目的: 研制首批1型单纯疱疹病毒(herpes simplex virus 1,HSV-1)溶瘤活性测定国家标准品。方法: 按2020年版《中华人民共和国药典》三部相关要求,分别进行HSV-1溶瘤活性测定液体标准品和冻干标准品的制备和质量检测,并使用热加速试验进行稳定性考察,以U-2 OS细胞/CCK-8法分别在3家实验室对标准品的溶瘤活性进行协作标定。对比研究液体标准品和冻干标准品,选择其中更为适用的1种作为国家标准品。结果: 制备的2种HSV-1溶瘤活性测定标准品的质量检测结果均符合要求,其中冻干标准品水分含量为1.09%,分装精度为0.15%。稳定性试验结果用阿伦尼乌斯公式计算,初步预测液体标准品溶瘤活性在-70 ℃下降低10%需7.7年;冻干标准品溶瘤活性在-70 ℃下降低10%需6.1×105年,其稳定性得到很大提高。3个实验室协作标定进行了共21次测定,液体标准品的溶瘤活性值几何均数为7.08×104 U·mL-1,冻干标准品的溶瘤活性值几何均数为1.82×104 U·支-1。HSV-1标准品原液在冻干后溶瘤活性由7.08×104 U·mL-1下降至3.03×104 U·mL-1,但因其溶瘤活性在预稀释100倍后仍然出现良好的S型量效关系曲线,并不影响它作为标准品使用的要求。将液体标准品作为样品,用冻干标准品作为标准品进行校准后,3家实验室结果的几何变异系数(GCV)由64.4%下降到29.2%,实验的精密度得到较大提高。结论: 该批HSV-1溶瘤活性测定冻干标准品各项指标均符合相关要求,与液体标准品相比更适合作为国家标准品使用,其溶瘤活性赋值为1.82×104 U·支-1。
秦玺, 胡金盼, 李永红, 史新昌, 丁有学, 毕华, 韩春梅, 郑红梅, 饶春明, 梁成罡
. 首批1型单纯疱疹病毒溶瘤活性测定国家候选标准品的研制*[J]. 药物分析杂志, 2024
, 44(4)
: 663
-670
.
DOI: 10.16155/j.0254-1793.2024.04.13
Objective: To establish the first national standard for oncolytic activity assay of herpes simplex virus type 1(HSV-1). Methods: According to the requirements in Chinese Pharmacopoeia(Volume Ⅲ, 2020 edition), the liquid and freeze-dried standard for oncolytic activity of HSV-1 were prepared and tested,of which the stability were evaluated by thermal acceleration test. The oncolytic activity of the standard was calibrated collaboratively by U-2 OS cells/CCK-8 method in 3 laboratories. The liquid standard was compared with the freeze-dried standard, and the more suitable one was selected as the national standard. Results: The prepared standard substance was all qualified, among which the moisture content of the freeze-dried standard was 1.09% and the dispensing accuracy was 0.15%. The results of stability test were calculated by Arrhenius formula. It was preliminarily predicted that it would take 7.7 years for the oncolytic activity of liquid standard to decrease by 10% at -70 ℃, and it would take 6.1×105 years for the oncolytic activity of lyophilized standard to decrease by 10% at -70 ℃. Compared with liquid standard, the stability of lyophilized standard was greatly improved. Twenty-one times of collaborative calibration tests by 3 laboratories showed that the oncolytic activity liquid standard was 7.08×104 U·mL-1 and the oncolytic activity liquid standard was 1.82×104 U·vial-1. After lyophilized, the oncolytic activity of the bulk of HSV-1 standard decreased from 7.08×104 U·mL-1 to 3.03×104 U·mL-1. However, the good S-shaped dose-response curve still appeared after 100 times of pre-dilution, which did not affect the requirements of its use as a standard. When the liquid standard was used as the sample and the freeze-dried standard was used as the standard for calibration, the geometric coefficient of variation (GCV) of the results of the 3 laboratories decreased from 64.4% to 29.2%, and the precision of the experiment was greatly improved. Conclusion: The batch of freeze-dried HSV-1 standards for oncolytic activity assay meets the relavant requirements, and is more suitable for use as a national standard than liquid standards. Its oncolytic activity is assigned a value of 1.82×104 U·vial-1.
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