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[摘要]
目的 建立定量测定重组人IL-12注射液中泊洛沙姆188的HPLC分析方法。方法 采用Waters Alliance系统、蒸发光检测器、赛分Poly RP-100色谱柱,以0.1% TFA水溶液(A)-0.1% TFA乙腈溶液(B)作为流动相梯度洗脱:0~5 min,50% A→50% A;5~6 min,50% A→5% A;6~12 min,5% A→5% A;12~13 min,5% A→50% A;13~20 min,50% A→50% A。体积流量1.0 mL/min,柱温30℃测定重组人IL-12注射液中泊洛沙姆188。结果 HPLC法测定泊洛沙姆188的保留时间RSD(n=6)为0.19%,峰面积RSD(n=6)为1.7%。泊洛沙姆188在低(50%)、中(100%)、高(150%)个浓度的回收率分别为93.3%、93.1%、93.8%,平均回收率为93.4%。泊洛沙姆188在1.5~9 μg(r>0.99)与峰面积呈良好的线性关系。结论 建立了重组人IL-12注射液中泊洛沙姆188含量检测的高效液相色谱法,并对建立的分析方法进行专属性、准确度、精密度、检测限、定量限、线性、范围和耐用性的验证,测定了3批重组人IL-12注射液中的泊洛沙姆188的量。
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[Abstract]
Objective To develop a HPLC method for potency determination of poloxamer 188 in recombinant human IL-12 injection. Methods A Waters Alliance 2695 system with a sepax poly RP-100 column was used for separation and subsequent detection by an evaporative light scattering detector(ELSD); 0.1%TFA-water and 0.1%TFA-acetonitrile were used as eluent A and B, and gradient elution conditions were as follows:0-5 min, 50%A→50%A; 5-6 min, 50%A→5%A; 6-12 min, 5%A→5%A; 12-13 min, 5%A→50%A; 13-20 min, 50%A→50%A. The flow rate was 1.0 mL/min; the column was maintained at 30℃. Results The RSD of retention time and peak area for poloxamer 188 were 0.19% and 1.7%, respectively (n=6); the recovery rates at low (50%), middle (100%) and high (150%) dose were 93.3%, 93.1% and 93.8%, and the average was 93.4%; the potency of poloxamer 188 between 1.5 μg and 9.0 μg was linearly related to peak area. Conclusions A HPLC for potency determination of poloxamer 188 in recombinant IL-12 injection was successfully developed and validated in specificity, accuracy, precion, limit of detection (LOD), limit of quantitation (LoQ), linearity, detection range and robustness. The developed method was finally applied to potency determination of poloxamer 188 in three recombinant IL-12 injections.
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[基金项目]
十三五科技重大专项课题“生物类似药质量相似性评价体系建设研究(2015ZX09501008)”资助项目