Chu Grace C, Chelius Dirk, Xiao Gang, Khor Hui K, Coulibaly Sururat, Bondarenko Pavel V
Department of Pharmaceutics, Amgen, Inc., One Amgen Center Drive, MS 8-1-C, Thousand Oaks, California 91320, USA.
Pharm Res. 2007 Jun;24(6):1145-56. doi: 10.1007/s11095-007-9241-4. Epub 2007 Mar 24.
The purpose of this paper was to identify the location of a succinimide and determine the rate of its formation and hydrolysis in a recombinant human monoclonal IgG2 antibody aged in mildly acidic buffers at elevated temperatures.
Cation exchange (CEX) HPLC separated multiple Main Peaks and high levels (up to 50%) of basic variants, the identification of which was an analytical challenge and required several complementary techniques. The relative abundance of the CEX basic variants was used to quantify the percentage of succinimide and to study the rates of its formation and hydrolysis.
Mass decrease by approximately 18 Da for intact antibodies from the CEX basic fractions suggested succinimide formation from aspartic acid as the major modification. Reversed-phase HPLC/MS of the reduced and trypsin-digested samples detected an isoaspartate 30 (isoD30) in the light chain peptide A25-R37. Direct evidence that isoD30 was from succinimide was obtained by performing succinimide hydrolysis in H2(18)O followed by tryptic digestion in H2(16)O.
Succinimide formation increased as pH became more acidic, whereas its hydrolysis was faster as pH became neutral and alkaline. Succinimide hydrolysis in a denatured sample was estimated to have completed in less than 2 h, but approximately three days for a similar pH but without denaturant. These observations suggest that protein conformation affects succinimide hydrolysis.
本文旨在确定琥珀酰亚胺的位置，并测定其在重组人单克隆IgG2抗体于温和酸性缓冲液中在高温下老化时的形成和水解速率。
阳离子交换（CEX）高效液相色谱法分离出多个主峰和高水平（高达50%）的碱性变体，对其进行鉴定是一项分析挑战，需要多种互补技术。CEX碱性变体的相对丰度用于定量琥珀酰亚胺的百分比，并研究其形成和水解速率。
来自CEX碱性馏分的完整抗体质量减少约18 Da，表明天冬氨酸形成琥珀酰亚胺是主要修饰。还原和胰蛋白酶消化样品的反相高效液相色谱/质谱法在轻链肽A25-R37中检测到异天冬氨酸30（isoD30）。通过在H2(18)O中进行琥珀酰亚胺水解，然后在H2(1６)O中进行胰蛋白酶消化，获得了isoD30来自琥珀酰亚胺的直接证据。
随着pH值变得更酸性，琥珀酰亚胺的形成增加，而随着pH值变为中性和碱性，其水解更快。变性样品中的琥珀酰亚胺水解估计在不到2小时内完成，但在类似pH值但无变性剂的情况下约需三天。这些观察结果表明蛋白质构象影响琥珀酰亚胺水解。
