While characterizing a therapeutic IgG4 monoclonal antibody (mAb), we observed a variant with a mass 1177 Da larger than the predominant mAb form that could not be ascribed to previously described modifications. Through successive rounds of experimentation, we localized the mass addition to the C-terminus of the heavy chain (HC). During this process we observed that when the mAb was broken down into separate domains, the Fc and the 1177 Da-modified Fc could be chromatographically separated. Separation allowed collection of native and modified Fc fractions for LC/MS peptide mapping. A unique peptide present in the modified fraction was de novo sequenced and demonstrated to be a modified form of the HC C-terminus lacking two native residues (GK) and gaining twelve additional non-native residues (EAEAASASELFQ). Aware of other mAb variants with genetic origins, we sought to understand whether this modification too had a genetic basis. In silico translation of the expression vector encoding the mAb demonstrated that a normally non-coding section of nucleotides in the + 1 reading frame relative to the HC C-terminal coding region could have led to a transcript with the non-native C-terminal extension. Two potential mechanisms for how this nucleotide sequence might have fused to the native HC coding region and led to expression of the extension product are presented.

在表征治疗性IgG4单克隆抗体（mAb）的同时，我们观察到质量比主要mAb形式大1177 Da的变体，该变体不能归因于先前描述的修饰。通过连续几轮实验，我们确定了重链（HC）C端的加成质量。在此过程中，我们观察到，当mAb分解为单独的结构域时，可以色谱分离Fc和1177 Da修饰的Fc。分离允许收集天然和修饰的Fc馏分用于LC / MS肽图分析。修饰级分中存在的独特肽是从头测序并证明是HC C末端的修饰形式，其缺乏两个天然残基（GK）并且获得了另外十二个非天然残基（EAEAASASELFQ）。意识到具有遗传起源的其他mAb变体后，我们试图了解这种修饰是否也具有遗传基础。编码mAb的表达载体的计算机翻译表明，相对于HC C末端编码区，+ 1阅读框中的核苷酸正常非编码部分可能导致了具有非天然C末端延伸的转录本。对于该核苷酸序列如何与天然HC编码区融合并导致延伸产物的表达，提出了两种潜在的机制。