A Universal Buffer System for Native LC-MS Analysis of Antibody-Based Therapeutics

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Liquid chromatography coupled to mass spectrometry (LC-MS) is crucial for analyzing biological macromolecules.
• A significant challenge in LC-MS is performing analyses at physiological pH under native conditions using volatile buffers.
• Traditional buffers like ammonium acetate lack sufficient buffering capacity in the physiological pH range (7.0-7.4).

Purpose of the Study:

• To develop and evaluate a novel volatile buffer system for native LC-MS applications.
• To assess the buffering capacity of fluorinated ethylamines across a wide pH range.
• To demonstrate the applicability of the new buffer system across various chromatographic modalities.

Main Methods:

• Evaluated fluorinated ethylamines (MFEA, DFEA, TFEA) for their buffering capacity and pKa values.
• Performed protein separations using strong cation- and anion-exchange chromatography with volatile mobile phases.
• Utilized hydrophobic interaction chromatography (HIC) with volatile mobile phases.
• Analyzed protein variants, glycoforms, and antibody-drug conjugates using LC-MS.

Main Results:

• Fluorinated ethylamines provide buffering capacity across the pH range of 4.5-9.8.
• Protein separations using volatile buffers were comparable to traditional nonvolatile buffers, with similar elution profiles.
• High-resolution mass spectra enabled the resolution of antibody glycoforms.
• HIC in volatile mobile phases resolved drug-to-antibody ratio (DAR) species.
• Reproducible results were obtained when comparing innovator and biosimilar antibodies.

Conclusions:

• Fluorinated ethylamines, combined with ammonium acetate, form a universal volatile buffer system for native LC-MS.
• This system is broadly applicable across major chromatographic modalities, including ion-exchange and HIC.
• The developed buffer system overcomes previous limitations, enabling more versatile native LC-MS analyses of biomolecules.