Fast and High Resolution Analysis of Intact and Reduced Therapeutic Monoclonal Antibodies (mAbs)

Significance of the Topic

Therapeutic monoclonal antibodies have become a cornerstone of modern biopharmaceuticals, with dozens of new candidates entering clinical trials each year. Robust analytical workflows are essential to ensure product consistency, support regulatory comparability studies, and meet quality control demands in biopharma development. High-resolution, high-throughput methods for both intact and reduced antibody forms accelerate development timelines and enhance confidence in biosimilar studies.

Objectives and Study Overview

This work presents a streamlined reverse phase HPLC approach for rapid, reproducible analysis of intact and TCEP-reduced rituximab (innovator and biosimilar). The goals were to (1) achieve baseline separation of full-length antibodies in under five minutes, (2) resolve light and heavy chains following reduction without method modifications, and (3) demonstrate precision suitable for QA/QC and comparability assessments.

Methodology and Instrumentation

All measurements were performed on an Agilent 1260 Infinity Bio-inert Quaternary LC system, featuring:

• Quaternary pump (G5611A) rated to 600 bar
• Bio-inert autosampler (G5667A)
• Thermostat (G1330B) and Thermostatted Column Compartment with click-in bio-inert heating (G1316C)
• Diode Array Detector with 60 mm high-sensitivity flow cell (G4212B option 33)
• AdvanceBio RP-mAb columns: Diphenyl and C4, 2.1 × 50 mm, 3.5 µm superficially porous particles, 450 Å pores

Chromatographic conditions for both intact and reduced analyses:

• Mobile phase A: water + 0.1 % TFA
• Mobile phase B: isopropanol/acetonitrile/water (70:20:10) + 0.09 % TFA
• Gradient: 15 % B to 60 % B over 4 min, hold 2 min post time
• Flow rate: 1.0 mL/min; column temperature: 80 °C; injection: 1 µL
• Detection wavelengths: 220 and 280 nm

Samples of intact mAb (2 mg/mL) and TCEP-reduced light/heavy chain mixtures were prepared in PBS and heated at 60 °C for 30 min.

Main Results and Discussion

Intact intact rituximab samples (innovator vs. biosimilar) were baseline-separated in approximately 4 min on both diphenyl and C4 phases. The superficially porous particles delivered narrow, symmetric peaks and high resolution. Diphenyl and C4 columns provided complementary selectivity, highlighting minor structural variations. Following reduction, light and heavy chains eluted as distinct peaks under the same gradient, also in under 4 min.

Precision studies (n = 6) yielded retention time RSDs below 1.5 % and peak area RSDs below 5 %, meeting typical QA/QC thresholds. Overlay plots confirmed high comparability between innovator and biosimilar materials.

Benefits and Practical Applications

• Rapid throughput reduces cycle time in routine QC workflows
• Bio-inert flow path eliminates metal interaction, preserving antibody integrity
• Superficially porous stationary phases offer high efficiency and short run times
• Single-method approach for intact and reduced analysis simplifies laboratory operations

Future Trends and Potential Applications

• Integration with mass spectrometry for multi-attribute monitoring
• Expansion to other antibody classes and complex biologics
• Development of novel stationary phases for enhanced selectivity
• Automation and high-throughput screening in biosimilar pipelines

Conclusion

The described bio-inert LC-UV method using Agilent AdvanceBio RP-mAb columns provides a fast, high-resolution, and reproducible solution for both intact and reduced mAb analysis. Its precision and simplicity make it ideal for comparability studies and routine QA/QC in biopharmaceutical laboratories.

Reference

• Navas N; et al. Anal Bioanal Chem. 2013;405:9351–9363.