Abstract

Background: Human Plasminogen (PLG, EC 3.4.21.7) is a single chain glycoprotein. This protein circulates in blood as an inactive zymogen. Due to post-translational modification two distinct glycoforms has been found: Glycoform I (GluI-PLG) and Glycoform II (GluII-PLG).A proteolytic activation with physiological relevance consists in the plasmin-catalyzed hydrolysis of the N-terminal amino acid sequence at position 77. This activation can occur on GluI-PLG or GluII-PLG producing respectively LysI-PLG or LysII-PLG. For a pharmaceutical company that produces a drug based on Glu-PLG it is important to develop a method that allows checking the presence of Lys-PLG to exclude some phenomena of pre-activation in the Drug Product.

Study design and methods: A new electrophoretic method to separate the Glu-PLG (I and II) forms from the Lys-PLG (I and II) one was developed. The Design of Experiment (DoE) approach has been employed to set the specific parameters of electrophoresis. The Voltage the Electrophoretic runs time and the loaded protein amount.

Results: Two batches of Plasminogen manufactured by Kedrion were analyzed for their content of Lys-PLG. No trace of this form has been highlighted. The Relative Fronts (RF) of the different protein bands was considered and the elaboration of the DoE results allowed obtaining a robust and reproducible separation of GluI, GluII, LysI and LysII protein band. In addition, the method limit of detection (LOD) was investigated.

Conclusion: The developed method is able to separate the Glu-PLG forms from the Lys-PLG one. The method can be used for checking the stability of a PLG-based drug during shelf life.