We report an online analytical platform based on the
coupling of
asymmetrical flow field-flow fractionation (AF4) and native mass spectrometry
(nMS) in parallel with UV-absorbance, multi-angle light scattering
(MALS), and differential-refractive-index (UV–MALS–dRI)
detectors to elucidate labile higher-order structures (HOS) of protein
biotherapeutics. The technical aspects of coupling AF4 with nMS and
the UV–MALS–dRI multi-detection system are discussed.
The “slot-outlet” technique was used to reduce sample
dilution and split the AF4 effluent between the MS and UV–MALS–dRI
detectors. The stability, HOS, and dissociation pathways of the tetrameric
biotherapeutic enzyme (anticancer agent)l-asparaginase (ASNase)
were studied. ASNase is a 140 kDa homo-tetramer, but the presence
of intact octamers and degradation products with lower molecular weights
was indicated by AF4–MALS/nMS. Exposing ASNase to 10 mM NaOH
disturbed the equilibrium between the different non-covalent species
and led to HOS dissociation. Correlation of the information obtained
by AF4–MALS (liquid phase) and AF4–nMS (gas phase) revealed
the formation of monomeric, tetrameric, and pentameric species. High-resolution
MS revealed deamidation of the main intact tetramer upon exposure
of ASNase to high pH (NaOH and ammonium bicarbonate). The particular
information retrieved from ASNase with the developed platform in a
single run demonstrates that the newly developed platform can be highly
useful for aggregation and stability studies of protein biopharmaceuticals.