Rekombinant Granülosit Koloni Uyarıcı Faktörün Biyopolimerlerle Enzimatik Konjugasyonu ve Proteinin Özellikleri Üzerindeki Etkilerinin Araştırılması Üzerinde Çalışmalar

Abstract

Second generation protein products are studied and products are developed in recent years. Aim of this thesis is to add a new alternative to second generation drug development approaches with filgrastim. For this purpose, enzymatic conjugation of filgrastim with transglutaminase is designed by using a polymer which is not used for this aim before. Conjugation studies are conducted with hydrolysates of chitosan, which is a natural polymer in the polysaccharide structure. Conjugate formation is shown by SEC-HPLC, RP-HPLC, Fourier transform infrared spectroscopy, mass spectroscopy, SDS-PAGE gel electrophoresis, isoelectric focusing and western blot analysis. In addition, HPLC, FTIR and mass spectral analysis showed that conjugate is formed of three similar molecular weight subfraction. After the conjugate formation, effects of parameters such as temperature, reaction period, pH and different ratios of components on the formation and stability of conjugates are studied. Polymer ratio is found to be the main factor on conjugate formation. Solution and lyophilized forms of conjugate is found stable at 5±3oC for four months. Bioactivity of the conjugate solution is studied on cell cultures complying with the European Pharmacopeia by using NFS-60 cells and conjugate is seen more active than reference filgrastim.