Greener, Lower Cost Organic Mobile Phase Solvents for the LC-MS Analysis of Intact Biotherapeutic Proteins

Importance of the Topic

Reversed-phase liquid chromatography coupled with mass spectrometry (RP LC-MS) is a cornerstone technique for characterizing intact biotherapeutic proteins and their subunits. Traditional organic mobile phases rely heavily on acetonitrile, which poses environmental, safety, and cost challenges. Evaluating greener, less toxic, and lower-cost alternatives such as methanol (MeOH) and isopropanol (IPA) can advance sustainable practices in pharmaceutical analysis and reduce overall operating expenses.

Objectives and Study Overview

This application note examines the feasibility of substituting acetonitrile with IPA or MeOH in RP LC-MS analysis of monoclonal antibodies (mAbs) and their IdeS-digested subunits. Key aims include comparing chromatographic performance, mass spectral quality, sensitivity, and system pressure across mobile phase formulations containing 0.1% formic acid in water with either acetonitrile, 95% MeOH, or 95% IPA.

Methodology and Instrumentation

Analyses were performed on a BioAccord LC-MS platform comprising ACQUITY UPLC I-Class PLUS, TUV detector (280 nm), and RDa accurate mass detector, controlled by waters_connect software.

• Column: BioResolve RP mAb Polyphenyl, 2.1×50 mm, 2.7 µm, 450 Å, at 80 °C.
• Flow rate: 0.4 mL/min; gradient length: 7 min for intact mAb, 5.5 min for subunits.
• MS settings: positive full scan (400–7000 m/z), 2 Hz, customized cone and capillary voltages, desolvation temperature up to 550 °C.
• Samples: NISTmAb intact standard (0.2 µg/µL, 4 µL injection) and IdeS-digested/reduced subunit standard (0.1 µg/µL, 4 µL).

Results and Discussion

Chromatographic peak shapes and retention times were comparable across all mobile phases, with IPA and acetonitrile yielding slightly narrower total ion chromatogram (TIC) peaks than MeOH (3.6 s vs. 4.8 s at half-height). Relative TIC intensity for MeOH was ~80% of IPA or acetonitrile in intact mAb runs. Mass spectral charge envelopes and glycoform resolution were similar, though IPA and acetonitrile exhibited marginally deeper inter-glycoform valleys, indicating more effective desolvation. Deconvoluted mass accuracy remained within 15 ppm for all major glycoforms and subunits. Subunit analysis (scFc, LC, Fd) delivered equivalent separation and signal quality, with IPA demonstrating ~18% higher TIC response compared to acetonitrile.

System backpressure was notably higher with IPA (~7500 psi) due to its greater viscosity, but remained within the 18 000 psi limit at 80 °C and 0.4 mL/min.

Benefits and Practical Applications

• Reduced toxicity: IPA and MeOH are classified as Class III solvents vs. Class II for acetonitrile, lowering health and disposal risks.
• Lower cost: Commercial LC-MS grade IPA and MeOH average one-third the price of acetonitrile.
• Comparable performance: Equivalent peak shape, mass accuracy, sensitivity, and proteoform resolution for intact mAbs and subunits.

Future Trends and Potential Applications

Ongoing efforts to green analytical workflows may explore other bio-compatible solvents, mixed solvent systems, or microflow formats to balance viscosity and backpressure. Integration with high-throughput automation, microfluidics, and orthogonal separation techniques could further enhance sensitivity and throughput. Broader adoption of sustainable mobile phases can align pharmaceutical QC/QA with environmental and economic imperatives.

Conclusion

This study demonstrates that IPA, and to a lesser extent MeOH, provide a viable, greener, and cost-effective alternative to acetonitrile for routine RP LC-MS analysis of intact biotherapeutic proteins and their subunits. Equivalent analytical performance, combined with reduced toxicity and lower solvent cost, supports transitioning to alcohol-based mobile phases in biopharmaceutical laboratories.

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