Liu Hong, Cheng Yong-Gang, Pan Hong-Chun, Xu Bo, Peng Li, Yang Hong-Tao, Guo Wei
School of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China.
Yao Xue Xue Bao. 2012 Mar;47(3):393-8.
The amino group PEGylation of rhIFNomega with monomethoxy polyethylene glycol succinimidyl succinate (mPEG-SS, 20 000) was investigated, and the modified mixture was separated and purified by ion exchange chromatography and gel filtration chromatography. Under the optimized purification conditions, the average content ofmono PEG-rhIFNomega in the collect liquid reached 182 microg x mL(-1). The average purified yield of mono PEG-rhIFNomega exceed to 22%, and the purity of mono PEG-rhIFNomega was greater than 98% by SDS-PAGE and RP-HPLC. Relative molecular mass of mono PEG-rhIFNomega was 43 790 detected by MALDI-TOF MS. The apparent molecular mass measured by SDS-PAGE was about 60 810. The purified PEG-rhIFNomega has the characteristics of typical PEGylated protein. Activity reservation rate of mono PEG-rhIFNomega was 15.0%, while the antigenicity decreased by at least 64 folds. In addition, the acid stability, thermal stability and stability in serum and trypsin solution of mono PEG-rhIFNomega were markedly better than those of the rhIFNomega. The pharmacological properties of mono PEG-rhIFNomega were significantly improved. The prepared PEG-rhIFNomega might be developed to a novel safe and long-acting interferon.
研究了用单甲氧基聚乙二醇琥珀酰亚胺琥珀酸酯（mPEG-SS，20000）对重组人ω干扰素（rhIFNomega）进行氨基聚乙二醇化，并通过离子交换色谱和凝胶过滤色谱对修饰后的混合物进行分离纯化。在优化的纯化条件下，收集液中单一聚乙二醇化重组人ω干扰素（mono PEG-rhIFNomega）的平均含量达到182μg·mL-1。mono PEG-rhIFNomega的平均纯化产率超过22%，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和反相高效液相色谱（RP-HPLC）测定，mono PEG-rhIFNomega的纯度大于98%。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）检测，mono PEG-rhIFNomega的相对分子质量为43790。通过SDS-PAGE测定的表观分子质量约为60810。纯化后的聚乙二醇化重组人ω干扰素（PEG-rhIFNomega）具有典型的聚乙二醇化蛋白特征。mono PEG-rhIFNomega的活性保留率为15.0%，而抗原性降低了至少64倍。此外，mono PEG-rhIFNomega在酸性、热稳定性以及在血清和胰蛋白酶溶液中的稳定性均明显优于rhIFNomega。mono PEG-rhIFNomega的药理性质得到显著改善。所制备的PEG-rhIFNomega有望开发成为一种新型安全长效干扰素。
