Protein primary structure is a potential critical quality attribute for biotherapeutics. Identifying and characterizing any sequence variants present is essential for product development. A sequence variant ~11 kDa larger than the expected IgG mass was observed by size-exclusion chromatography and two-dimensional liquid chromatography coupled with online mass spectrometry. Further characterization indicated that the 11 kDa was added to the heavy chain (HC) Fc domain. Despite the relatively large mass addition, only one unknown peptide was detected by peptide mapping. To decipher the sequence, the transcriptome of the manufacturing cell line was characterized by Illumina RNA-seq. Transcriptome reconstruction detected an aberrant fusion transcript, where the light chain (LC) constant domain sequence was fused to the 3ʹ end of the HC transcript. Translation of this fusion transcript generated an extended peptide sequence at the HC C-terminus corresponding to the observed 11 kDa mass addition. Nanopore-based genome sequencing showed multiple copies of the plasmid had integrated in tandem with one copy missing the 5ʹ end of the plasmid, deleting the LC variable domain. The fusion transcript was due to read-through of the HC terminator sequence into the adjacent partial LC gene and an unexpected splicing event between a cryptic splice-donor site at the 3ʹ end of the HC and the splice acceptor site at the 5ʹ end of the LC constant domain. Our study demonstrates that combining protein physicochemical characterization with genomic and transcriptomic analysis of the manufacturing cell line greatly improves the identification of sequence variants and understanding of the underlying molecular mechanisms.

蛋白质的一级结构是生物治疗的潜在关键质量属性。鉴定和表征存在的任何序列变体对于产品开发至关重要。通过尺寸排阻色谱和二维液相色谱结合在线质谱法观察到比预期的IgG质量大约11 kDa的序列变体。进一步表征表明11 kDa已添加至重链（HC）Fc域。尽管增加了相对较大的质量，但通过肽图分析仅检测到一种未知肽。为了解密该序列，用Illumina RNA-seq表征了生产细胞系的转录组。转录组重建检测到异常的融合转录本，其中轻链（LC）恒定域序列与HC转录本的3′末端融合。该融合转录本的翻译在HC C末端产生了延伸的肽序列，其对应于观察到的11 kDa质量增加。基于纳米孔的基因组测序表明，该质粒的多个拷贝已串联整合，其中一个拷贝缺失了质粒的5'末端，从而删除了LC可变域。融合转录本是由于HC终止子序列通读到相邻的部分LC基因中，并且是在HC 3'端的隐蔽剪接供体位点与在5'端的剪接受体位点之间发生了意外的剪接事件。 LC恒定域。