HL60-IL6法在注射用胶原酶和尿激酶热原检测中的应用研究

Application Study on HL60-IL6 in the Pyrogen Detection of Collagenase and Urokinase for Injection

关键词:  HL60-IL6单核细胞活化反应法  胶原酶  尿激酶

Key words:  HL60-IL6 monocyte activation test  collagenase  urokinase

摘要:

目的 探讨HL60-IL6单核细胞活化反应法在注射用胶原酶和尿激酶热原检测中的应用。方法 参照中国药典2015年版通则1193，确定注射用胶原酶和尿激酶最小有效稀释浓度。通过细胞增殖抑制试验、热原干扰试验和白介素-6（interleukin-6，IL-6）干扰试验探讨对HL60细胞生长，对热原和IL-6测定无影响的供试品浓度，进而确定适用于注射用胶原酶和尿激酶体外热原测定的HL60-IL6方法。用该方法对2批注射用胶原酶、19批注射用尿激酶供试品进行热原测定。结果 注射用胶原酶最小有效稀释浓度为0.04 U·mL^-1，当浓度≤ 60 U·mL^-1时对HL60细胞生长无影响，浓度≤ 6 U·mL^-1时对IL-6和热原测定无影响。注射用尿激酶最小有效稀释浓度为100 U·mL^-1，当浓度≤ 1 000 U·mL^-1时对HL60细胞生长无影响，浓度≤ 500 U·mL^-1时对IL-6和热原测定无影响。因此，适合HL60-IL6单核细胞活化反应法的注射用胶原酶和尿激酶浓度分别为6，500U·mL^-1。用该方法对供试品进行热原测定，结果均未检出热原。结论 HL60-IL6方法适用于注射用胶原酶和尿激酶热原测定。

Abstract:

OBJECTIVE To explore the application of HL60-IL6 method in the pyrogen detection for collagenase and urokinase for injection. METHODS According to the general rule 1193 of Chinese Pharmacopoeia(the 2015 Edition), the minimum effective dilution concentration of collagenase and urokinase for injection was determined. The max concentrations of samples which had no effect on HL60 proliferation, pyrogen test and IL-6 test were determined by the methods of proliferation-inhibition test, pyrogen interference and IL-6 interference test. According to the results, the in vitro pyrogen test for collagenase and urokinase for injection was established. Then, the pyrogen of 2 batches of collagenase for injection and 19 batches of urokinase for injection were determined with the method. RESULTS For collagenase, the minimum effective dilution concentration was 0.04 U·mL^-1, the sample which was ≤ 60 U·mL^-1 had no effect on HL60 proliferation, and ≤ 6 U·mL^-1 had no effect on pyrogen and IL-6 test. For urokinase, the minimum effective dilution concentration was 100 U·mL^-1, the sample which was ≤ 1 000 U·mL^-1 had no effect on HL60 proliferation, and ≤ 500 U·mL^-1 had no effect on pyrogen and IL-6 test. As a result, the suitable concentration was 6 U·mL^-1 for collagenase and 500 U·mL^-1 for urokinase respectively. Then, the suitable the in vitro pyrogen test for collagenase and urokinase for injection was established. There were no pyrogen in all samples. CONCLUSION The method is suitable for in vitro pyrogen test of collagenase and urokinase for injection.