EAS: 2D Analysis of Protein Therapeutics and Amino Acid Excipients with Combined UV and Charged Aerosol Determination

Importance of the Topic

Protein-based therapeutics are a rapidly growing class of biopharmaceuticals that require careful formulation to maintain stability, efficacy, and safety during manufacturing, storage, and administration. Aggregation, pH variations, and temperature shifts can compromise protein integrity. Amino acid excipients and surfactants are widely used to stabilize these formulations by acting as buffers, bulking agents, or antioxidants. A robust analytical method capable of simultaneously separating intact proteins and underivatized amino acid excipients addresses critical quality control challenges in formulation development and regulatory compliance.

Objectives and Study Overview

The primary objective of the study was to develop and demonstrate a two-dimensional (2D) UHPLC method for the concurrent separation and detection of therapeutic proteins and underivatized amino acid excipients. Specific goals included:

• Establishing heart-cut 2D chromatography conditions that resolve proteins on the first dimension and amino acids on the second.
• Leveraging dual detection with diode array (UV) and charged aerosol detectors (CAD) for comprehensive coverage of chromophoric and non-chromophoric species.
• Validating method precision, linearity, and applicability to a mock protein formulation containing surfactant, amino acids, ions, and protein.

Methodology and Instrumentation

A 2D LC approach employed:

1. Dimension 1: Reverse-phase separation of intact protein on a C4 column using a water–acetonitrile gradient with 0.1% TFA.
2. Heart-cut transfer (0.5–0.8 min) of early-eluting polar fraction containing amino acids.
3. Dimension 2: HILIC separation of underivatized amino acids and selected ions on a mixed-mode Acclaim Trinity P1 column using a gradient of acetonitrile, water, formic acid, and ammonium formate.

Instrumentation Used

• UHPLC system: Thermo Scientific UltiMate 3000 Dual Gradient with DGP-3600RS pump and WPS-3000TRS autosampler.
• Column oven and switching valve: TCC-3000RS with six-port column switching.
• Detectors: Diode array detector (DAD-3000RS) at 10 Hz, Charged Aerosol Detector (Veo RS CAD) at 20 Hz.
• Dimension 1 column: Thermo Scientific Accucore 150 C4, 2.6 µm, 3.0 × 50 mm.
• Dimension 2 column: Thermo Scientific Acclaim Trinity P1, 3.0 µm, 3.0 × 50 mm and 3.0 × 100 mm in series.

Main Results and Discussion

The 2D method successfully resolved protein therapeutic peaks in dimension 1 and a suite of underivatized amino acids (Pro, Met, Gly, Glu, Asp, His, Lys, Arg) plus buffer ions in dimension 2. Key findings:

• Precision: RSD for amino acids ranged from 0.45% to 2.91% (n=6) after heart-cut transfer, with slightly elevated RSD for early-eluting species.
• Linearity: CAD calibration curves for amino acids over 100–1000 ng on column yielded R² values > 0.985, demonstrating reliable quantification.
• Mock formulation test: Simultaneous analysis of bovine serum albumin, Polysorbate 80, sodium chloride, phosphate, and histidine confirmed the method’s ability to detect protein via UV and excipients via CAD in a single 2D run.

Benefits and Practical Applications

• Comprehensive detection: Combines UV and universal CAD to cover both chromophoric proteins and non-chromophoric excipients without derivatization.
• Improved formulation screening: Enables rapid assessment of excipient effects on protein stability and aggregation propensity.
• Quality control: Offers an orthogonal, high-throughput workflow for in-process monitoring and final product release testing in biopharmaceutical manufacturing.

Future Trends and Opportunities

• Integration with mass spectrometry for detailed impurity profiling alongside universaldetection.
• Automation and online data analytics using machine-learning algorithms to predict formulation stability trends.
• Extension to other biologics, including peptides, monoclonal antibodies with post-translational modifications, and complex multi-component vaccines.
• Adoption of miniaturized and high-resolution multi-dimensional LC platforms for resource-efficient process development.

Conclusion

This study demonstrates a robust 2D UHPLC approach that integrates reverse-phase protein separation with HILIC amino acid analysis using dual UV and CAD detection. The method exhibits excellent precision, linearity, and applicability to complex formulation matrices. It provides a powerful tool for biopharmaceutical formulation development and quality control by enabling simultaneous quantification of therapeutic proteins and key excipients in a single analytical workflow.

Reference

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