SEC-HPLC法测定溶瘤腺病毒注射液的纯度

目的： 建立检测溶瘤腺病毒注射液纯度的分子排阻高效液相色谱法（SEC-HPLC法）。方法： 采用XBridge BEH SEC分析柱和Waters 2695/2489高效液相色谱系统，以含150 mol·L^-1氯化钠的磷酸盐缓冲溶液（pH 7.0）为流动相进行等度洗脱，流速0.5 mL·min^-1，检测波长260 nm，采用峰面积归一法检测溶瘤腺病毒注射液纯度，并对方法的专属性、线性、重复性、中间精密度、检测下限和定量下限等指标进行考察。结果： 空白溶剂无干扰峰出现；未经纯化和热处理的溶瘤腺病毒溶液中溶瘤腺病毒峰与其他杂质峰分离度均大于2.0；溶瘤腺病毒的进样量在2×10⁸~2×10¹⁰病毒颗粒（VP）范围内，浓度与吸收峰的线性关系良好（R²=0.998 5）；6次进样测得的峰面积的RSD为0.78%；中间精密度试验的RSD为4.7%；检测下限为2.3×10⁶ VP，定量下限为6.7×10⁶ VP。3批溶瘤腺病毒注射液的纯度分析结果分别为99.84%、99.80%、99.64%。结论： SEC-HPLC法可用于检测溶瘤腺病毒注射液的纯度。

Objective: To develop an assay for the purity determination of oncolytic adenovirus injection by SEC-HPLC. Methods: An SEC-HPLC method was used to determine the purity of oncolytic adenovirus injection with XBridge BEH SEC column and HPLC system(Waters 2695/2489). The phosphate buffer solution(pH 7.0) containing 150 mol·L^-1 sodium chloride was used as mobile phase for isocratic elution. The flow rate was 0.5 mL·min^-1,and the detection wavelength was 260 nm. The purity of oncolytic adenovirus injection was determined by peak area normalization method. The assay was subsequently validated for its specificity,linearity,repeatability,intermediate precision,limit of detection(LOD) and limit of quantitation (LOQ). Results: There was no interference peak in the blank solvent;the resolution between the peak of oncolytic adenovirus and other impurities in the unpurified and heat-treated oncolytic adenovirus solution was greater than 2.0;the linear relationship between the concentration and the absorption peak was good in the range of 2×10⁸-2×10¹⁰ viral particle(VP),with R² being 0.998 5;peak area RSD of six injections was 0.78%;RSD of intermediate precision was 4.7%;the LOD was 2.3×10⁶ VP,and the LOQ was 6.7×10⁶ VP. The purity analysis results of three batches of oncolytic adenovirus injections were 99.84%,99.80%,and 99.64%,respectively. Conclusion: The SEC-HPLC assay can be applied to determine the purity of oncolytic adenovirus injection.

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