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采用直链PEG-琥珀酰亚胺琥珀酸酯(mPEG-SS)选择性修饰重组人干扰素ω(rhIFNω),离子交换色谱和凝胶过滤色谱组合分离纯化单链PEG修饰产物(PEG-rhIFNω),基质辅助激光解吸附飞行时间质谱(MALDI-TOF MS)测定单链PEG-rhIFNω的相对分子量,并利用RP-HPLC和SDS-PAGE对修饰产物进行分析。在优化的工艺条件下,分离纯化收集液的单链PEG-rhIFNω,平均含量达182μg.mL-1,分离纯化收率超过22%,纯度大于98%,SDS-PAGE法测得表观分子质量为60 810,MALDI-TOF MS法测得相对分子质量为43 790;单链PEG-rhIFNω具有典型PEG修饰蛋白的特性,抗病毒活性保留率为15.0%,抗原性降低了64倍,酸稳定性、抗胰酶水解能力、血清稳定性和热稳定性均显著提高。单链PEG-rhIFNω的药学性质获得显著改善,有望开发为安全、长效的新型干扰素。
The recombinant human interferon ω (rhIFNω) was selectively modified with linear PEG-succinimidyl succinate (mPEG-SS), and PEG-rhIFNω was isolated and purified by ion-exchange chromatography and gel filtration chromatography. , Matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS) were used to determine the relative molecular weight of single-stranded PEG-rhIFNω. The modified products were analyzed by RP-HPLC and SDS-PAGE. Under optimal conditions, single-stranded PEG-rhIFNω was separated and purified, with an average content of 182 μg.mL-1. The purification yield was over 22% and the purity was more than 98%. The apparent molecular weight Was 60 810. The relative molecular mass of MALDI-TOF MS method was 43 790. Single-stranded PEG-rhIFNω had the characteristic of typical PEG-modified protein with antiviral activity retention rate of 15.0%, antigenicity decreased by 64-fold, and acid stability , Anti-pancreatic enzymatic hydrolysis, serum stability and thermal stability were significantly increased. Pharmacological properties of single-stranded PEG-rhIFNω have been significantly improved and are expected to be developed as safe, long-acting novel interferons.