Evaluation of Modern and Legacy HPLC Systems for the Size-Exclusion Analysis of Aggregation and Fragmentation in a Monoclonal Antibody Drug

Importance of the Topic

The reliable characterization of monoclonal antibody (mAb) drugs is essential for ensuring their safety and efficacy. Size-exclusion chromatography (SEC) is the primary tool to monitor critical quality attributes such as aggregation and fragmentation. However, conventional HPLC systems introduce extra-column dispersion and non-specific adsorption, leading to peak broadening and tailing that can obscure low-level impurities. Advances in bio-compatible instrumentation aim to address these challenges and improve the accuracy of mAb purity assessments.

Study Objectives and Overview

This work compares three high-performance liquid chromatography systems for SEC analysis of a therapeutic mAb (infliximab):

• Alliance iS Bio HPLC System (modern, bio-compatible, MaxPeak HPS technology)
• Alliance e2695 HPLC System (legacy model)
• Competitive Bio-Inert HPLC System (System Y)

The study evaluates system dispersion and chromatographic performance using a protein standard mix and infliximab samples to assess aggregation (high-molecular weight species) and fragmentation (low-molecular weight species).

Methodology and Instrumentation

Sample Preparation:

• BEH™ 200 SEC Protein Standard Mix, reconstituted in mobile phase and filtered.
• Remicade™ (infliximab) diluted to 10 mg/mL, frozen aliquots analyzed past expiry.

Instrumentation Section:

• Alliance iS Bio HPLC System with MaxPeak High-Performance Surfaces Technology (bio-inert flow path)
• Alliance e2695 HPLC System
• Competitive Bio-Inert HPLC System (System Y)
• UV detection at 280 nm (10 points/s)
• XBridge™ Premier Protein SEC column (4.6 × 300 mm, 250 Å, 2.5 µm)
• Mobile phase: phosphate buffer with KCl at pH 6.2, isocratic flow at 0.3 mL/min

Data Processing:

• Empower™ CDS software versions 3.8.0.1 (iS Bio) and 3.7 (others)

Main Results and Discussion

System Dispersion:

• Alliance iS Bio exhibited the lowest extra-column dispersion (4σ), followed by System Y and the Alliance e2695.
• Lower dispersion correlates with sharper peaks and reduced band broadening.

Peak Tailing and Resolution:

• Protein standards showed significantly less USP tailing on the iS Bio system versus legacy systems.
• Monomer peaks on the Alliance e2695 and System Y displayed broader shapes and higher tailing due to both dispersion and surface interactions.

Monoclonal Antibody Analysis:

• High-molecular weight species (HMWS) were similarly quantified across systems, but better peak/valley separation was seen on iS Bio.
• Low-molecular weight species (LMWS) (~0.2% relative area) were clearly resolved only on the Alliance iS Bio System.
• Legacy systems either buried the LMWS peaks within monomer tailing (e2695) or overestimated their abundance (System Y).

The improved performance of the iS Bio system is attributed to its reduced dispersion and MaxPeak HPS modification, which minimizes non-specific adsorption of charged protein patches.

Benefits and Practical Applications

Key advantages of the Alliance iS Bio HPLC System include:

• Enhanced biocompatibility with non-ferrous, inert materials throughout the flow path
• Reduced extra-column dispersion for sharper peaks
• MaxPeak HPS surfaces to prevent off-target analyte interactions and peak tailing

These features lead to more accurate quantitation of mAb aggregates and fragments, supporting robust quality control and regulatory compliance in biopharmaceutical workflows.

Future Trends and Potential Applications

Advances in SEC instrumentation will likely focus on further minimizing dispersion and enhancing surface chemistries to handle increasingly complex biologics. Integration with multidimensional separations, high-throughput automation, and inline detection methods (e.g., multi-angle light scattering, mass spectrometry) may expand analytical capabilities. Ongoing development of software algorithms for peak deconvolution and impurity profiling will also improve data fidelity.

Conclusion

The Alliance iS Bio HPLC System outperforms legacy and competitive bio-inert systems for SEC analysis of mAb aggregation and fragmentation. Its low dispersion and MaxPeak HPS-enabled inert flow path deliver superior peak shapes, enabling reliable detection of trace impurities. This makes it a valuable tool for biopharmaceutical characterization and quality control.

Reference

1. Vázquez-Rey, M.; Lang, D. A. Aggregates in Monoclonal Antibody Manufacturing Processes. Biotechnol. Bioeng. 108(7), 1494–1508 (2011).
2. Vlasak, J.; Ionescu, R. Fragmentation of Monoclonal Antibodies. mAbs 3(3), 253–263 (2011).
3. Koza, S. M.; Reed, C.; Chen, W. Impact of LC System Dispersion on SEC Analysis of Proteins. Waters Application Note (2019).
4. Koza, S. M.; Reed, C. E.; Chen, W. Impact of LC System Dispersion on SEC Analysis of mAb Aggregates. Waters Application Note 720006336 (2019).