A Study on the Mechanism of Aggregation of Therapeutic Reteplase Protein by Using the Monomer-Loss Model

Document Type : Original Article


Biotechnology Group, School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran


Aggregation of pharmaceutical proteins reduces the efficiency and increases the cost of production. It can also lead to the reduced efficacy of drug or cause side effects on the patient’s body. Investigating how to create them plays an important role to find agents that prevents the aggregation. This study was allocated for understanding the mechanism of formation of the reteplase protein using thermal stimulation. Aggregation was studied by ultraviolet spectrometry, and observation at 4, 25, 50 and 70°C, the concentration of protein monomer was measured by using a spectrum of 360 nm and 280 nm. At 4°C, there was no significant change in monomer concentration for a month. By increasing the temperature to 25˚C, aggregation process was slow, but at 70°C, the reaction was carried out at a rapid rate less than 2 hours. In order to investigate the mechanism of reteplase aggregation, some kinetics that was presented in of monomer-loss models were used. Experimental data was fitted in three “pre balance core”, “self-catalytic” and “slow start” models using MATLAB. The best fit was obtained using optimization methods. Best fit for self-catalytic model is (R2> 0.98). For other two models (R2 <0.9) occurred. The best fit for the pre balance core and a slow start model was occurred in n = 2. These results could indicate that the core is formed by connecting two reteplase monomers together. The reaction rate constants were calculated too.  The results showed that increasing the temperature increases the reaction rate constant. With increasing temperature from 25 ˚C up to 70˚C, both of K1 and K2 increased from 4.7±0.1*10-11K1(min-1) to 1.6±0.2*10-7K1(min-1) and from 1.04±0.2*10-5 K2(M -1 min-1) to 1.5± 0.3*10-4 K2(M -1min-1),  respectively for autocatalytic model.  Limitation step of the reaction is the nucleation. K1<K2 demonstrates this fact. In addition, an existence of disulfide bonds in aggregate protein was guessed. These bonds are strong covalent that cannot be easily broken.