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

Liquid chromatography
coupled to mass spectrometry (LC–MS)
is a powerful analytical technique for analyzing biological macromolecules.
A long-standing challenge has been applying LC–MS at physiological
pH under native conditions using volatile buffers. The predominant
“buffer” used, ammonium acetate (AmAc, pKa 4.75 for acetic acid and 9.25 for ammonium), does not
offer sufficient buffering capacity in the physiological pH range
of 7.0–7.4. To address this, we evaluated a set of fluorinated
ethylamines, 2-fluoroethylamine (MFEA), 2,2-difluoroethylamine (DFEA),
and 2,2,2-trifluoroethylamine (TFEA), producing conjugate acids with
pKa values of 8.9, 7.2, and 5.5, respectively,
that together provide buffering across the 4.5–9.8 pH range.
We show that protein separations on strong cation- and anion-exchange
resins in these volatile mobile phases perform comparably to traditional
nonvolatile buffers, with similar elution profiles and analyte elution
ranking, albeit with slightly broader peaks. Using fully volatile
gradients of pH or ionic strength, we chromatographically resolved
charge variants of protein analytes such as mAbs and bovine serum
albumin. For many of the eluting LC peaks, we obtained high-resolution
mass spectra capable of resolving glycoforms of antibodies. Hydrophobic
interaction chromatography (HIC) in volatile mobile phases preserved
native separation order and further resolved drug-to-antibody ratio
(DAR) species of the antibody-drug conjugate brentuximab-vedotin.
For each chromatography modality we further compare innovator and
biosimilar antibodies, demonstrating the reproducibility of results
in the proposed volatile compounds. Together, our results establish
fluorinated ethylamines, in combination with ammonium acetate, as
a universal volatile buffer system for native LC–MS, broadly
applicable across major chromatographic modalities.