Optimization Of Trypsin Drying Technology And Development Of Spray Freeze Equipment

In recent years,Manufacturing solid dosage in powder form of therapeutic agents with high biological activity,such as antibody,vaccine and bio-enzyme,etc has attracted numerous research&design(R&D)efforts in pharmaceutical research and industries.Dry formulation and precursor solution formulation play an important role in the bioactivity of medicaments.In this study,we have selected trypsin as the model enzyme,and trehalose and mannitol as the excipient.Spray drying(SD)and Spray freeze drying(SFD)techniques both equipped with a specially designed micro-fluidic aerosol nozzle(MFAN)able to generate monodisperse droplets have been used to fabricate uniform trypsin-doped microparticles.The effects of precursor formula and manufacturing means on particle size,morphology,structure and enzymatic actiivity have been investigated.SEM images showed that SD microparticles of pure trehalose were spherical,and turned crumpled with addition of trypsin,probably due to appreciation of surface active trypsin on the droplet/air interface inducing deformation while drying droplets into particles,whereas the trypsin/mannitol composite microparticles were spherical with smooth surface,maybe because the mannitol had low solubility and was easy to crystallize;SFD microparticles were of spherical and porous morphology independent with precursor formulations.However,SFD microparticles containing more trypsin had more solid skin,regardless of which excipient were used,also indicating that trypsin may enrich on the surface.For the thermostability of samples,the trehalose could improve the thermostability of trypsin/trehalose composite microparticles,and SD is better than SFD for improved,probably because the trypsin and trehalose molecules could interact each other strongly through intra-molecular forces,but the cause of the improvement of the thermostability of trypsin/mannitol composite microparticles might be the crystallized mannitol.In comparison with SD,SFD produced pure trypsin microparticles with higher enzyme activity(SD:79.9 ± 0.7%;SFD:87.7 ± 3.4%)assayed by the kinetic degradation method with ELIAS A.It can be ascribed to the heat treatment of SD unfolding trypsin secondary structure to a lager extent,as characterized via FTIR.The addition of trehalose could protect the enzyme activity,reaching 97.7 ± 2.6%and 97.3 ± 1.6%with the mass ratio of 1:1 for SD and SFD,respectively.Nevertheless,as far as mannitol is concerned,the bioactivity hardly seemed an obvious improves.In terms of process operability,SD offers a rather simple means to produce microparticles while achieving similarly good protection over trypsin activity with the appropriate dosage of trehalose.Furthermore,the effect of spray drying temperature on the trypsin and trypsin/excipient microparticles has been investigated.For the pure trypsin,higher the inlet temperature affects the trypsin bioactivity to a more extent.Upon addition of trehalose in the trypsin precursor formulation,independent with the mass ratio of trypsin/trehalose,there is a significant recovery of original trypsin bioactivity,while there was no effect on the bioactivity of trypsin by using mannitol as excipient,no matter the inlet temperature of spray drying,the reason may be caused by crystallized mannitol and mannitol unable to form hydrogen bonds with trypsin to fix the second structure.Comprehensive analysis of this series,it will provide theoretical meaning and practical value for the preparation of high bioactivity microparticles via spray drying or spray freeze drying.Finally,we have developed a novel temperature-controlled spray freeze tower.On the one hand,the advantage of this equipment is control the temperature,range from 0?80?,to study the effects of icing temperature;on the other hand,it avoids the waste of refrigerant,and its characteristics including energy conservation,being good for environmental protection.