Zhen Jing, Lee Jennifer, Wang Yueyang, McLaughlin Lena, Yang Fei, Li Zhengjian, Wang Jihong
Department of Analytical Sciences, U.S. Technical & Biologics Development, Horizon Therapeutics, Rockville, MD 20850, USA.
Antibodies (Basel). 2023 Sep 19;12(3):59. doi: 10.3390/antib12030059.
This study presents a novel degradation pathway of a human immunoglobulin G (IgG) molecule featuring a light chain N-terminal asparagine. We thoroughly characterize this pathway and investigate its charge profiles using cation exchange chromatography (CEX) and capillary isoelectric focusing (cIEF). Beyond the well-documented asparagine deamidation into isoaspartic acid, aspartic acid, and succinimide intermediate, a previously unreported clipping degradation pathway is uncovered. This newly identified clipped N-terminal IgG variant exhibits a delayed elution in CEX, categorized as a "basic variant", while retaining the same main peak isoelectric point (pI) in cIEF. The influence of temperature and pH on N-terminal asparagine stability is assessed across various stressed conditions. A notable correlation between deamidation percentage and clipped products is established, suggesting a potential hydrolytic chemical reaction underlying the clipping process. Furthermore, the impact of N-terminal asparagine modifications on potency is evaluated through ELISA binding assays, revealing minimal effects on binding affinity. Sequence alignment reveals homology to a human IgG with the germline gene from Immunoglobulin Lambda Variable 6-57 (IGLV6-57), which has implications for amyloid light-chain (AL) amyloidosis. This discovery of the N-terminal clipping degradation pathway contributes to our understanding of immunoglobulin light chain misfolding and amyloid fibril deposition under physiological conditions.
本研究提出了一种以轻链N端天冬酰胺为特征的人免疫球蛋白G（IgG）分子的新型降解途径。我们全面表征了该途径，并使用阳离子交换色谱（CEX）和毛细管等电聚焦（cIEF）研究了其电荷分布。除了已充分记录的天冬酰胺脱酰胺生成异天冬氨酸、天冬氨酸和琥珀酰亚胺中间体之外，还发现了一种以前未报道的剪切降解途径。这种新鉴定的剪切N端IgG变体在CEX中表现出延迟洗脱，归类为“碱性变体”，而在cIEF中保留相同的主峰等电点（pI）。在各种应激条件下评估了温度和pH对N端天冬酰胺稳定性的影响。建立了脱酰胺百分比与剪切产物之间的显著相关性，表明剪切过程背后存在潜在的水解化学反应。此外，通过ELISA结合试验评估了N端天冬酰胺修饰对效力的影响，结果显示对结合亲和力的影响最小。序列比对揭示了与来自免疫球蛋白λ可变区6-57（IGLV6-57）的种系基因的人IgG具有同源性，这对淀粉样轻链（AL）淀粉样变性具有重要意义。N端剪切降解途径的这一发现有助于我们理解生理条件下免疫球蛋白轻链的错误折叠和淀粉样纤维沉积。
