Protein Analytical Chemistry, Genentech, South San Francisco, California 94080-4990, USA.
Anal Chem. 2009 Nov 15;81(22):9282-90. doi: 10.1021/ac901541h.
Translation errors in protein biosynthesis may result in low level amino acid misincorporation and contribute to product heterogeneity of recombinant protein therapeutics. We report the use of peptide map analysis by reversed-phase high-performance liquid chromatography and high-resolution mass spectrometry to detect and identify mistranslation events in recombinant monoclonal antibodies expressed in mammalian cell lines including Chinese hamster ovary (CHO) cells. Misincorporation of an asparagine residue at multiple serine positions was detected as earlier-eluting peptides with masses 27.01 Da higher than expected. The exact positions at which misincorporation occurred were identified by tandem mass spectrometry of the asparagine-containing variant peptides. The identified asparagine misincorporation sites correlated with the use of codon AGC but with none of the other five serine codons. The relative levels of misincorporation ranged from 0.01%-0.2% among multiple serine positions detected across three different antibodies by targeted analysis of expected and variant peptides. The low levels of misincorporation are consistent with published predictions for in vivo translation error rates. Our results demonstrate that state-of-the-art mass spectrometry with a combination of high sensitivity, accuracy, and dynamic range provides a new ability to discover and characterize low level protein variants that arise from mistranslation events.
蛋白质生物合成中的翻译错误可能导致低水平氨基酸错配，并导致重组蛋白治疗药物的产品异质性。我们报告了使用反相高效液相色谱和高分辨率质谱的肽图分析来检测和鉴定在包括中国仓鼠卵巢（CHO）细胞在内的哺乳动物细胞系中表达的重组单克隆抗体中的翻译错误事件。在多个丝氨酸位置检测到天冬酰胺残基的错配，其洗脱时间比预期早，分子量高出 27.01 Da。通过含天冬酰胺的变异肽的串联质谱鉴定了错配发生的确切位置。鉴定的天冬酰胺错配位点与密码子 AGC 的使用相关，但与其他五个丝氨酸密码子均不相关。通过对预期和变异肽的靶向分析，在三种不同抗体中检测到的多个丝氨酸位置的错配水平范围为 0.01%-0.2%。低水平的错配与体内翻译错误率的已发表预测一致。我们的结果表明，结合高灵敏度、准确性和动态范围的最先进质谱技术为发现和表征由翻译错误事件引起的低水平蛋白质变异体提供了新的能力。
