Isolation and identification of cyclic imide and deamidation products in heat stressed pramlintide injection drug product
Isolation and identification of cyclic imide and deamidation products in heat stressed pramlintide injection drug product
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DOI：
10.1016/s0731-7085(99)00075-8
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发表时间：
1999-09-01
影响因子：
3.4
通讯作者：
Williams, JD
中科院分区：
文献类型：
--
作者：
Hekman, CM;DeMond, WS;Williams, JD
本报告总结了从40℃下应激45天的普拉林肽最终产品样品中鉴定出六个环酰亚胺[ASU]和两个脱酰胺化产物。采用阳离子交换高效液相色谱(HPLC)和反相高效液相色谱(RP-HPLC)分离普拉林肽降解产物。对分离出的组分进行了MS、MS/MS鉴定，必要时还进行了酶(热裂解)消化、LC/MS和序列分析。分离得到的产物分别鉴定为[ASU(14)]-普拉林肽、[ASU(21)]-普拉林肽、[ASU(22)]-普拉林肽、[ASU(35)]-普拉林肽、[1-21]-琥珀酰亚胺-普拉林肽和[1-22]-琥珀酰亚胺-普拉林肽。还鉴定了[Asp(35)]-普拉林肽和[Tyr(37)-OH]-普拉林肽，[Asp(35)]-Pramlinide是普拉林肽在ASN(35)上的脱酰胺产物，以及[Tyr(37)-OH]-Pramlinide是酰胺化的普拉林肽C-末端Tyr的脱酰胺产物。总之，这些数据支持前面提出的(C.Hekman等人，分离和鉴定热应激普拉林肽注射药物产品的多肽降解产物)。药典1998；15：650-9)表明普拉林肽在该pH 4.0配方中的主要降解机制是脱酰胺作用，观察到8个可能的脱酰胺部位中有6个发生了脱酰胺作用。Gln-10和ASN-31是仅有的两个可进行脱酰胺化的残基，没有观察到任何残基。数据表明，环酰亚胺产物约占总热降解的20%，脱酰胺化产物约占热降解产物的20%。剩余的降解是由于肽骨架的水解。(C)1999 Elsevier Science B.V.保留所有权利。
This report summarizes the identification of six cyclic imide [Asu] and two deamidation products from a sample of pramlintide final drug product that had been stressed at 40 degrees C for 45 days. The pramlintide degradation products were isolated by cation exchange high-performance liquid chromatography (HPLC) followed by reversed-phase HPLC. The isolated components were characterized by mass spectrometry (MS), tandem MS (MS/MS) and when necessary, by enzymatic (thermolysin) digestion followed by liquid chromatography/mass spectrometry (LC/MS) and sequence analysis. The isolated products were identified as [Asu(14)]-pramlintide, [Asu(21)]-pramlintide, [Asu(22)]-pramlintide, [Asu(35)]-pramlintide, [1-21]-succinimide-pramlintide, and [1-22]-succinimide-pramlintide. Also identified were [Asp(35)]-pramlintide, the deamidation product of pramlintide at Asn(35), and [Tyr(37)-OH]-pramlintide, the deamidation product of the pramlintide amidated C-terminal Tyr. Together these data support those presented earlier (C. Hekman et al., Isolation and identification of peptide degradation products of heat stressed pramlintide injection drug product. Pharm Res 1998;15:650-9) indicating that the primary mechanism of degradation for pramlintide in this pH 4.0 formulation is deamidation, with six of the eight possible deamidation sites observed to undergo deamidation. Gln-10 and Asn-31 are the only two residues subject to deamidation for which none is observed. The data indicate that the cyclic imide products account for approximate to 20% of the total thermal degradation while the deamidation products account for approximate to 64%. The remaining degradation is due to peptide backbone hydrolysis. (C) 1999 Elsevier Science B.V. All rights reserved.