Application of Charged Aerosol HPLC Detection in biopharmaceutical Analysis

Importance of the Topic

Advances in biopharmaceutical analysis demand sensitive and universal detection techniques for compounds that lack UV chromophores or are formulated in transparent excipients. Charged aerosol detection (CAD) fills this gap by offering a mass-based, near-universal response to nonvolatile analytes, enabling label-free quantification of glycans, proteins, surfactants and other critical components in drug development and quality control.

Objectives and Study Overview

This work illustrates the versatility of HPLC-CAD for biopharmaceutical applications. Key aims include:

• Demonstrating label-free detection of released N- and O-linked glycans
• Quantifying sialic acids, adjuvants and amino acids
• Measuring nonchromophoric formulation excipients such as polysorbates and PEGs
• Evaluating sensitivity and precision across diverse analyte classes

Methodology

A combination of reversed-phase and hydrophilic interaction chromatography (HILIC) separations was employed. Gradient elution with aqueous buffers and organic modifiers (acetonitrile, methanol or THF) resolved analytes ranging from small amino acids to large glycoprotein fragments. Detection was achieved using a corona-charged aerosol detector configured for constant power operation, exploiting gas-phase charging and electrometric measurement to produce signals proportional to analyte mass.

Instrumentation Used

• High-performance liquid chromatographs equipped for HILIC and RP-LC
• Thermo Scientific Charged Aerosol Detector (VCAD) operated at 50–70 °C, PF 1.0, 5 Hz–10 Hz
• Columns: GlycanPac AXR-1 and AXH-1, Hypercarb, Betasil C1, Accucore PFP and C4, Acclaim Surfactant Plus, Trinity P2
• Mobile phases: water, acetonitrile, ammonium formate (50–100 mM, pH ~4.4), formic acid or TFA modifiers
• Optional coupling to mass spectrometry and UV or fluorescence detectors

Main Results and Discussion

CAD enabled detection limits in the low nanogram range with excellent peak area precision (<2% RSD). Label-free analysis of fetuin and alpha1-acid glycoprotein confirmed efficient measurement of mono- to tetra-sialylated glycans. O-linked mucin glycans and free sialic acids were resolved under HILIC conditions. Amino acids and proteins such as BSA exhibited consistent mass-based signals. Key excipients including Tween 80, Tween 20, PEG standards (MW 300–1000) and DPPC lipids were quantified without derivatization. Orthogonal data from CAD and UV or CAD and MS provided comprehensive sample characterization.

Benefits and Practical Applications

• Universal detection of nonvolatile analytes irrespective of chromophores
• Label-free quantification of carbohydrates and excipients simplifies sample preparation
• Applicability to quality control of drug substances and formulations
• Compatibility with multiple chromatographic modes and detector hyphenation

Future Trends and Opportunities

Ongoing developments include integration of CAD with high-resolution mass spectrometry for structural elucidation, application to lipidomics and metabolomics, and incorporation into automated QC workflows. Emerging high-throughput platforms and microfluidic interfaces may further expand CAD utility in process analytical technology for continuous manufacturing.

Conclusion

Charged aerosol detection delivers a sensitive, universal, label-free approach for HPLC analysis of a wide range of biopharmaceutical analytes and excipients. Its robust performance and compatibility with orthogonal detectors make it a valuable tool for research, development and quality assurance in the pharmaceutical industry.

References

No formal literature references were provided in the source document.