血管内皮生长因子抑制剂生物学活性检测方法的建立

目的建立血管内皮生长因子抑制剂(VEGF Trap)生物学活性检测方法。方法利用HEK293/D9/Flt-18R(al-pha)/Flt-IL18R(beta),clone V3H9细胞系,通过荧光素酶检测系统(Steady-Glo~ Luciferase Assaysystem)进行VEGF Trap的生物学活性检测,用VEGF Trap参考品计算供试品的相对百分效价,并对该方法进行精密性和准确性验证。结果VEGF Trap供试品及参考品在该方法中均存在量效关系,且符合四参数方程式:y=(A-D)/1+(X/C)B]+D。3批VEGF Trap原液和6批成品经3次测定,相对百分效价的平均值在(90.00±2.40)%～(116.77±16.50)%之间,变异系数均小于15%。1批VEGF Trap原液及成品经3次测定,回收率分别为(90.40±2.67)%和(117.20±18.12)%。结论已成功建立VEGF Trap生物学活性检测方法,该方法重复性好,准确性高,可作为VEGFTrap生物学活性的常规检测方法。

Development of A Method for Determination of Biological Activity of Vascular Endothelial Growth Factor Trap

Objective To develop a method for determination of biological activity of vascular endothelial growth factor (VEGF)Trap. Methods The biological activity of VEGF Trap was determined by Steady-Glo～ Luciferase Assay System using HEK293 / D9 / Flt-18R (alpha)/ Flt-IL18R (beta), clone V3H9 cell line, and relative percent potency was calculated. The developed method was verified for precision and accuracy. Results Either test sample or reference of VEGF Trap showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation: y =(A D)/1 +(X / C)B]+ D. Three batches of bulks and six batches of final products of VEGF Trap were determined for 3 times, and the result showed mean relative percent potencies of(90. 00 ± 2. 40)% ～(116. 77 ± 16. 50)%, with a variation coefficient of less than 15%. One batch of bulk and one batch of final product were determined for 3 times, and the result showed recovery rates of (90. 40 ± 2. 67)% and (117. 20 ± 18. 12)% respectively. Conclusion A method for determination of biological activity of VEGF Trap was successfully developed, which showed good reproducibility and high accuracy and might be used routinely.