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Robust manufacturing processes resulting in consistent glycosylation are critical for the efficacy and safety of biopharmaceuticals. Information on glycosylation can be obtained by conventional bottom–up methods but is often limited to the glycan or glycopeptide level. Here, we apply high-resolution native mass spectrometry (MS) for the characterization of the therapeutic fusion protein Etanercept to unravel glycoform heterogeneity in conditions of hitherto unmatched mass spectral complexity. Higher spatial resolution at lower charge states, an inherent characteristic of native MS, represents a key component for the successful revelation of glycan heterogeneity. Combined with enzymatic dissection using a set of proteases and glycosidases, assignment of specific glycoforms is achieved by transferring information from subunit to whole protein level. The application of native mass spectrometric analysis of intact Etanercept as a fingerprinting tool for the assessment of batch-to-batch variability is exemplified and may be extended to demonstrate comparability after changes in the biologic manufacturing process. The specific glycosylation patterns of biological drugs often impact the efficacy and safety of the therapeutic product. Here the authors describe a native mass spectrometry approach that allows the resolution of highly complex glycosylation patterns on large proteins, which they apply to the therapeutic Fc-fusion protein Etanercept.

An analytical workflow was developed for the absolute quantification of uridine diphosphate (UDP)-sugars in plant material in order to compare their metabolism both in wild-type Arabidopsis thaliana and mutated plants ( ugd2,3 ) possessing genetic alterations within the UDP-glucose dehydrogenase genes involved in UDP-sugar metabolism. UDP-sugars were extracted from fresh plant material by chloroform-methanol-water extraction and further purified by solid-phase extraction with a porous graphitic carbon adsorbent with extraction efficiencies between 80 ± 5 % and 90 ± 5 %. Quantitative determination of the UDP-sugars was accomplished through HPLC separation with a porous graphitic carbon column (Hypercarb TM ) which was interfaced to electrospray ionization Orbitrap mass spectrometry. The problem of instable retention times due to redox processes on the stationary phase were circumvented by grounding of the column effluent and incorporation of a column regeneration procedure using acetonitrile-water containing 0.10 % trifluoroacetic acid. The method was calibrated using external calibration and UDP as internal standard. Calibration functions were approximated by first- or second-order regression analysis for concentrations spanning three orders of magnitude. Upon injecting sample volumes of 2.65 μL, the limits of detection for the UDP-sugars were in the 70 nmol L −1 range. Six different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-arabinose, UDP-xylose, UDP-glucuronic acid, and UDP-galacturonic acid were found in concentrations of 0.4 to 38 μg/g plant material. Data evaluation by analysis of variance (ANOVA) revealed statistically significant differences in UDP-sugar concentrations between wild-type and mutant plants, which were found to conclusively mirror the impaired metabolic pathways in the mutant plants. Figure ᅟ