Agilent BioHPLC Columns - Characterization of NIST Monoclonal Antibody Critical Quality Attributes - Application Compendium

Significance of the Topic

Biotherapeutic monoclonal antibodies (mAbs) exhibit complex physicochemical heterogeneity that directly impacts safety, efficacy, and regulatory compliance. Reliable characterization of critical quality attributes (CQAs) such as aggregation, charge variants, intact mass, subunit composition, glycosylation, and peptide‐level modifications is essential during development, manufacturing, and quality control of mAb therapeutics.

Objectives and Overview of the Study

This compendium from Agilent Technologies, built around the NISTmAb Reference Material (8671), presents a suite of optimized liquid chromatography solutions for comprehensive mAb CQA monitoring. It collates applications, quick-start guides, SOPs, and application notes covering Size Exclusion Chromatography (SEC), Ion Exchange Chromatography (IEX), Reversed-Phase Chromatography for intact/subunit analysis, hydrophobic interaction chromatography, peptide mapping, and glycan analysis.

Methodology and Instrumentation

Agilent offers dedicated column chemistries and LC/MS platforms optimized for each CQA:

• SEC: AdvanceBio SEC columns (1.9–2.7 µm, 120–200 Å) on the 1260 Infinity II Bio-inert LC system with UV/DAD detection; buffer-advisor software for gradient screening.
• IEX: Bio MAb (PEEK, nonporous, 5 µm) and Bio IEX columns on the 1260 Infinity II Bio-inert LC with quaternary pump, enabling pH- or salt-gradient separation of charge variants.
• Intact/Subunit Mass: AdvanceBio RP-mAb, ZORBAX RRHD and PLRP-S columns coupled to the 6545XT AdvanceBio LC/Q-TOF; high‐temperature, formic acid mobile phases, and rapid 4–8 min gradients.
• Automation: AssayMAP Bravo for affinity capture, on-cartridge PNGase F deglycosylation, IdeS digestion, and subunit preparation prior to LC/Q-TOF analysis.

Main Results and Discussion

SEC method development using 1.9 µm AdvanceBio SEC 200 Å columns and Buffer Advisor delivered symmetrical monomer peaks, baseline resolution of aggregates, and < 3 % high molecular weight species at optimal pH 7.4 and 150 mM phosphate. IEX using Bio MAb columns with pH-gradient elution achieved high-resolution separation of acidic, main, and basic variants with %RSD < 1.6 % (area) over multiple runs. Reversed-phase LC/MS on the 6545XT LC/Q-TOF enabled intact mAb mass measurement with single-ppm accuracy across major glycoforms, rapid ADC drug-to-antibody ratio (DAR) determination, and mirror-plot comparison of innovator versus biosimilar samples. AssayMAP Bravo automation streamlined affinity purification, on-cartridge PNGase F and IdeS treatments, and subunit generation with high reproducibility.

Benefits and Practical Applications

• Comprehensive CQA coverage—from aggregates to glyco- and charge variants—using a single vendor platform for method consistency and streamlined training.
• Significantly reduced method development time with quaternary pumps, buffer-advisor software, and rapid screening capabilities.
• High throughput: sub-5 min gradients for intact mass, < 30 min SEC and IEX analyses support large sample sets for process development and QC.
• Automation at the sample prep level (AssayMAP) minimizes manual handling, lowers variability, and increases walk-away time.

Future Trends and Potential Uses

Emerging applications include native SEC-MS for direct aggregate identification, multi-dimensional LC workflows combining IEX and RP for deeper glyco- and charge variant profiling, and integration with high-resolution MS/MS for peptide-level mapping. Advances in superficially porous particles and bio-inert system components will continue to elevate sensitivity, speed, and method robustness for next-generation biotherapeutic characterization.

Conclusion

Agilent’s comprehensive LC and LC/MS solutions, validated on the NISTmAb standard, provide robust, high-resolution workflows for in-depth monoclonal antibody CQA monitoring. From SEC and IEX to intact/subunit mass analysis and automated sample prep, these integrated platforms deliver accuracy, throughput, and reproducibility needed to support biotherapeutic development, QC, and biosimilar comparability studies.

References

• Schiel JE et al. State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization. ACS Publications, Volumes 1–3, 2014-2015.
• Turner A et al. Anal. Bioanal. Chem. 410(8):2079–2095 (2018).
• Mouchahoir T and Schiel JE. Anal. Bioanal. Chem. 410(8):2111–2126 (2018).
• Qin V. Size Exclusion Chromatography Method Development of NISTmAb Using AdvanceBio SEC 200 Å 1.9 µm Column (Agilent Application Note 5994-0876EN).
• He Y et al. J. Sep. Sci. 34:548–555 (2011).