人纤维蛋白原干热病毒灭活效果及灭活前后的质量变化

Inactivation effect of human fibrinogen by dry heat and quality change before and after inactivation

目的 评价人纤维蛋白原干热病毒灭活效果及灭活前后的质量变化。方法 取3批人纤维蛋白原样品,按样品与病毒9∶1的体积比分别加入Sindbis病毒和脑心肌炎病毒(encephalomyocarditis virus,EMCV),冻干后,于(99.5±0.5)℃下干热灭活30 min。96孔微量细胞病变法测定病毒滴度降低量,考察人纤维蛋白原干热灭活效果;并对干热前/后样品进行可见异物、稳定性、纯度、凝固活力、SDS-PAGE、高效液相分子排阻色谱(high performance liquid chromatography-size exclusion chromatography,HPLC-SEC)、圆二色光谱、荧光光谱、差示扫描荧光(differential scanning fluorimetry,DSF)分析,以评价其质量变化。结果 3批人纤维蛋白原样品干热30 min,Sindbis病毒滴度降低量分别为6.13、5.88、6.00 LgTCID_(50)/0.1 mL,EMCV滴度降低量分别为≥4.94、≥4.68、≥5.00 LgTCID_(50)/0.1 mL。3批人纤维蛋白原样品干热前/后,可见异物、稳定性试验均合格;SDS-PAGE分析可见相对分子质量约65 000、56 000和47 000的目的蛋白条带,未见裂解条带;纯度均值分别为(87.7±0.9)%和(87.7±1.5)%,活力均值分别为(18±0.8)和(18±0.5)s,HPLC纯度均值分别为(86.16±1.60)%和(86.36±0.67)%,最大荧光发射光谱均值分别为(341.9±0.2)和(342.1±0.2)nm,热转变中点温度1(mid point tem-perature of thermal transition 1,Tmid 1)均值分别为(47.84±0.09)和(47.36±0.36)℃,Tmid 2均值分别为(52.34±0.15)和(52.19±0.33)℃,差异均无统计学意义(P> 0.05);α螺旋、β折叠、β转角、无规卷曲结构比例均值差异无统计学意义(P> 0.05)。结论 干热灭活可对人纤维蛋白原进行有效病毒灭活,灭活后质量未发生变化。

Objective To investigate the inactivation effect of human fibrinogen by dry heat and the quality change before and after inactivation. Methods Samples were taken from three batches of human fibrinogen,and added with Sindbis virus and encephalomyocarditis virus(EMCV)at a volume ratio of 9 ∶ 1 respectively,lyophilized and inactivated by dry heat at(99. 5 ± 0. 5)℃ for 30 min. The decrease of viral titer was determined by micro-CPE method on a 96-well microtiter plate to evaluate the inactivation effect of dry heat. The samples before and after inactivation were subjected to the inspection on visible foreign matters,tests for stability,purity and coagulation activity,SDS-PAGE,high performance liquid chromatography-size exclusion chromatography(HPLC-SEC),circular dichroism spectrum,fluorescence spectrum and differential scanning fluorimetry(DSF)to evaluate the quality change. Results After the three batches of samples were heated for 30 min,the titers of Sindbis virus decreased by 6. 13,5. 88 and 6. 00 LgTCID_(50)/0. 1 mL,while the EMCV titers decreased by not less than 4. 94,not less than 4. 68 and not less than 5. 00 LgTCID_(50)/0. 1 mL respectively. All the three batches of samples before and after inactivation were qualified for inspection on visible foreign matters and test for stability. Protein bands with relative molecular masses of about 65 000,about 56 000 and about 47 000 were observed on SDS-PAGE profiles of samples before and after inactivation. The mean purities of samples before and after inactivation were(87. 7 ± 0. 9)% and(87. 7 ± 1. 5)%,while the mean activities were(18 ± 0. 8)and(18 ± 0. 5)s,mean HPLC purities were(86. 16 ± 1. 60)% and(86. 36 ± 0. 67)%,the maximum mean fluorescence spectra were(341. 9 ± 0. 2)and(342. 1 ± 0. 2)nm,the mean mid point temperatures of thermal thansition 1(Tmid 1) were(47. 84 ± 0. 09) and(47. 36 ± 0. 36)℃,and the mean Tmid 2 were(52. 34 ± 0. 15)and(52. 19 ± 0. 33)℃,respectively,which showed no significant difference(P > 0. 05). However,the mean proportions of α helix,β sheet,β corner and random coli in samples before and after inactivation showed no significant difference(P > 0. 05). Conclusion Dry heat was effective for inactivation of human fibrinogen,and the quality of samples remains unchanged before and after inactivation.