重组人胸腺素β4滴眼液蛋白含量反相高效液相色谱检测方法的建立及验证  

Development and verification of a reversed-phase high-performance liquid chromatography method for determination of protein content in recombinant human thymosin beta 4 eye drops

作　　者：张波[1,2] 王艺诺 孙进 ZHANG Bo;WANG Yinuo;SUN Jin(Shenyang Pharmaceutical University,Shenyang 110016,Liaoning Province,China)

机构地区：[1]沈阳药科大学,辽宁沈阳110016 [2]北京诺思兰德生物技术股份有限公司,北京100085

出　　处：《中国生物制品学杂志》2025年第1期96-101,共6页Chinese Journal of Biologicals

基　　金：政府间国际科技创新合作项目(2021YFE0115300)。

摘　　要：目的建立重组人胸腺素β4(recombinant human thymosin beta 4,rh-Tβ4)滴眼液中蛋白含量检测的反相高效液相色谱(reversed-phase high-performance liquid chromatography,RP-HPLC)法,并进行验证,以期对rh-Tβ4滴眼液中蛋白含量进行质量控制。方法用ZORBAX 300SB-C18(4.6 mm×250 mm,5μm)色谱柱,以超纯水、乙腈、三氟乙酸为流动相进行梯度洗脱,紫外检测波长为210 nm,进样量为100μL,流速为1.0 mL/min,柱温为25℃,样品室温度为5℃,以标准曲线法计算滴眼液中蛋白含量;对方法进行系统适用性、专属性、线性与范围、准确性、重复性、中间精密度、溶液稳定性、耐用性验证。用建立的RP-HPLC法检测4批rh-Tβ4滴眼液蛋白含量。结果系统适用性主峰理论塔板数分别为192039、185819、186348,均不低于5000,主峰与相邻峰分离度分别为3.88、3.79、3.76,均大于1.5,系统适用性合格;空白溶剂、空白对照均未影响重组rh-Tβ4主峰积分,对检测结果无干扰,方法专属性良好;蛋白含量在50~150μg/mL范围内标准曲线线性良好,相关系数(R^(2))为0.9923,大于0.99;高、中、低浓度标准品回收率分别为98.01%、99.17%、97.70%;同浓度6份样品蛋白含量的RSD为1.38%;不同实验人员不同日期检测同一样品蛋白含量的RSD为1.05%;在0、6、12、18、24 h内,同一份供试品蛋白含量的RSD为0.36%;供试品在23、25和27℃柱温下蛋白含量的RSD为0.45%。4批rh-Tβ4滴眼液蛋白含量分别为0.51、0.51、0.49、0.50 mg/mL,均与标示量一致。结论建立了rh-Tβ4滴眼液蛋白含量检测的RP-HPLC法,经验证准确可靠,可对rh-Tβ4滴眼液中蛋白含量进行质量控制。Objective To develop and verify a reversed-phase high-performance liquid chromatography(RP-HPLC)method for the determination of protein content in recombinant human thymosin beta 4(rh-Tβ4)eye drops for the quality control of protein content in rh-Tβ4 eye drops.Methods A ZORBAX 300SB-C18(4.6 mm×250 mm,5μm)chromatographic column was used for gradient elution with ultrapure water,acetonitrile and trifluoroacetic acid as mobile phases.The UV detection wavelength was 210 nm,the injection volume was 100μL,the flow rate was 1.0 mL/min,the column temperature was 25℃,and the sample room temperature was 5℃.The protein content in the eye drops was calculated by standard curve method.The system applicability,specificity,linearity and range,accuracy,repeatability,intermediate precision,solution stability and durability of the method were verified.The protein contents of four batches of rh-Tβ4 eye drops was detected by using the developing RP-HPLC.Results The theoretical number of trays for the main peak of system applicability was 192039,185819 and 186348 respectively,all of which were not less than 5000.The separation degree between the main peak and adjacent peaks was 3.88,3.79 and 3.76 respectively,all greater than 1.5,indicating that the system applicability was qualified.The blank solvent and blank control did not affect the main peak integration of recombinant rh-Tβ4,and did not interfere with the detection results,indicating that the specificity was good.The standard curve for protein content within the range of 50μg/mL to 150μg/mL showed good linearity,with the correlation coefficient(R^(2))of 0.9923,greater than 0.99.The recovery rates of high,medium and low concentration standards were 98.01%,99.17%and 97.70%respectively.The RSD of protein content in 6 samples with the same concentration was 1.38%.The RSD of protein content in the same sample detected by different experimenters on different dates was 1.05%.Within 0,6,12,18 and 24 h,the RSD of protein content in the same test sample was 0.36%.The RSD of

关 键 词：反相高效液相色谱法 重组人胸腺素β4滴眼液 蛋白含量 

分 类 号：R917[医药卫生—药物分析学]