Gao Kai, Wang Jun-zhi, Rao Chun-ming, Wu Xiao-bing
National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China.
Yao Xue Xue Bao. 2003 Sep;38(9):684-9.
To establish quality control requirements and methods for recombinant adeno-associated virus(rAAV) type 2/human blood coagulation factor IX (rAAV-2/hFIX).
Identification of rAAV genome fragments, potential contaminants including wild type AAV(wtAAV) and helper virus, were detected by PCR. Purity of rAAV-2/hFIX was analyzed by cation-exchange HPLC and SDS-PAGE. Virus partical numbers were performed by dot blot assay. hFIX expression was demonstrated by ELISA and potency of hFIX was verified by APTT.
Identity of rAAV-2/hFIX was proved. Residues of wtAAV and helper virus were conformed to requirements. Purity of rAAV-2/hFIX were more than 98%. Partical numbers of rAAV-2/hFIX were more than 1.0 x 10(15) VG.L-1. hFIX expression was more than 20.0 micrograms.L-1. hFIX potency was verified by APTT following rAAV-2/hFIX injected to FIX gene knockout mice, potency results conformed to requirements.
The methods and requirements had been established for quality control of rAAV-2/hFIX.
建立重组腺相关病毒2型/人凝血因子IX（rAAV-2/hFIX）的质量控制要求和方法。
采用聚合酶链反应（PCR）检测rAAV基因组片段、包括野生型腺相关病毒（wtAAV）和辅助病毒在内的潜在污染物。通过阳离子交换高效液相色谱法（HPLC）和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析rAAV-2/hFIX的纯度。采用斑点印迹法测定病毒颗粒数。通过酶联免疫吸附测定（ELISA）证明hFIX表达，并通过活化部分凝血活酶时间（APTT）验证hFIX的效价。
证实了rAAV-2/hFIX的一致性。wtAAV和辅助病毒的残留符合要求。rAAV-2/hFIX的纯度大于98%。rAAV-2/hFIX的颗粒数大于1.0×10¹⁵vg·L⁻¹。hFIX表达大于20.0μg·L⁻¹。将rAAV-2/hFIX注射到FIX基因敲除小鼠体内后，通过APTT验证hFIX效价，效价结果符合要求。
已建立rAAV-2/hFIX质量控制的方法和要求。
