PLRP-S Polymeric Reversed-Phase Column for LC/MS Separation of mAbs and ADC

Significance of the Topic

The analysis of monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) is critical for ensuring the safety, efficacy, and quality of modern biotherapeutics. High-resolution LC/MS characterization at both intact and fragment levels provides detailed insight into molecular heterogeneity, glycoforms, drug-load distributions, and post-translational modifications. Traditional silica-based reversed-phase columns often struggle under formic acid conditions, resulting in compromised chromatographic performance and MS sensitivity. A polymeric reversed-phase approach addresses these limitations and supports routine, robust LC/MS workflows.

Objectives and Study Overview

This study evaluated the performance of the Agilent PLRP-S polymeric reversed-phase column for the characterization of mAbs and ADCs. Key aims included:

• Assessing intact separation quality under formic acid mobile phases
• Resolving major glycoforms and drug-load species in ADC samples
• Demonstrating fragment-level separations following reduction, IdeS digestion, and papain digestion
• Comparing chromatographic metrics such as peak width, resolution, and signal-to-noise ratios

Methodology and Used Instrumentation

A polymeric PLRP-S column (2.1 × 50 mm, 5 µm, 1000 Å) was employed to achieve stable, high-resolution separations across a wide pH range without silanol activity. Sample preparation included dilution of intact mAbs/ADC in 0.1% formic acid/ACN, chemical reduction with DTT, and enzymatic digestion (IdeS and papain). Key LC and MS conditions:

• Mobile phases: 0.1% formic acid in water (A) and acetonitrile (B)
• Gradient protocols tailored for intact (20–90% B over 11 min) and fragment (20–85% B over 11 min) analyses
• Column temperature: 80 °C; flow rate: 0.6 mL/min; injection: 1 µL
• Mass spectrometry: Agilent 6530 Q-TOF in positive mode, mass range 600–6000 m/z

Main Results and Discussion

Intact mAb/ADC analysis delivered narrow total ion chromatogram peaks (FWHM ≤ 0.1 min for mAbs, 0.25 min for ADC) using formic acid–based mobile phases. Deconvoluted spectra revealed:

• Five major glycoforms for mAb1 and four for mAb2, each clearly resolved
• Stepwise drug-load distribution (D0–D8) for lysine-conjugated ADC

Fragment-level separations after reduction, IdeS, and papain digestion showed well-resolved heavy chain, light chain, Fc, F(ab′)2, ScFc, and multi-drug fragments. The polymeric column maintained peak shape and MS sensitivity without TFA, overcoming traditional column limitations.

Benefits and Practical Applications

The PLRP-S polymeric reversed-phase column offers:

• Strong mechanical and chemical stability across broad pH
• Excellent compatibility with formic acid for MS-friendly analyses
• High reproducibility and minimal silanol interference
• Enhanced resolution for intact heterogeneity and fragment profiling
• Suitability for routine QC, biopharma research, and drug-development labs

Future Trends and Opportunities for Use

Emerging directions include column optimization for higher throughput, integration with automation platforms, adaptation to novel biotherapeutic formats (bispecifics, fusion proteins), and coupling to high-resolution MS/MS for deeper structural characterization. Advances in polymer chemistry may further improve selectivity, while miniaturized formats could reduce sample and solvent consumption.

Conclusion

The Agilent PLRP-S polymeric reversed-phase column demonstrates superior chromatographic performance for intact and fragment analysis of mAbs and ADCs under formic acid conditions. It delivers sharp peaks, high MS sensitivity, and accurate mass determination, making it a robust choice for biotherapeutic characterization.

References

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