Small Molecule Analytical Chemistry, Genentech, Inc., So. San Francisco, CA 94080, United States.
Small Molecule Process Chemistry, Genentech, Inc., So. San Francisco, CA 94080, United States.
J Pharm Biomed Anal. 2019 Sep 10;174:518-524. doi: 10.1016/j.jpba.2019.06.020. Epub 2019 Jun 19.
Identification and localization of modifications in peptides containing multiple disulfide bonds is challenging due to inefficient fragmentation in mass spectrometry (MS) analysis. In cases where MS fragmentation techniques such as electron capture dissociation (ECD), electron transfer dissociation (ETD), and ultraviolet photodissociation (UVPD) fail to achieve efficient fragmentation, off-line disulfide bond reduction techniques are typically employed prior to MS analysis. Some commonly used reducing agents include dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP). In this work, we describe the detection and identification of an unexpected impurity that formed during the reduction of Peptide A, containing multiple disulfide bonds, while using DTT or TCEP as reducing agents and acetonitrile as a co-solvent. The DTT reduced products were found to be a mixture of the expected linear Peptide A (fully reduced) and an unknown product (>50%) with a mass corresponding to linear Peptide A plus 41 Da ([reduced-M + 41]). A series of experiments were subsequently performed to investigate the identity and origin of this impurity. Disulfide bond reduction with DTT was performed in aqueous mixtures containing acetonitrile, methanol, and deuterated acetonitrile; and with TCEP in aqueous mixtures containing acetonitrile. Additionally, glycine amino acid was used as a surrogate to investigate the mechanism. The liquid chromatography-mass spectrometry (LCMSMS) results demonstrated that the [reduced-M + 41] impurity was an acetonitrile addition on the peptide's N-terminal glycine. The corresponding impurity [M + 41] was also found in the native Peptide A (non-reduced), suggesting that small amounts of this impurity may also be generated during the synthesis in the upstream process steps. By understanding the formation of this process-related impurity [M + 41], one could potentially reduce or eliminate its presence in Peptide A through chemical controls. Finally, this observation provides caution against using acetonitrile as a co-solvent during DTT- or TCEP-promoted reduction of peptides with an uncapped N-terminus primary amine.
由于在质谱（MS）分析中碎片化效率低下，因此鉴定和定位含有多个二硫键的肽中的修饰是具有挑战性的。在 MS 碎片化技术（如电子俘获解离（ECD）、电子转移解离（ETD）和紫外光解离（UVPD））未能实现有效碎片化的情况下，通常在 MS 分析之前采用离线二硫键还原技术。一些常用的还原剂包括二硫苏糖醇（DTT）和三（2-羧乙基）膦（TCEP）。在这项工作中，我们描述了在使用 DTT 或 TCEP 作为还原剂和乙腈作为共溶剂还原含有多个二硫键的肽 A 时，形成的一种意想不到的杂质的检测和鉴定。发现 DTT 还原产物是预期线性肽 A（完全还原）和一种未知产物（>50%）的混合物，其质量对应于线性肽 A 加上 41 Da([还原-M+41])。随后进行了一系列实验来研究该杂质的性质和来源。在含有乙腈、甲醇和氘代乙腈的水混合物中以及在含有乙腈的水混合物中用 TCEP 进行二硫键还原。此外，还使用甘氨酸氨基酸作为替代物来研究其机制。液相色谱-质谱（LCMSMS）结果表明，[还原-M+41]杂质是肽的 N 端甘氨酸上的乙腈加成物。在天然肽 A（未还原）中也发现了相应的杂质[M+41]，这表明在合成过程中的上游步骤中也会产生少量这种杂质。通过了解这种与过程相关的杂质[M+41]的形成，人们可以通过化学控制来降低或消除肽 A 中该杂质的存在。最后，这一观察结果告诫人们，在用 DTT 或 TCEP 促进具有无帽 N 端伯胺的肽还原时，避免使用乙腈作为共溶剂。
