Characterization of Biotherapeutics: ACQUITY UPLC H-Class Bio with 2D Part 1 of 3: On-line Desalting of Biotherapeutic Samples for Increased Productivity

Significance of the Topic

Characterizing charge variants and removing interfering buffer components are critical steps in the analysis of biotherapeutic proteins. Online desalting and automated fractionation accelerate workflows, improve reproducibility, and enable seamless coupling to mass spectrometry or other downstream assays.

Objectives and Overview

• Demonstrate automated on-line fractionation and desalting of biotherapeutic samples using 2D UPLC.
• Enable efficient heart-cut transfers from ion exchange to reversed-phase trapping columns.
• Lay groundwork for coupling ion exchange separations to mass spectrometry in subsequent parts.

Methodology and Instrumentation

The study employed the ACQUITY UPLC H-Class Bio System with 2D Technology, featuring synchronized quaternary and binary solvent managers and dual valves for heart-cut operation. A strong cation exchange Protein-Pak Hi Res SP column (4.6×100 mm, 7 µm) served as the first dimension, and a BEH C4 trapping column (2.1×50 mm, 1.7 µm, 300 Å) as the second dimension.

• Pump configuration: IEX gradient (25–65 mM NaCl in 20 mM MES, pH 6.5) at 0.5 mL/min; heart-cut flow to RPLC at 0.25 mL/min.
• Column temperatures: 25 °C for IEX, 80 °C for reversed-phase.
• Detection: UV at 280 nm (TUV first dimension, PDA second dimension).
• Software: MassLynx v4.1 for data acquisition and control.

Main Results and Discussion

Heart-cut events were programmed in the UPLC Column Manager to capture the central portion of selected charge variant peaks. The diverted fraction was retained on the second-dimension C4 column, where on-line desalting removed salts under aqueous conditions. Subsequent organic gradient elution yielded desalted peptide or protein fractions compatible with downstream MS or enzymatic digestion. The process demonstrated reproducible trapping, desalting, and elution of the +0 lysine variant of infliximab.

Benefits and Practical Applications

• Automated fractionation reduces manual collection and offline processing.
• Online desalting streamlines buffer exchange, saving time and consumables.
• Compatibility with MS and peptide mapping workflows improves identification and characterization of charge variants.

Future Trends and Opportunities

Integration of multidimensional chromatographic strategies will expand to real-time monitoring of product quality attributes. Further automation and miniaturization may enable parallel processing of multiple targets. Coupling with high-resolution mass spectrometry and advanced data analytics will enhance variant identification and quantitation in complex biologics batches.

Conclusion

The ACQUITY UPLC H-Class Bio System with 2D Technology and heart-cut capability offers a robust, automated platform for on-line fractionation and desalting of biotherapeutic samples. This approach increases productivity, reduces manual handling, and prepares samples for downstream MS or peptide analysis without additional offline steps.

References

1. Liu Y, Beck A, Wakankar A, et al. Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. mAbs. 2012;4(5):578–585.
2. Rea JC, Hadjidj S, Pinto JF, et al. Validation of a pH gradient-based ion-exchange chromatography method for high-resolution monoclonal antibody charge variant separations. J Pharm Biomed Anal. 2011;54:317–323.
3. Annesley TM. Ion Suppression in Mass Spectrometry: Clinical Chemistry. 2003;49(7):1041–1044.