光阻法检测治疗性抗体不溶性微粒的取样方式探讨

目的：考察不同取样方式对光阻法检测小装量治疗性抗体不溶性微粒的影响。方法：采用光阻法对体积小于25 mL的19个靶点65个批次治疗性抗体中≥ 2 μm、≥ 10 μm和≥ 25 μm的不溶性微粒进行检测，将抗体分为3组，取样方式分别为单支样品不同取样体积（0.5、1、3 mL）检测与合并样品（取样体积5 mL）检测，合并样品不同取样体积（0.5、1、3、5 mL）检测，以及合并样品后稀释（稀释20、10、5倍）检测与合并样品原倍检测。结果：对于≥ 2 μm、≥ 10 μm、≥ 25 μm的不溶性微粒，不管是采用单支样品不同取样体积还是采用合并样品后不同取样体积，与合并样品后每次取样5 mL的不溶性微粒检测结果相比，均没有统计学意义上的差异；且≥ 2 μm不溶性微粒的取样体积变异小于≥ 10 μm、≥ 25 μm的不溶性微粒。合并样品后稀释检测的结果则与合并样品检测的结果具有统计学差异，且稀释后的结果要高于未稀释的结果，稀释倍数越大，与未稀释结果的差异也越大。结论：采用光阻法对体积小于25 mL的小装量治疗性抗体进行不溶性微粒检测时，为节省样品并减少外源微粒污染，不管单支取样还是合并取样，均可首先考虑选择小于5 mL的单次取样体积进行检测，而对合并后稀释检测的方式应慎重选择。

Objective: To explore the effect of different sampling methods on sub-visible particle testing of smallvolume therapeutic antibodies by light obscuration method.Methods: Sub-visible particles in 65 lots of therapeutic antibodies against 19 targets were detected using light obscuration method. The volumes of adopted antibodies were all below 25 mL.The detection channels of ≥ 2 μm, ≥ 10 μm and ≥ 25 μm were included. The antibodies were divided into 3 groups, and the corresponding sampling methods were single dose with different sampling volumes (0.5,1,3 mL)and combined doses(sample volume 5 mL); combined doses with different sampling volumes(0.5, 1, 3, 5 mL); combined doses with different dilutions(20 times, 10 times, 5 times)and combined doses without dilution.Results: For sub-visible particles of ≥ 2 μm, ≥ 10 μm and ≥ 25 μm, the testing results of different sampling volumes with single dose or combined doses were not significantly different with combined doses with sampling volume of 5 mL; and the result variation of ≥ 2μm particles was smaller than that of ≥ 10 μm and ≥ 25 μm particles. However, there was significant difference between results of combined doses with different dilutions and combined doses without dilution. And the results of diluted samples were higher than those of without dilution; the larger the dilution multiple, the higher the result is.Conclusion: When light obscuration method is applied in testing sub-visible particles of therapeutic antibodies with volumes of less than 25 mL, small sampling volume of less than 5 mL was the prior consideration in order to save samples and reduce exogenous particles, for both single dose and combined doses; while method of combined doses with different dilutions should be used with caution.