Jan Bechmann ^1* Ira Schmid ¹ Simone Brand ¹ Felix Miller ¹ Chengzhi Zhang ²

• ¹ Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
• ² The 1st Affiliated Hospital of Chongqing Medical University, Chongqing, China

In the last decades, the recombinant tissue plasminogen activator alteplase has been the standard fibrinolytic treatment of acute myocardial infarction, pulmonary embolism, and acute ischemic stroke. An optimized version of alteplase, tenecteplase, has been developed by exchanging three amino acids to increase half-life, achieve higher fibrin selectivity and increase resistance to plasminogen activator inhibitor-1. The aim of this study was to compare the fibrinolytic activity and overall product quality of the 25 mg/vial presentation of tenecteplase originator Metalyse ® (Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim, Germany) to the 16 mg/vial formulation of the tenecteplase copy Mingfule ® (CSPC Recomgen Pharmaceutical, Guangzhou, Co., Ltd.). We have systematically analyzed and evaluated the biochemical and fibrinolytic differences between Metalyse ® and Mingfule ® using a wide range of routine quality testing assays, supplemented by mass spectrometry analysis and surface plasmon resonance assays. Additional host cell protein quantification and clot lysis testing following plasmin incubation over time were performed. Several key differences in biochemical composition and clot lysis activity were observed between the two tenecteplase variants. Versus Metalyse ® , Mingfule ® exhibited lower clot lysis activity and contained less of the two-chain form of tenecteplase. In addition, there were differences in sialic acid content, galactosylation, and fucosylation patterns, with Mingfule ® exhibiting more bi-and less tri-and tetraantennary glycosylation, leading to a different charge and size heterogeneity profile. Furthermore, Mingfule ® displayed highly dissimilar binding to the three clearance receptors (LRP-1, ASGR, and MR) compared with Metalyse ® . Purity analysis showed that Mingfule ® contained a lower monomer content and, in contrast to Metalyse ® , substantial amounts of host cell protein.Taken together, these data demonstrate that the tenecteplase copy Mingfule ® has several meaningful fibrinolytic and biochemical differences compared with Metalyse ® . This raises the question of whether data from clinical studies with one of the products can be generalized for all tenecteplase variants.