Size-exclusion chromatography employing aqueous mobile phases with volatile salts at neutral pH combined with electrospray-ionization mass spectrometry (SEC-ESI-MS) is a useful tool to study proteins in their native state. However, whether the applied eluent conditions actually prevent protein−stationary phase interactions, and/or protein denaturation, often is not assessed. In this study, the effects of volatile mobile phase additives on SEC retention and ESI of proteins were thoroughly investigated. Myoglobin was used as the main model protein, and eluents of varying ionic strength and pH were applied. The degree of interaction between protein and stationary phase was evaluated by calculating the SEC distribution coefficient. Protein-ion charge state distributions obtained during offline and online native ESI-MS were used to monitor alterations in protein structure. Interestingly, most of the supposedly mild eluent compositions induced nonideal SEC behavior and/or protein unfolding. SEC experiments revealed that the nature, ionic strength, and pH of the eluent affected protein retention. Protein−stationary phase interactions were effectively avoided using ammonium acetate at ionic strengths above 0.1 M. Direct-infusion ESI-MS showed that the tested volatile eluent salts seem to follow the Hofmeister series: no denaturation was induced using ammonium acetate (kosmotropic), whereas ammonium formate and bicarbonate (both chaotropic) caused structural changes. Using a mobile phase of 0.2 M ammonium acetate (pH 6.9), several proteins (i.e., myoglobin, carbonic anhydrase, and cytochrome c) could be analyzed by SEC-ESI-MS using different column chemistries without compromising their native state. Overall, with SEC-ESI-MS, the effect of nonspecific interactions between protein and stationary phase on the protein structure can be studied, even revealing gradual structural differences along a peak.
Native Size-exclusion chromatography (SEC) employing aqueous mobile phases with volatile salts at neutral pH combined with native mass spectrometry (nMS) is a useful tool for the characterization of proteins in their native state. However, in many cases the conditions needed to realize the hyphenation of SEC with MS require relative high activation energy and therefore hinder the analysis of labile protein complexes. In this work, we are investigating the advantages of narrow SEC columns (1 mm internal diameter) operated at 15 μL/min flow rates coupled directly to nMS for the characterization of proteins, labile protein complexes and their higher-order structures (HOS). Reducing the flow rate, allowed for a significant increase of the MS sensitivity and ionization efficiency, facilitating detection of low-abundant impurities and HOS (up to the limit of the Orbitrap-MS used, i.e. 230 kDa). More-efficient solvent evaporation could be achieved, allowing using softer MS conditions (e.g. lower gas temperature, lower activation energy) that ensured (little or) no structural alterations or denaturation of the proteins and their HOS during their transfer to the gas phase. Furthermore, high-ionic-strength conditions of volatile salts (200-400 mM), are often necessary to ensure (almost) interaction-free SEC analysis of proteins, such as antibodies (mAbs). With this approach the salt tolerance of the MS was much improved. Because of the reduced column dimensions, band broadening effects resulting from the injection volume became more critical. At high injection volumes (exceeding 3% of the column volume) of more dilute samples, the peak shape and width was affected. Therefore, a new set-up was developed to pre-concentrate the injected proteins on an anion and cation-exchange mixed bed trap column prior to SEC-nMS analysis. This “trap-and-elute” set-up was able to eliminate adverse injection-volume effects in SEC and provide additional desalting, while improving MS detection limits.
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 UVabsorbance, multi-angle light scattering (MALS), and differentialrefractive-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.
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