Pegylation Of Recombinant Hirudin And Its Reaction Kinetics

Therapeutic proteins have several advantages over small-molecule drugs, such as high activity, high specificity, and clear biological function. Development of therapeutic proteins has become a hotspot in the biopharmaceutical industry. However, therapeutic proteins show several limitations in clinical use. such as short plasma half-lives, immunogenicity, and poor stability. Therefore, development of long-effective therapeutic proteins has become a new trend. PEGylation is one of the most successful strategies to develop long-effective therapeutic proteins. The therapeutic efficacy of PEGylated therapeutic proteins can be improved through prolonging their circulating half-life, reducing their immunogenicity and proteolysis, and increasing their stability and solubility. However, PEGylation of therapeutic proteins is still suffering from the problems of the reduction of PEG reagent usage and the control of the PEGylation site and degree. To solve these problems, recombinant hirudin was chosen as a model therapeutic protein to be PEGylated. A reaction kinetics model was constructed for the PEGylation of recombinant hirudin. The effects of solvent environment, in situ PEGylation on ion exchange column, and different PEG agents on the PEGylation of recombinant hirudin were investigated. The main results are as follows:(1) A kinetics model was constructed to describe the PEGylation reaction of recombinant hirudin. Moreover, the established model was used to optimize the reaction conditions of PEGylation. Several important process parameters including ymax, mcrit and tmax, and their mathematical equations were obtained to determine the key factors to achieve the mono-PEGylated recombinant hirudin at the desired yield. The results indicated that the proposed reaction kinetics model can provide a possible interpretation of mechanism for real PEGylation reactions and optimize efficiently the PEGylation process.(2) The effects of mixed aqueous-organic solutions on the PEGylation of recombinant hirudin were investigated. Recombinant hirudin structure, hydrolysis kinetics of PEG reagent, and PEGylation kinetics of recombinant hirudin were analyzed. The results revealed that solvent effects including PEGylation acceleration and PEG hydrolysis inhibition can enhance the PEGylation efficiency in mixed aqueous-organic solutions. According to the dominant solvent effect, the selected mixed aqueous-organic solutions can be divided into three different types (PEGylation-driven, PEG hydrolysis-driven, both PEGylation and PEG hydrolysis-driven). In aqueous-DMSO solutions, the optimal usage of PEG reagent to achieve the desired yield (approximately 50%) of mono-PEGylated recombinant hirudin can decrease 3-5 folds, compared with that of PEGylation in pure aqueous solution.(3) An integrated process was developed for in situ PEGylation of recombinant hirudin on anion exchange column. PEGylation reaction and separation can be efficiently integrated into one unit in this process. Effect of different ion exchange resins on the in situ PEGylation was investigated. The results showed that the pores and internal surface structures such as the combination of polymer dextran of different resins have significant impact on the yield of mono-PEGylated recombinant hirudin. Moreover, effect of different PEG sizes on the in situ PEGylation was also investigated. The results showed that in situ PEGylation on anion exchange column (solid-phase PEGylation) is diffusion-driven process. The yield of mono-PEGylated recombinant hirudin decreases as PEG size increases, which is contrary to liquid-phase PEGylation. In vitro and in vivo activity analysis of mono-PEGylated recombinant hirudin showed that in situ PEGylation on anion exchange column could enhance the selectivity of PEGylation. In vitro and in vivo anticoagulant activities of mono-PEGylated recombinant hirudin derived from in situ PEGylation on anion exchange column are greater than those from liquid-phase PEGylation.(4) PEGylation of recombinant hirudin with branched mPEG2-NHS was performed to achieve the mono-PEGylated recombinant hirudin with high yield and active retention. Compared with linear mPEG-SC, branched mPEG2-NHS displayed higher PEGylation rates, but lower PEG reagent deactivation rate. Thus, higher PEG/recombinant hirudin molar ratio was required to achieve the mono-PEGylated recombinant hirudin at the desired yield. In vitro and in vivo activity analysis of mono-PEGylated recombinant hirudin showed that branched mPEG2-NHS could easier shield the active region of the protein from PEGylation to achieve higher in vitro and in vivo anticoagulant activities of the mono-PEGylated recombinant hirudin compared with those from linear mPEG-SC.(5) Site-specific PEGylation of the N-terminus of recombinant hirudin was performed by using mPEG-ALD. The PEGylation reactions were optimized by response surface analysis. The results showed that mono-PEGylated recombinant hirudin derived from site-specific PEGylation of recombinant hirudin with mPEG-ALD had more homogeneous PEGylation site and higher yield, compared with those of random PEGylation with mPEG-SC. N-terminal mono-PEGylated recombinant hirudin retained approximately 20% of the in vitro anticoagulant activity of unmodified recombinant hirudin.In summary, the reduction of PEG reagent usage and the control of PEGylation site and degree were achieved through the study of the effects of micro-environmental factors on the PEGylation of therapeutic proteins based on the reaction kinetics model. The results of this study demonstrated effective strategies for the development of PEGyalted therapie proteins.