified, and separated by CE-LIF on the PA 800 Plus with the EZ-CE cartridge in under 5 minutes. Glycan species were assigned automated GU values and confirmed via exoglycosidase sequencing.
- Charge variant profiling: Capillary zone electrophoresis (CZE) was performed on the EZ-CE cartridge (20 cm effective length) at 12 kV and 20 °C to resolve basic, main, and acidic charge variants in a rapid assay.
- Purity determination: Reduced and non-reduced assays were executed on the EZ-CE cartridge to quantify glycan occupancy, monomeric purity, and detect fragment impurities. Run times were 12 minutes for the reduced assay and 18 minutes for the non-reduced assay, with relative abundances calculated from corrected peak areas.

Main Results and Discussion

• Glycan profile: Twenty-six distinct N-glycan species were separated and identified in under 5 minutes; the most abundant were G0F (41.5%), G1F (26.9%), G1′F (8.9%), and G2F (6.1%).
• Charge variants: CZE resolved basic variants (11.7%), the main peak (73.0%), and acidic variants (15.3%), consistent with NIST reference data.
• Purity: Glycan occupancy was 99.4% and monomeric purity reached 94.5%, with low inter-assay variability (RSD < 0.5% for peak area and migration time).
• All assays showed high robustness and repeatability, matching or improving upon published NIST results.

Benefits and Practical Applications

• Consolidation of glycan, charge, and purity analyses on a single CE platform reduces instrument footprint and resource requirements.
• Fast Glycan Technology and the EZ-CE cartridge simplify sample preparation and accelerate throughput.
• High resolution and reproducibility support biopharmaceutical process development, quality control, and regulatory submissions.

Future Trends and Potential Applications

Advancements may include further automation of sample handling, integration with mass spectrometry for orthogonal confirmation, expansion to other biotherapeutics, and implementation of real-time monitoring during production. Enhanced data analytics and AI-driven peak assignment could further streamline workflows.

Conclusion

The SCIEX PA 800 Plus system combined with Fast Glycan Technology and the EZ-CE cartridge provides a robust, rapid, and comprehensive solution for mAb characterization. It delivers performance on par with traditional multi-instrument methods while simplifying workflows and reducing analysis time.

References

1. Guttman A. et al. Fast Glycan Labeling and Analysis: High-Resolution Separation and Identification in Minutes. SCIEX Technical Note (2017).
2. Santos M. Analysis of Monoclonal Antibody Charge Variants by Capillary Zone Electrophoresis. Beckman Coulter Technical Note (2012).
3. IgG Purity/Heterogeneity. SCIEX PA 800 Plus Pharmaceutical Analysis System Application Guide.
4. Gallegos-Perez J. IgG Purity/Heterogeneity and SDS-MW Assays with High Speed Separation Method. SCIEX Technical Note (2015).
5. Guttman A. et al. Fast Glycan Sequencing Using a Fully Automated Carbohydrate Sequencer. SCIEX Technical Note (2017).
6. National Institute of Standards and Technology. Report of Investigation: Reference Material 8671 NISTmAb. (2016).
7. Michaels DA et al. Separation Methods and Orthogonal Techniques. In Schiel JE et al., Biopharmaceutical Characterization: The NISTmAb Case Study. ACS (2015).