Determination And Removal Of The Biomass Impurities In Transplantation-grade Chitosan

Chitosan as wall material for microcapsule has been extensively studied as a device for cell transplantation,but the therapeutic effect is limited for the impurities in the materials used.Chitosan is natural biopolymer and the impurities such as proteins and bacterial endotoxins in it,the main contaminants that cause biocompatibility are varied with chitosan of different origins or batches.So,it is necessary to determine and control the content of impurities in chitosan in order to guarantee the security and reproducibility of microcapsules.The aim of this study is to develop methods for assay of the impurities in chitosan,to study the effects of purification methods and conditions of chitosan and to provide references for the quality standard of transplant grade chitosan.Firstly,coomassie brilliant blue was used to determine the amount of protein compounds in chitosan,and kinetic turbidimetric assay was used to determine the bacterial endotoxin content of the samples.Proteins,bacterial endotoxins and other impurities in chitosan were determined by these methods.The results show that the impurities in chitosan could be quantified accurately by the methods developed in the paper.Secondly,removal of protein impurities by different methods such as filtering and alcohol washing,activated carbon adsorption,HCl treatment,NaOH trentment,Sevag method,surfactant treatment,et al were tried in the article.The results show that removal ratio of protein from chitosan by filtering and alcohol washing,activated carbon adsorption and Sevag method was less than 10%.Removal ratio of protein from chitosan by NaOH treatment,SDS methods was 15%to 20%.Tween20 method could remove the impurities most effectively,and the removal ratio of proteins was up to 73%when pH=3.00,the ratio of tween20 and chitosan was 5:2.Furthermore,filtration,activated carbon adsorption,NaOH treatment,and surfactant treatment were studied for removal of bacterial endotoxin.The results show that filtration,activated carbon adsorption and surfactant method could effectively remove the bacterial endotoxin.Bacterial endotoxin content of chitosan can be reduced to 3118EU/g by NaOH method and the removal ratio was 94%.Bacterial endotoxin content can be reduced to 625EU/g by improve method which combined with practical methods,such as combined method of activated carbon adsorption and surfactant treatment,or combined method of NaOH treatment and surfactant method.