目的 建立定量測定重組人IL-12注射液中泊洛沙姆188的HPLC分析方法。方法 采用Waters Alliance系統(tǒng)、蒸發(fā)光檢測器、賽分Poly RP-100色譜柱，以0.1% TFA水溶液（A）-0.1% TFA乙腈溶液（B）作為流動相梯度洗脫：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。體積流量1.0 mL/min，柱溫30℃測定重組人IL-12注射液中泊洛沙姆188。結果 HPLC法測定泊洛沙姆188的保留時間RSD（n=6）為0.19%，峰面積RSD（n=6）為1.7%。泊洛沙姆188在低（50%）、中（100%）、高（150%）個濃度的回收率分別為93.3%、93.1%、93.8%，平均回收率為93.4%。泊洛沙姆188在1.5～9 μg（r>0.99）與峰面積呈良好的線性關系。結論 建立了重組人IL-12注射液中泊洛沙姆188含量檢測的高效液相色譜法，并對建立的分析方法進行專屬性、準確度、精密度、檢測限、定量限、線性、范圍和耐用性的驗證，測定了3批重組人IL-12注射液中的泊洛沙姆188的量。

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.

十三五科技重大專項課題“生物類似藥質量相似性評價體系建設研究（2015ZX09501008）”資助項目