重组人组织型纤溶酶原激活剂改构体质控方法的建立

目的建立重组人组织型纤溶酶原激活剂(tissue type plasminogen activator,tPA)改构体(TNK-tPA)的质控方法。方法采用气泡法测定TNK-tPA原液的生物学活性,非还原SDS-PAGE和RP-HPLC法测定其纯度,还原型SDS-PAGE确定其相对分子质量,HPLC-SEC法测定其单链含量,胰酶裂解法分析其肽图,Edman降解法测定其N-末端氨基酸序列,毛细管等电聚焦电泳法测定其等电点,Lowrry法测定其蛋白质含量,地高辛法测定其外源DNA残留量。按《中国药典》三部(2005版)要求,对TNK-tPA成品进行鉴别试验及外观、pH值、水分含量、无菌、装量、异常毒性等检测。结果 3批TNK-tPA原液的生物学活性为(2.09～2.16)×106U/ml,比活性均大于3.5×105U/mg,因此确定TNK-tPA原液的比活性不得低于3.0×105U/mg。3批TNK-tPA原液的非还原型SDS-PAGE纯度均大于98.0%,RP-HPLC纯度均大于97.0%;经还原型SDS-PAGE分析可见两条带,相对分子质量分别为68 000和35 000;样品原液的单链含量为75.7%;3批TNK-tPA原液的肽图酶切图谱与理化参考品一致;TNK-tPA原液的N-末端氨基酸序列、等电点、蛋白质含量、外源DNA残留量及成品的鉴别试验结果、外观、pH值、水分含量、无菌、装量、异常毒性等均符合《中国药典》三部(2005版)要求。结论建立的质控方法可保证产品安全、有效、质量可控,可用于TNK-tPA产品的常规检定。

Development of methods for quality control of recombinant human Tenecteplase

Objective To develop methods for quality control of recombinant human Tenecteplase(TNK-tPA).Methods The bulk of TNK-tPA was determined for biological activity by bubble assay,for purity by non-reduced SDS-PAGE and RP-HPLC,for relative molecular mass by reduced SDS-PAGE,for single chain content by HPLC-SEC,for peptide map by trypsin digestion,for amino acid sequence at N-terminus by Edman degradation,for isoelectric point by capillary isoelectric focusing electrophoresis,for protein content by Lowrry method,and for residual exogenous DNA content by Digoxin method.Identity,appearance,pH value,moisture content,sterility,filling quantity and abnormal toxicity of final product of TNK-tPA were tested according to the requirements in Chinese Pharmacopoiea(Volume III,2005 edition).Results The biological activities of three batches of bulks of TNK-tPA were(2.09 ~ 2.16) × 106 U / ml,while the specific activities were more than 3.5 × 105 U / mg,thus the specific activity of bulk of TNK-tPA was defined as not less than 3.0 × 105 U / mg.All the non-reduced SDS-PAGE purities of three batches of bulk of TNK-tPA were more than 98.0%,while the RP-HPLC purities were more than 97.0%.Two bands were observed on reduced SDS-PAGE profile,with relative molecular masses of 68 000 and 35 000 respectively.The single chain content in bulk was 75.7%.All the peptide maps of three batches of bulks were consistent with those of physiochemical references.The amino acid sequence at N-terminus,isoelectric point,protein content,residual exogenous DNA content of bulk,as well as the test results of Identity,appearance,pH value,moisture content,sterility,filling quantity and abnormal toxicity of final product,met the requirements in Chinese Pharmacopoiea(Volume III,2005 edition).Conclusion The developed methods ensured the safety,effectiveness and controllability of TNK-tPA,which might be used for routine control tests.