目的：对特定反应条件下的酰化修饰产物进行研究,利用RP-HPLC 建立德谷胰岛素（IDeg）修饰产物的质控方法。方法：对德谷胰岛素酰化修饰产物进行质谱相对分子质量分析,确定重点研究的修饰副产物。制备相应修饰副产物对照品,利用Waters Acquity H-Class/Xevo G2-XS QTof 系统对各对照品进行结构确认（质谱相对分子质量、肽图覆盖率）。建立德谷胰岛素修饰产物质控方法,采用Welch Ultimate XB-C18（250 mm×4.6 mm,5 μm,300 Å）色谱柱,以0.1% 三氟乙酸溶液-0.1% 三氟乙酸乙腈溶液为流动相,流速1.0 mL·min-1,检测波长214 nm,柱温35 ℃,进样量20 μL。结果：确定了3种重点研究的修饰副产物IDeg-A1、IDeg-B1、IDeg-A1B29。IDeg、IDeg-A1、IDeg-B1、IDeg-A1B29 相对分子质量均与理论值一致;覆盖率均为100% 且修饰位点正确。对修饰未完全样品、修饰完全样品进行检测,修饰未完全样品、修饰完全样品含量分别为5.45、7.13 mg·mL-1,IDeg 纯度分别为46.651%、63.325%。对建立的德谷胰岛素修饰产物质控方法进行方法学验证,IDeg 质量浓度在0.20~0.75 mg·mL-1 范围内与峰面积线性关系良好（R2=1.000 0,n=5）,回收率范围为98.3%~100.5%（n=9）;有关物质IDeg-A1、IDeg-B1、IDeg-A1B29、D30的检测下限分别为1.50、1.83、1.40、4.00 μg·mL-1;定量下限分别为3.00、3.65、4.20、5.00 μg·mL-1。结论：德谷胰岛素酰化修饰产物中的修饰副产物主要包括IDeg-A1、IDeg-B1、IDeg-A1B29。建立了IDeg修饰产物的质控方法,该方法准确度、精密度、专属性、耐用性良好,可用于IDeg的含量测定及纯度分析;为进行C16-谷氨酸-NHS 修饰位点的专一性研究、优化修饰条件、制定质量标准提供支持。
Objective: To study on the composition of acylated modified products under specific reaction conditions,and establish a quality control method for modified products of insulin degludec（IDeg） by RP-HPLC. Methods: The acylated modified products of IDeg were analyzed by relative molecular mass spectrometry,and the modified by-products were determined. The Waters Acquity H-Class/Xevo G2-XS QTof system was used to confirm the structure（relative molecular mass of mass spectrum,peptide coverage） of each control. A RPHPLC method was established to control the modified products. The chromatographic separation was achieved on Welch Ultimate XB-C18column（250 mm×4.6 mm,5 μm）,using 0.1% aqueous trifluoroacetic acid-0.1% trifluoroacetic acid acetonitrile as mobile phase. The flow rate was 1.0 mL·min-1. The detection was performed at 214 nm,and the column temperature was 35 ℃. The injection volume was 20 μL. Results: Three byproducts, including IDeg-A1,IDeg-B1,IDeg-A1B29,were identified. The relative molecular mass of IDeg, IDeg-A1,IDeg-B1 and IDeg-A1B29 was consistent with the theoretical value,the coverage rates were 100% and the modified sites were correct. The incompletely modified sample and the completely modified sample were tested. The contents of the incompletely modified sample and the completely modified sample were 5.45 mg·mL-1 and 7.13 mg·mL-1. The purities of IDeg were 46.651% and 63.325%,respectively. According to the method validation,IDeg had a good linear relationship with peak area within the range of 0.20-0.75 mg·mL-1 （R2=1.000 0,n=5）. The recovery ranged from 98.28% to 100.51%（n=9）. The limits of detection of related substances IDeg-A1,IDeg-B1,IDeg-A1B29,D30 were 1.50,1.83,1.40,4.00 μg·mL-1,respectively. And the limits of quantification were 3.00,3.65,4.20,5.00 μg·mL-1,respectively. Conclusion: Main modified by-products of the acylated modified products of IDeg are IDeg-A1,IDeg-B1 and IDeg-A1B29. A quality control method for modified products of IDeg is established. The developed method has good accuracy,precision,specificity and durability. It is suitable for the determination of the content and purity analysis of IDeg. It provides support for the specificity study of C16-glutamic acid-NHS modification site,optimization of modification conditions and development of quality standards.
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