摘要
目的:建立重组 GM-CSF/IL-3融合蛋白的质控标准和检测方法,以用于该产品的常规质量控制。方法:用 TF-1/MTT 细胞增殖试验定量测定 GM-CSF/IL-3融合蛋白体外生物学活性;用抗 GM-CSF 和 IL-3的抗体分别进行 WesternBlot 进行鉴别试验;用 C_8-HPLC 和 SDS～PAGE 测定蛋白纯度;用胰蛋白酶消化/C_(18)-HPLC 进行肽图分析;用顶空进样气相色谱法检测甲醇残留量;还原型 SDS-PAGE 测定融合蛋白相对分子质量;其他原液和成品检测项目参照2005年版中国药典三部的方法进行。结果:GM-CSF/IL-3融合蛋白的原液及成品与 TF-1细胞的反应呈典型的 S 型曲线,符合四参数方程 y=(A-D)/[1+(x/C)(?)B)]+D,相关系数大于0.99,成品和原液的回收率均大于94%,RSD 均小于20%;与 GM-CSF 和 IL-3抗体进行的 Western Blot 均为阳性;原液纯度大于95.0%;甲醇残留量小于0.005%,其他项目均符合2005年版中国药典三部要求。结论:建立的重组 GM-CSF/IL-3融合蛋白质控标准和检测方法可以有效控制该制品的质量,并确保制品的安全与有效,已用于该制品的常规检定。
Objective：To establish the requirements and methods for quality control of recombinant human GM - CSF/IL -3 fusion protein. Methods：The MTF cell proliferation assay of TF - 1 was used to evaluate the bioaetivity of GM- CSF/IL-3 in vitro;protein purity was tested by C8 -HPLC （214 nm detective wavelength） and SDS- PAGE; Western Blot was assay using anti - GM - CSF and anti - IL - 3 antibody ;Peptide mapping was tested by trypsine digestion/C8 -HPLC;methanol residue was determined by headspace gas chromatography;relative molecular mass was determined by reduced SDS -PAGE ;other items all were referred ChP Volm. Result：Dose -response relationship of TF- 1 cell proliferation/MTT staining assay for bulk and drug products of GM -CSF/IL -3 fusion protein all displayed typical S curve and were complied with four -parameter equation y = （A -D）/[ 1 ＋ （x/C）^B） ] ＋ D, the relative coefficient was more than 0. 99;and the average recovery rate for bulk and drug products were all more than 94%, RSD were less than 20% ; Western blot results were positive using antibody of GM - CSF and IL - 3 individually; protein purity was more than 95.0% and the residual methanol was less than 0. 005% ; the other test results were all compliance with ChP Vol Ⅲ of 2005 edition. Conclusion：The specifications and analysis methods for recombinant GM - CSF/IL - 3 fusion protein had the ability to control the recombinant protein＇s quality and ensure the drug＇s safety and stability;it had already been used for GM -CSF/IL-3 routine analysis and lot release.
出处
《药物分析杂志》
CAS
CSCD
北大核心
2008年第10期1605-1608,共4页
Chinese Journal of Pharmaceutical Analysis
基金
国家863高技术发展计划项目资助(2003AA2Z3481)
