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An etanercept biosimilar, TuNEX ® , was compared to the innovator drug, Enbrel ® , for its reaction to stress-induced oxidation and deamidation, which may affect drug efficacy. A tryptic peptide map of both etanercept products was generated by liquid chromatography (LC) using mass spectrometry (MS) and ultraviolet (UV) spectrophotometry detection methods. The sequence of each modified or non-modified peptide peak was assigned based on accurate measurement of the mass of the protein and analysis utilizing tandem MS. Similar profiles of intrinsic oxidation on methionine (M) and deamidation on asparagine (N) were obtained for the two products, regardless of a two-amino acid (AA) residue variance in the heavy chain (Fc) between them. The level of oxidative stress exerted by tert -butyl hydroperoxide (tBHP), and alkaline stress exerted by a pH 10.4 solution, was examined using an LC-UV method. The results indicated that TuNEX ® demonstrated a similar stress-induced modification profile compared to that of Enbrel ® . For both products, oxidative stress increased the oxidation from an intrinsically low (0–6.9%) to moderate or high (42–100%) level for almost all M residues (M30, M174, M187, M223, M272, and M448); alkaline stress increased the deamidation level of N404 from a low (0.0 or 1.7%) to moderate (19–26%) level. Based the results of a cell-based bioactivity assay, TuNEX ® also exhibited a similar level of bioactivity as Enbrel ® in unstressed, oxidative-stressed, or alkaline-stressed conditions. The bioactivity of both products remained unaltered by oxidative stress but was reduced by alkali stress. In conclusion, our data indicated that TuNEX ® exhibits a similar chemical stress profile as that of Enbrel ® in terms of oxidation and deamidation as well as bioactivity. [ABSTRACT FROM AUTHOR]

We reported an improved combinatorial approach for identifying site-specific O-glycosylation using both glycan cleaved and non-cleaved methods. In this approach, a non-reducing β-elimination kit coupled with non-specific enzymes performed efficient digestion, O-glycan cleavage, and partial dephosphorylation without significant side reactions, thus enabling an automatic database search for the cleaved O-glycosylation or serine/threonine (S/T) phosphorylation sites. From the same sample concurrently prepared without β-elimination, the corresponding intact O-glycopeptides were mapped by accurate precursor ion mass using an in-house glycan database majorly composed of GalNAc (mucin-type) core and the retention-time shift (Δ R t ). Each glycopeptide assignment was verified by the detection of glycan-specific fragments using collision-induced dissociation (CID) to estimate False Discovery Rate (FDR). Using fetuin as a model, all identified S/T elimination sites were matched to multiple intact glycopeptides with a 31% FDR. This considerably reduced to 0% FDR by Δ R t filtering. This approach was then applied to a protein mixture composed of therapeutic Factor IX and Enbrel ® mixed with fetuin and kappa-casein. A total of 26 glycosylation sites each of which corresponds to 1–4 glycans were positively mapped and confirmed. The FDR decreased from 33% to 3.3% by Δ R t filtering and exclusion of repeated peptide tags that covered the same glycosylation sites. Moreover, the phosphorylation and O-glycosylation on the same site such as T159 of Factor IX and T170 of kappa-casein were able to be unambiguously differentiated. Thus, our approach is useful for in-depth characterization of site-specific O-glycosylation of a simple mixture such as protein-based therapeutics. [ABSTRACT FROM AUTHOR]