Hydrophobic interaction chromatography(HIC)as an indispensable method for protein purification has attracted considerable attentions of researchers as well as biopharmaceutical industries.However,the low binding capac...Hydrophobic interaction chromatography(HIC)as an indispensable method for protein purification has attracted considerable attentions of researchers as well as biopharmaceutical industries.However,the low binding capacity and slow adsorption rate of the currently available HIC media lead to a little supply and high price of the highly purified proteins.Herein,nanofibrous membranes with hydrophobic binding sites were developed for HIC by directly coupling phenyl glycidyl ether on the hydrolyzed cellulose acetate nanofiber membrane(cellulose-phenyl NFM).Scanning electron microscope(SEM),water contact angle(WCA),Fourier transform infrared(FTIR),thermogravimetric analysis(TGA),Brunauer-Emmett-Teller(BET)surface area analysis and capillary flow porometer(CFP)were applied to evaluate the physically and chemically structural transformation.The obtained cellulose-phenyl NFMs showed a proper hydrophilcity(WCA=37°),a relatively high BET surface area(3.6 times the surface area of commercial fibrous membranes),and tortuous-channel structure with through-hole size in the range of 0.25-1.2μm,which led to a little non-specificity adsorption,high bovine serum albumin adsorption capacity of 118 mg g^(−1),fast adsorption process within 12 h,good long-term stability and reusability.Moreover,compared with traditional modification methods which always include activation and graft two steps,direct coupling method is more efficient for HIC media fabrication.Therefore,cellulose-phenyl NFMs with outstanding protein adsorption performance could be a kind of promising candidate for HIC.展开更多
重组人干扰素2b作为一种上市药物,被广泛用于治疗慢性丙型肝炎、慢性乙型肝炎、恶性黑色素瘤等多种疾病的治疗,产生了良好的临床疗效和经济效益。目前国内厂家生产的重组人干扰素以大肠杆菌基因工程表达为主。大肠杆菌表达的人干扰素2b...重组人干扰素2b作为一种上市药物,被广泛用于治疗慢性丙型肝炎、慢性乙型肝炎、恶性黑色素瘤等多种疾病的治疗,产生了良好的临床疗效和经济效益。目前国内厂家生产的重组人干扰素以大肠杆菌基因工程表达为主。大肠杆菌表达的人干扰素2b以包涵体的形式表达,包涵体产物需通过变性复性以及其后的纯化才能获得生物活性。干扰素复性得率低,纯化时需要经过四步色谱处理,工艺繁琐,成本高,而且无法去除未完全复性的中间产物,从而影响产品质量和疗效。建立可溶性重组人干扰素α2b的高密度发酵工艺,并在此基础上,建立其高效、快速和稳定的纯化工艺。通过发酵工艺优化获得可溶性重组人干扰素α2b的高密度发酵,收集菌体后,通过高压破菌、硫酸铵分级沉淀、phenyl Sepharose Fast Flow疏水层析、Q Sepharose Fast Flow阴离子交换层析、SP Sepharose Fast Flow阳离子交换层析、Octyl Sepharose Fast Flow疏水层析进行分离纯化,并对纯化产物进行生物活性分析。重组人干扰素α2b工程菌的发酵密度A_(600)达到38.5,可溶性重组人干扰素α2b占菌体总蛋白的14.0%;100克湿菌可平均纯化获得240 mg纯度接近100%的重组人IFNα2b,比活为1.81×10^(8) IU/mg。建立的重组人干扰素α2b原液生产工艺具有良好的效率、回收率和活性,产品质量远高于国家药典规定的质量标准。因此,本文所建立生产工艺可用于重组人干扰素α2b工业化生产。展开更多
基金This work was supported by the National Natural Science Foundation of China(Grant Nos.51673037,51873029,and 81771338)the Science and Technology Commission of Shanghai Municipality(Grant No.18511109500).
文摘Hydrophobic interaction chromatography(HIC)as an indispensable method for protein purification has attracted considerable attentions of researchers as well as biopharmaceutical industries.However,the low binding capacity and slow adsorption rate of the currently available HIC media lead to a little supply and high price of the highly purified proteins.Herein,nanofibrous membranes with hydrophobic binding sites were developed for HIC by directly coupling phenyl glycidyl ether on the hydrolyzed cellulose acetate nanofiber membrane(cellulose-phenyl NFM).Scanning electron microscope(SEM),water contact angle(WCA),Fourier transform infrared(FTIR),thermogravimetric analysis(TGA),Brunauer-Emmett-Teller(BET)surface area analysis and capillary flow porometer(CFP)were applied to evaluate the physically and chemically structural transformation.The obtained cellulose-phenyl NFMs showed a proper hydrophilcity(WCA=37°),a relatively high BET surface area(3.6 times the surface area of commercial fibrous membranes),and tortuous-channel structure with through-hole size in the range of 0.25-1.2μm,which led to a little non-specificity adsorption,high bovine serum albumin adsorption capacity of 118 mg g^(−1),fast adsorption process within 12 h,good long-term stability and reusability.Moreover,compared with traditional modification methods which always include activation and graft two steps,direct coupling method is more efficient for HIC media fabrication.Therefore,cellulose-phenyl NFMs with outstanding protein adsorption performance could be a kind of promising candidate for HIC.
文摘重组人干扰素2b作为一种上市药物,被广泛用于治疗慢性丙型肝炎、慢性乙型肝炎、恶性黑色素瘤等多种疾病的治疗,产生了良好的临床疗效和经济效益。目前国内厂家生产的重组人干扰素以大肠杆菌基因工程表达为主。大肠杆菌表达的人干扰素2b以包涵体的形式表达,包涵体产物需通过变性复性以及其后的纯化才能获得生物活性。干扰素复性得率低,纯化时需要经过四步色谱处理,工艺繁琐,成本高,而且无法去除未完全复性的中间产物,从而影响产品质量和疗效。建立可溶性重组人干扰素α2b的高密度发酵工艺,并在此基础上,建立其高效、快速和稳定的纯化工艺。通过发酵工艺优化获得可溶性重组人干扰素α2b的高密度发酵,收集菌体后,通过高压破菌、硫酸铵分级沉淀、phenyl Sepharose Fast Flow疏水层析、Q Sepharose Fast Flow阴离子交换层析、SP Sepharose Fast Flow阳离子交换层析、Octyl Sepharose Fast Flow疏水层析进行分离纯化,并对纯化产物进行生物活性分析。重组人干扰素α2b工程菌的发酵密度A_(600)达到38.5,可溶性重组人干扰素α2b占菌体总蛋白的14.0%;100克湿菌可平均纯化获得240 mg纯度接近100%的重组人IFNα2b,比活为1.81×10^(8) IU/mg。建立的重组人干扰素α2b原液生产工艺具有良好的效率、回收率和活性,产品质量远高于国家药典规定的质量标准。因此,本文所建立生产工艺可用于重组人干扰素α2b工业化生产。
