Characterization of the Glycosylation Occupancy and the Active Site in the Follow-on Protein Therapeutic: TNK-Tissue Plasminogen Activator

Abstract

Summary 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 (N103 and N448) and one O-linked fucose (T61) sites were fully glycosylated in both innovator and follow-on products. The other N-linked site (N184) was partially glycosylated and exhibited a ~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 innovator and biosimilar were shown at M207, M445, M490 and N58, N184. The observation of different extent 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.

Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars

“…For example, a study comparing the glycosylation pattern of an innovator tissue-type plasminogen activator (tPA) and its biosimilar demonstrated a ~2.5-fold greater amount of glycosylation at one N -linked site in the innovator material versus the biosimilar material 34 . Differences between an innovator monoclonal antibody, trastuzumab (Herceptin; Genentech/Roche), and a bio-similar were readily detected with liquid chromatography–MS (LC–MS) 35 , including changes in glycosylation and amino acid mutations in the heavy chain.…”

Semi-Automated Identification of N-Glycopeptides by Hydrophilic Interaction Chromatography, nano-Reverse-Phase LC–MS/MS, and Glycan Database Search

“…PNGaseF detachment of N-glycans boosts the abundance of glycopeptides’ peptide substrate by aggregating the glycopeptides’ glycoforms on a single peptide species. The enzymatic deglycosylation leads to a substitution of Asn for Asp (mass delta +0.98 Da), providing a mechanism for identifying a glycoproteins’ N-linked glycosylation sites 37,38 . Analysis of detached N-glycans can provide important insights into the heterogeneity of protein glycosylation at the expense of the ability to characterize the glycans’ protein or attachment site 36,39 .…”

Therapeutic plasma proteins – application of proteomics in process optimization, validation, and analysis of the final product

“…In a group of recent publications coming from Hancock's and Karger's laboratory, the use of proteomics technology for pharmacokinetic studies of protein drugs [160,161], and for quality control and quality assurance in production of followon (generic) recombinant proteins [162,163] was also demonstrated. This work opens new perspectives for the use of proteomics in quality assurance and preclinical testing of new products that can be either recombinant or pd.…”