Evaluating 2D-RP/RP Fractionation Capabilities of the ACQUITY UPLC M-Class System with 300-μm I.D. Configuration

Importance of the topic

The detection and quantification of low-abundance host cell proteins (HCPs) in biotherapeutics is critical for product safety and regulatory compliance. Extending chromatographic dynamic range through orthogonal two-dimensional reversed-phase separation enhances resolution and sensitivity for trace protein analysis.

Study objectives and overview

This work evaluates the fractionation performance of the ACQUITY UPLC M-Class System with 2D Technology in a 300-µm internal diameter configuration. Two peptide mixtures—a four-protein tryptic digest (MIX-1) and an enolase digest (ENL)—were separated using a five-step high-pH/low-pH RP/RP scheme. The goal was to assess peak capacity, fractionation efficiency and applicability to HCP quantitation.

Methodology and instrumentation

A first-dimension RP column operating at pH 10 (XBridge Peptide BEH C18, 1.0 × 50 mm) performed step-gradient elution at five discrete acetonitrile concentrations. Eluted fractions were trapped on an on-line Symmetry C18 guard column (300 µm × 25 mm), diluted and transferred to a second-dimension HSS T3 analytical column (300 µm × 150 mm) at pH 2.5 for high-resolution separation. Fraction transfer and dilution were automated within the UPLC fluidic setup.

Used instrumentation

• ACQUITY UPLC M-Class System with 2D Technology (300-µm ID)
• XBridge Peptide BEH C18 first-dimension column (300 Å, 5 µm, 1.0 × 50 mm)
• Symmetry C18 trap column (5 µm, 300 µm × 25 mm)
• HSS T3 analytical column (100 Å, 1.8 µm, 300 µm × 150 mm)
• SYNAPT G2-S HDMS or Xevo G2-XS QTof mass spectrometer with ESI+ and MS
• MassLynx Software v4.1 for data management

Main results and discussion

Base peak chromatograms and extracted ion traces demonstrated clear separation of 11 MIX-1 peptides across five fractions and 12 ENL peptides similarly distributed. Peak widths at 10 % height averaged 12 s in the second dimension, corresponding to a 2D peak capacity of approximately 1,000. This performance underlines the system’s ability to resolve complex peptide mixtures and detect low-level analytes.

Benefits and practical applications

• High-resolution fractionation improves detection of trace HCPs in therapeutic proteins.
• Orthogonal pH-based RP/RP separation extends dynamic range and peak capacity.
• On-line trapping and dilution reduce manual handling and sample loss.
• Compatible with high-resolution QTof instruments for confident peptide identification and quantitation.

Future trends and potential applications

Advances may include further miniaturization to increase sensitivity, integration with ion mobility separation for additional resolution, and application to broader biopharmaceutical and proteomics targets. Automated method development and real-time data interpretation using AI could streamline HCP monitoring in production environments.

Conclusion

The ACQUITY UPLC M-Class System with 2D Technology in a 300-µm ID format delivers robust two-dimensional RP/RP performance with high peak capacity and efficient fractionation. It is well suited for the sensitive analysis of low-abundance host cell proteins and other complex peptide samples.

References

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