Characterization of Biotherapeutics: ACQUITY UPLC H-Class Bio with 2D Part 2 of 3: Rendering a Viable Interface for IEX with ESI-MS Analysis

Significance of the Topic

Charge heterogeneity such as C-terminal lysine variants influences monoclonal antibody quality and is sensitive to manufacturing.
Combining ion exchange chromatography with mass spectrometry provides in-depth variant identification but is challenged by nonvolatile salts.
Automated and on-line desalting using two-dimensional UPLC enhances productivity and data quality in biotherapeutic characterization.

Objectives and Study Overview

Demonstrate the viability of the ACQUITY UPLC H-Class Bio System with 2D Technology to interface ion exchange chromatography (IEX) with electrospray ionization mass spectrometry (ESI-MS).
Use the therapeutic monoclonal antibody infliximab as a model to evaluate heart-cut fractionation, on-line desalting, and MS analysis of charge variants.

Methodology and Used Instrumentation

• 2D UPLC system: ACQUITY UPLC H-Class Bio with 2D Technology and Auto•Blend Plus for reproducible IEX buffer blending
• Columns: Protein-Pak Hi Res SP SCX (7 µm, 4.6×100 mm) and ACQUITY UPLC BEH C4 (1.7 µm, 2.1×50 mm)
• Detectors: ACQUITY UPLC TUV (1st dimension) and PDA (2nd dimension)
• Mass spectrometer: Xevo G2 QTof with ESI source (capillary 3 kV, cone 45 V, source 150 °C, desolvation 500 °C)
• LC conditions: IEX heart-cut with MES buffer gradient (25–65 mM NaCl, pH 6.5) at 0.5 mL/min; RP gradient (5–85% acetonitrile, 0.1% formic acid) at 0.25–0.5 mL/min
• Software: MassLynx v4.1 for data acquisition and processing

Main Results and Discussion

The heart-cut mechanism directed individual IEX peaks into the second dimension for on-line desalting and subsequent RP-MS analysis.
Deconvoluted masses of infliximab variants were determined as 148 511 Da (Lys0), 148 640 Da (Lys+1), and 148 767 Da (Lys+2), confirming the addition of one and two lysine residues (~128 Da each) and consistent glycosylation profiles across variants.
The two-valve configuration allowed independent control of flow paths for efficient salt removal and seamless elution to MS.

Benefits and Practical Applications

• Enhanced productivity through automated heart-cut fractionation and on-line desalting
• Improved reproducibility and reduced manual buffer preparation using Auto•Blend Plus
• Orthogonal separation and detection on-line for comprehensive charge variant profiling
• Applicability to high-throughput QA/QC of therapeutic proteins during development and manufacturing

Future Trends and Potential Applications

Expansion of 2D UPLC/MS workflows for broader biotherapeutic formats such as ADCs and fusion proteins.
Integration with advanced MS technologies for increased sensitivity and resolution.
Greater automation and data integration with laboratory information management systems for real-time process monitoring and control.

Conclusion

The ACQUITY UPLC H-Class Bio System with 2D Technology delivers a robust, automated platform for interfacing IEX with ESI-MS, enabling efficient, high-throughput characterization of monoclonal antibody charge variants without offline desalting.

References

1. Cheng et al. Biotechnology and Bioengineering 2008, 100(6): 1132–1143.
2. Brorson and Jia. Current Opinion in Biotechnology 2014, 30: 140–146.
3. Nechansky et al. Journal of Chromatography B 2007, 852(1–2): 250–256.
4. Barnes and Lim. Mass Spectrometry Reviews 2007, 26(3): 370–388.
5. Annesley. Clinical Chemistry 2003, 49(7): 1041–1044.