Gucinski Ashley C, Boyne Michael T
U.S. Food and Drug Administration, CDER/OPS/OTR Division of Pharmaceutical Analysis, 10903 New Hampshire Ave, Silver Spring, MD, 20993, USA.
Rapid Commun Mass Spectrom. 2014 Aug 15;28(15):1757-63. doi: 10.1002/rcm.6957.
Protamine sulfate is a peptide drug product consisting of multiple basic peptides. As traditional high-performance liquid chromatography (HPLC) separation methods may not resolve these peptides, as well as any possible peptide-related impurities, a method utilizing top-down mass spectrometry was developed for the characterization of complex peptide drug products, including any low-level impurities, which is described in this study.
Herring protamine sulfate was used as a model system to demonstrate the applicability of the method. Direct infusion mass spectrometry and tandem mass spectrometry (MS/MS) on a high-resolution, mass accurate instrument with electron transfer dissociation (ETD) were used to identify all the species present in the herring protamine sulfate sample. Identifications were made based on mass accuracy analysis as well as MS/MS fragmentation patterns.
Complete sequence coverage of the three abundant herring protamine peptides was obtained using the top-down ETD-MS/MS method, which also identified a discrepancy with the published herring protamine peptide sequences. Additionally, three low-abundance related peptide species were also identified and fully characterized. These three peptides had not previously been reported as herring protamine peptides, but could be related to the published sequences through amino acid additions and/or substitutions.
A method for the characterization of protamine, a complex peptide drug product, was developed that can be extended to other complex peptide or protein drug products. The selectivity and sensitivity of this method improves a regulator's ability to identify peptide impurities not previously observed using the established methods and presents an opportunity to better understand the composition of complex peptide drug products.
硫酸鱼精蛋白是一种由多种碱性肽组成的肽类药物产品。由于传统的高效液相色谱（HPLC）分离方法可能无法分离这些肽以及任何可能的肽相关杂质，因此开发了一种利用自上而下质谱法来表征复杂肽类药物产品（包括任何低水平杂质）的方法，本研究对此进行了描述。
以硫酸鲱鱼精蛋白作为模型系统来证明该方法的适用性。在配备电子转移解离（ETD）的高分辨率、质量精确的仪器上，使用直接进样质谱法和串联质谱法（MS/MS）来鉴定硫酸鲱鱼精蛋白样品中存在的所有物种。基于质量精确分析以及MS/MS碎片模式进行鉴定。
使用自上而下的ETD-MS/MS方法获得了三种丰富的鲱鱼精蛋白肽的完整序列覆盖，该方法还发现了与已发表的鲱鱼精蛋白肽序列存在差异。此外，还鉴定并完全表征了三种低丰度相关肽物种。这三种肽以前未被报道为鲱鱼精蛋白肽，但可能通过氨基酸添加和/或取代与已发表序列相关。
开发了一种用于表征鱼精蛋白（一种复杂肽类药物产品）的方法，该方法可扩展到其他复杂肽或蛋白质药物产品。该方法的选择性和灵敏度提高了监管机构识别以前使用既定方法未观察到的肽杂质的能力，并为更好地了解复杂肽类药物产品的组成提供了机会。
