SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor-binding domain (RBD) are necessary for seropositivity assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein’s structure and function, and thus, comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid chromatography coupled to mass spectrometry has been widely used to characterize post-translational modifications in proteins, including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogeneous glycans at the intact protein level. Herein, we evaluated several online separation techniques: (1) C2 reverse-phase liquid chromatography (RPLC), (2) capillary zone electrophoresis (CZE), and (3) acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down mass spectrometry (MS). Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation and N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogeneous glycoproteins for facile comparison of biosimilars in quality control applications.
In this study, we have prepared thermally initiated polymeric monolithic stationary phases within discrete regions of 3Dprinted titanium devices. The devices were created with controllable hot and cold regions. The monolithic stationary phases were first locally created in capillaries inserted into the channels of the titanium devices. The homogeneity of the monolith structure and the interface length were studied by scanning a capacitively coupled conductivity contactless detector (C 4 D) along the length of the capillary. Homogeneous monolithic structures could be obtained within a titanium device equipped with a hot and cold jacket connected to two water baths. The confinement method was optimized in capillaries. The sharpest interfaces (between monolith and empty channel) were obtained with the hot region maintained at 70 °C and the cold region at 4 or 10 °C, with the latter temperature yielding better repeatability. The optimized conditions were used to create monoliths bound directly to the walls of the titanium channels. The fabricated monoliths were successfully used to separate a mixture of four intact proteins using reversed-phase liquid chromatography. Further chromatographic characterization showed a permeability (K f ) of ∼4 × 10 −15 m 2 and a total porosity of 60%.
In this study, we optimized a polymerization mixture to synthesize poly(acrylamide- co - N , N ′-methylenebisacrylamide) monolithic stationary phases for hydrophilic-interaction chromatography (HILIC) of intact proteins. Thermal polymerization was performed, and the effects of varying the amount of cross-linker and the porogen composition on the separation performance of the resulting columns were studied. The homogeneity of the structure and the different porosities were examined through scanning electron microscopy (SEM). Further characterization of the monolithic structure revealed a permeable ( K f between 2.5 × 10 –15 and 1.40 × 10 –13 m 2 ) and polar stationary phase suitable for HILIC. The HILIC separation performance of the different columns was assessed using gradient separation of a sample containing four intact proteins, with the best performing stationary phase exhibiting a peak capacity of 51 in a gradient of 25 min. Polyacrylamide-based materials were compared with a silica-based particulate amide phase (2.7 μm core–shell particles). The monolith has no residual silanol sites and, therefore, fewer sites for ion-exchange interactions with proteins. Thus, it required lower concentrations of ion-pair reagent in HILIC of intact proteins. When using 0.1% of trifluoroacetic acid (TFA), the peak capacities of the two columns were similar (30 and 34 for the monolithic and packed column, respectively). However, when decreasing the concentration of TFA to 0.005%, the monolithic column maintained similar separation performance and selectivity (peak capacity 23), whereas the packed column showed greatly reduced performance (peak capacity 12), lower selectivity, and inability to elute all four reference proteins. Finally, using a mobile phase containing 0.1% formic acid and 0.005% TFA, the HILIC separation on the monolithic column was successfully hyphenated with high-resolution mass spectrometry. Detection sensitivity for protein and glycoproteins was increased and the amount of adducts formed was decreased in comparison with separations performed at 0.1% TFA.
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.
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