Proteins provide the molecular basis for cellular structure, catalytic activity, signal transduction, and molecular transport in biological systems. Recombinant protein expression is widely used to prepare and manufacture novel proteins that serve as the foundation of many biopharmaceutical products. However, protein translation bioprocesses are inherently prone to low-level errors. These sequence variants caused by amino acid misincorporation have been observed in both native and recombinant proteins. Protein sequence variants impact product quality, and their presence can be exacerbated through cellular stress, overexpression, and nutrient starvation. Therefore, the cell line selection process, which is used in the biopharmaceutical industry, is not only directed towards maximizing productivity, but also focuses on selecting clones which yield low sequence variant levels, thereby proactively avoiding potentially inauspicious patient safety and efficacy outcomes. Here, we summarize a number of hallmark studies aimed at understanding the mechanisms of amino acid misincorporation, as well as exacerbating factors, and mitigation strategies. We also describe key advances in analytical technologies in the identification and quantification of sequence variants, and some practical considerations when using LC-MS/MS for detecting sequence variants.

蛋白质为生物系统中细胞结构，催化活性，信号转导和分子运输提供了分子基础。重组蛋白表达被广泛用于制备和生产新型蛋白，这些蛋白是许多生物制药产品的基础。但是，蛋白质翻译生物过程固有地容易发生低级错误。在天然蛋白和重组蛋白中都观察到了由氨基酸错误掺入引起的这些序列变异。蛋白质序列变异会影响产品质量，并且它们的存在会因细胞应激，过度表达和营养缺乏而加剧。因此，在生物制药行业中使用的细胞系选择过程不仅旨在最大程度地提高生产力，但也要着重于选择产生低序列变异水平的克隆，从而主动避免潜在的不安全的患者安全性和疗效。在这里，我们总结了许多标志性研究，旨在了解氨基酸错误掺入的机制以及加剧因素和缓解策略。我们还描述了分析技术在鉴定和定量序列变异中的关键进展，以及使用LC-MS / MS检测序列变异时的一些实际考虑。