Impact of Instrument and Detector Characteristics on Size Exclusion Chromatography Method Transfer Across Biocompatible UHPLC Systems

Significance of the Topic

An accurate size-exclusion chromatography (SEC) method is critical for monitoring protein aggregation and degradation in monoclonal antibody therapeutics. Aggregates and low molecular weight (LMW) fragments can affect safety and efficacy, making reproducible quantification essential across different biocompatible UHPLC systems.

Objectives and Study Overview

This study evaluates the transferability of an SEC method for trastuzumab across two biocompatible UHPLC platforms and compares detector performance. Key aims include:

• Assessing chromatographic consistency between ACQUITY Arc Bio System and Agilent 1260 Infinity Bio-inert System
• Comparing repeatability of retention times and peak areas
• Evaluating sensitivity and precision of photodiode array (PDA) versus tunable UV (TUV) detectors

Methodology

Trastuzumab was diluted to 2.1 mg/mL in a 25 mM sodium phosphate, 400 mM sodium chloride buffer (pH 6.7). A 10 µL injection was performed at 0.7 mL/min using an XBridge Protein BEH SEC column (7.8×300 mm, 3.5 µm, 200 Å) under ambient conditions. Data were acquired in Empower 3, converting Agilent OpenLab data as needed.

Used Instrumentation

• ACQUITY Arc Bio System: QSM-R Bio pump, FTN-R Bio injector, Waters 2998 PDA and 2489 TUV detectors
• Agilent 1260 Infinity Bio-inert System: Bio Quat pump, HiP Bio autosampler, 1260 DAD VL+ detector
• Column: XBridge Protein BEH SEC, 200 Å, 3.5 µm, 7.8×300 mm
• Software: Empower 3 CDS, Agilent OpenLab CDS Chemstation

Main Results and Discussion

Retention times for high molecular weight (HMW), monomer, and LMW peaks differed by approximately 0.1 min across systems, with RSDs below 0.2%. Peak area percentages were comparable (HMW ~0.40–0.42%, monomer ~99.53–99.55%, LMW ~0.05%). The ACQUITY Arc Bio System showed slightly lower RSDs for both retention times and peak areas.

Comparing detectors on the ACQUITY Arc Bio System, TUV detection yielded improved precision (HMW RSD 0.59% vs 0.79% PDA; LMW RSD 1.74% vs 4.85% PDA) and higher signal-to-noise ratios (HMW s/n 205 vs 105; LMW s/n 50 vs 24).

Benefits and Practical Applications

• Reliable method transfer ensures consistent aggregate quantification across UHPLC platforms
• Biocompatible system compatibility minimizes sample corrosion and oxidation
• TUV detectors enhance sensitivity for low-abundance degradants

Conclusion

The SEC method for trastuzumab transfers effectively between biocompatible UHPLC systems with minimal retention time shifts and robust repeatability. Detector choice further improves method performance, with TUV offering superior sensitivity and precision compared to PDA.

Future Trends and Applications

Advancements may include integration of multi-angle light scattering or mass spectrometry for detailed aggregate characterization, increased automation for high-throughput analyses, and data-driven method optimization using artificial intelligence.

References

• Rosenberg AS. Immunologic effects of protein aggregates. AAPS Journal. 2006;8:501–507.
• Xiang T et al. Structural impact of hinge region peptide hydrolysis on mAb. J Chromatogr B. 2007;858:254–262.
• Koza S et al. SEC method development for trastuzumab degradation. Application Note. 2012;720004416EN.
• Waters Operator’s Guides for 2489 TUV and 2998 PDA detectors.