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.
WANG Fang, NIU Gang, ZHENG Wei, XU Ming-bo, ZHENG Feng
. Study on acylation modification products of insulin degludec and establishment of detection method[J]. Chinese Journal of Pharmaceutical Analysis, 2021
, 41(4)
: 645
-654
.
DOI: 10.16155/j.0254-1793.2021.04.11
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