Department of Analytical and Formulation Sciences, Amgen, Seattle, Washington 98119, USA.
Protein Sci. 2010 Nov;19(11):2152-63. doi: 10.1002/pro.495.
Novel acetone and aldimine covalent adducts were identified on the N-termini and lysine side chains of recombinant monoclonal antibodies. Photochemical degradation of citrate buffers, in the presence of trace levels of iron, is demonstrated as the source of these modifications. The link between degradation of citrate and the observed protein modifications was conclusively established by tracking the citrate decomposition products and protein adducts resulting from photochemical degradation of isotope labeled (13)C citrate by mass spectrometry. The structure of the acetone modification was determined by nuclear magnetic resonance (NMR) spectroscopy on modified-free glycine and found to correspond to acetone linked to the N-terminus of the amino acid through a methyl carbon. Results from mass spectrometric fragmentation of glycine modified with an acetone adduct derived from (13)C labeled citrate indicated that the three central carbons of citrate are incorporated onto protein amines in the presence of iron and light. While citrate is known to stoichiometrically decompose to acetone and CO(2) through various intermediates in photochemical systems, it has never been shown to be a causative agent in protein carbonylation. Our results point to a previously unknown source for the generation of reactive carbonyl species. This work also highlights the potential deleterious impact of trace metals on recombinant protein therapeutics formulated in citrate buffers.
新型丙酮和亚胺共价加合物在重组单克隆抗体的 N 末端和赖氨酸侧链上被鉴定出来。在痕量铁存在的情况下，证明了柠檬酸盐缓冲液的光化学降解是这些修饰的来源。通过跟踪柠檬酸盐分解产物和同位素标记的（13）C 柠檬酸盐光化学降解产生的蛋白质加合物，通过质谱法，明确建立了柠檬酸盐降解与观察到的蛋白质修饰之间的联系。通过对修饰的游离甘氨酸进行核磁共振（NMR）光谱分析，确定了丙酮修饰的结构，发现它对应于通过甲基碳与氨基酸 N 末端相连的丙酮。由（13）C 标记的柠檬酸衍生的丙酮加合物修饰的甘氨酸的质谱裂解结果表明，在铁和光的存在下，柠檬酸的三个中心碳原子被掺入到蛋白质胺中。虽然已知柠檬酸通过光化学系统中的各种中间体化学计量地分解为丙酮和 CO（2），但从未证明它是蛋白质羰基化的原因。我们的结果指向了一种以前未知的活性羰基物种生成的来源。这项工作还强调了痕量金属对柠檬酸缓冲液配方中的重组蛋白治疗剂的潜在有害影响。
