Core determinant of the in vitro enzyme activity of tenecteplase: primary structure over glycosylation modifications

Tenecteplase (TNK), a novel genetically modified variant of tissue-type plasminogen activator (rt-PA), holds great promise as a first-line thrombolytic agent for thrombotic diseases owing to its convenient administration and favorable pharmacological properties. However, glycosylation heterogeneity derived from distinct production processes has resulted in industry-wide inconsistencies in enzyme activity determination methods, reference materials, and activity units, severely impairing the accuracy of clinical medication. Compounded by its narrow therapeutic window, minor dosage deviations of TNK can not only drastically reduce thrombolytic efficacy but also trigger severe hemorrhagic adverse reactions. This study aimed to establish an accurate and standardized in vitro enzymatic activity assay for TNK, and to elucidate the dominant determinant of TNK in vitro enzyme activity between primary structure and glycosylation modifications.

An in vitro enzyme activity assay was established using an automatic coagulation analyzer and subjected to systematic methodological validation. The primary structures of two TNK products (TNK A and TNK B) were confirmed via LC-MS/MS, while their glycosylation profiles were characterized using HILIC coupled with mass spectrometry. Comparative activity assays were conducted under various conditions, including human serum albumin (HSA) protection, dithiothreitol (DTT) disruption, and single-chain to two-chain conversion.

Methodological validation demonstrated that the automated assay possesses high accuracy (relative bias -0.24%-0.27%), precision (CV < 2.0%), and robustness, with results highly consistent with the traditional bubble-rising method. Characterization confirmed that TNK A and TNK B share identical amino acid sequences but exhibit significant differences in glycan distribution. Under stable conditions (using HSA-containing buffers), no statistically significant difference in clot lysis activity was observed between the two products (P > 0.05). Experimental data revealed that previously reported activity discrepancies were likely artifacts of buffer composition (lack of HSA) and environmental sensitivity rather than direct glycan-driven functional changes.

The primary structure is the decisive factor determining the in vitro enzymatic activity of TNK, while glycosylation modifications appear to exert minimal impact under the specific stable in vitro assay conditions tested. The established automated coagulation analysis provides a reliable tool for quality control and the unification of clinical activity units for recombinant tissue-type plasminogen activator-based agents.