TNK-tPA products from the innovator and follow-on manufacturers were characterized and compared. All tryptic peptides including N-terminal, C-terminal, and mutated peptides as well as the disulfide-linked peptides were identified, with the demonstration of the same primary sequence and disulfide linkages between the innovator and follow-on products. The three N-linked and one O-linked fucose glycosylation sites were identified. The two N-linked fucose sites (N103 and N448) and one O-linked fucose site (T61) were fully glycosylated in both innovator and follow-on products. The other N-linked site (N184) was partially glycosylated and exhibited a approximately 2.5-fold difference between the innovator (60% occupancy) and follow-on (25% occupancy) products. Since the glycosylation occupancy at this site is known to affect biological activity in the clot lysis assay, this observed difference could cause a concern as to their bioequivalence. The cleavage site for the conversion of the zymogen form to active enzyme was also identified between R275 and I276, with a cleavage of 40% for the innovator and 10% for the follow-on products. Both the glycosylation occupancy (%) and the chain cleavage (%) were determined by two independent approaches, starting from either the peptide or intact protein separation, with consistent results by both methods. Subtle differences of modifications such as deamidation and oxidation between the innovator and biosimilar products were shown at M207, M445, M490 and N58, N184. The observation of different extents of oxidation at M207 and deamidation at N184, which could influence the clot lysis activity, were also of potential concern in drug efficacy between the follow-on and innovator products.