ADVANCING ANALYTICAL APPROACHES FOR ROBUST EVALUATION OF LARGE MOLECULE BIOLOGICS USING LIQUID CHROMATOGRAPHY

Significance of the Topic

Large-molecule biologics such as monoclonal antibodies demand precise characterization to ensure safety and efficacy. Size-exclusion chromatography (SEC) and ion-exchange chromatography (IEX) are key techniques for evaluating aggregation, fragmentation and charge variants. Robust analytical platforms reduce variability and support regulatory compliance in biopharmaceutical development and quality control.

Objectives and Study Overview

This study benchmarks the Alliance iS Bio HPLC System against a legacy Alliance e2695 and a competitive bio HPLC system using infliximab (Remicade) as a model monoclonal antibody. The SEC evaluation focuses on monomer, high-molecular weight species (HMWS) and low-molecular weight species (LMWS). The IEX assessment investigates system performance over 30 days in a high-salt gradient to monitor charge variant stability.

Methodology and Instrumentation

• SEC Analysis: Three systems (Alliance iS Bio, Alliance e2695, Competitive Bio) with XBridge Premier Protein SEC column (4.6×300 mm, 250 Å, 2.5 µm); mobile phase: 60.3 mM K2HPO4, 140.3 mM KH2PO4, 249.5 mM KCl (pH 6.2); flow rate: 0.3 mL/min; ambient column temperature; 6 °C sample temperature; 10 µL injection.


• IEX Analysis: Alliance iS Bio HPLC System with BioResolve SCX mAb column (4.6×100 mm, 3 µm); mobile phases: 100 mM MES monohydrate (A), 100 mM MES sodium salt (B), 1 M NaCl (C), water (D); flow rate: 0.5 mL/min; column temperature: 30 °C; sample temperature: 6 °C; 10 µL injection; high-salt gradient over 50 min.

Main Results and Discussion

In the SEC comparison, the Alliance iS Bio System exhibited the lowest system dispersion, yielding superior peak shapes and the highest resolution between monomer and HMWS. Quantitative analysis showed HMWS at 0.47% area, monomer at 99.44% and LMWS at 0.09%. The e2695 and competitive systems demonstrated slightly higher dispersion and variability.

During the 30-day IEX robustness study (1,290 injections), the Alliance iS Bio System achieved relative area %RSDs below 0.4% for major charge variant peaks and retention time %RSDs under 1.5%. Acidic and basic lysine variants remained consistent, confirming long-term stability under high-salt conditions.

Benefits and Practical Applications

The reduced dispersion and enhanced resolution of the Alliance iS Bio System enable more accurate monitoring of aggregation and fragmentation in monoclonal antibodies. Its sustained performance in high-salt IEX workflows supports reliable detection of charge heterogeneity, critical for research, development and quality control in biologics manufacturing.

Future Trends and Opportunities

Advancements in chromatographic materials and microfluidic platforms may further improve resolution and throughput for large-molecule analysis. Integration of mass spectrometry offers deeper structural insights into charge and glyco-variants. Machine-learning algorithms applied to chromatographic data can streamline peak identification, deconvolution and trend analysis, driving automation in biopharmaceutical analytics.

Conclusion

The Alliance iS Bio HPLC System demonstrates superior performance for SEC and IEX evaluation of monoclonal antibodies, delivering low dispersion, precise quantitation and robust long-term stability. These attributes make it a reliable tool for comprehensive characterization of large-molecule biologics in R&D and QC laboratories.