Complementary Methodologies for Analysis of NISTmAb Impurities

Significance of the Topic

Size heterogeneity in monoclonal antibodies is a critical quality attribute affecting both the safety and therapeutic efficacy of biopharmaceuticals. Aggregation and fragmentation can alter pharmacokinetics, immunogenicity, and stability. Regulatory guidelines mandate comprehensive characterization of high molecular weight (HMW) aggregates and low molecular weight (LMW) fragments to ensure product quality and compliance.

Objectives and Study Overview

This study compares Size Exclusion Chromatography (SEC) and nonreducing Capillary Electrophoresis–Sodium Dodecyl Sulfate (CE-SDS) as complementary approaches for evaluating monomeric purity of the NISTmAb reference standard. The goal is to highlight each technique’s strengths in resolving HMW and LMW impurities and to benchmark results against NIST reference values.

Methodology and Instrumentation

The NISTmAb (RM 8671, Lot 14HB-D-002) was prepared at 2 mg/mL in phosphate-buffered saline under reducing and nonreducing conditions. SEC analyses employed an Agilent 1290 Infinity II Bio LC system fitted with a 1.9 µm AdvanceBio SEC column (2.1 × 150 mm), using 150 mM phosphate buffer (pH 7.0) at 0.1 mL/min, 30 °C column temperature, and UV detection at 280 nm. CE-SDS separations were performed on an Agilent ProteoAnalyzer system with the Protein Broad Range P240 kit. Samples were denatured with SDS and DTT, labeled by heat incubation, and separated under nonreducing conditions to resolve individual LMW fragments. Reduced CE-SDS runs provided quantitation of heavy and light chain species.

Main Results and Discussion

SEC clearly resolved HMW species into dimer and trimer peaks, the monomer peak, and a single coeluting LMW peak. Monomeric purity by SEC was 97.83% (NIST reference 96.63%), with HMW at 2.05% and LMW at 0.12%. In contrast, nonreducing CE-SDS separated distinct LMW peaks (light chain, heavy chain, Fab, HC:LC, HC:HC, HC:HC:LC) and yielded 98.18% monomer purity (NIST reference 98.47%) and 1.82% total LMW fragments. The marked difference in LMW quantitation reflects SEC’s limited resolution of small fragments versus CE-SDS’s denaturing separation power. HMW quantitation by CE-SDS is not applicable, as SDS disrupts noncovalent aggregates.

Benefits and Practical Applications

• SEC offers reliable quantitation of HMW aggregates and overall size distribution under native conditions.
• CE-SDS provides high-resolution separation and precise quantitation of individual LMW fragments.
• Combined application of SEC and CE-SDS delivers a comprehensive profile of antibody size heterogeneity, aligning with regulatory requirements for critical quality attributes.

Future Trends and Opportunities

Next-generation UHPLC columns, microfluidic capillaries, and multi-attribute mass spectrometry will enhance resolution, throughput, and structural insight. Automated sample handling, integrated data analytics, and real-time monitoring tools are expected to streamline workflows. Emerging native mass spectrometry and multi-detector platforms may enable deeper characterization and in-process control.

Conclusion

SEC and CE-SDS are orthogonal, complementary methods for monoclonal antibody size heterogeneity analysis. SEC excels at quantifying native HMW species, while CE-SDS accurately resolves LMW degradation products. Their combined use establishes a robust, regulatory-compliant workflow for comprehensive mAb characterization.

References

• Turner A.; Yandrofski K.; Telikepalli S.; King J.; Heckert A.; Filliben J.; Ripple D.; Schiel J.E. Anal. Bioanal. Chem. 2018, 410, 2095–2110.
• United States Pharmacopeia General Chapter <129> Analytical Procedures for Recombinant Therapeutic Monoclonal Antibodies; USP-NF, 2024.
• Michels D.; Parker M.; Salas-Solano O. Electrophoresis 2012, 33, 815–826.
• Agilent Application Note 5994-2709EN, 2020.
• Agilent ProteoAnalyzer Brochure 5994-6716EN, 2023.
• NIST Reference Material 8671: NISTmAb, Lot 14HB-D-002; Certificate, 2023.
• Agilent Technical Overview 5994-6960EN, 2023.
• Agilent OpenLab Software Version 2.5 Manual.