Peptide Synthesis Laboratory, Physics and Chemistry Department, Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana, Cuba.
Mass Spectrometry Laboratory, Department of Proteomics, Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana, Cuba.
J Pept Sci. 2018 Jun;24(6):e3081. doi: 10.1002/psc.3081. Epub 2018 Apr 20.
CIGB-300 is a first-in-class synthetic peptide-based drug of 25 amino acids currently undergoing clinical trials in cancer patients. It contains an amidated disulfide cyclic undecapeptide fused to the TAT cell-penetrating peptide through a beta-alanine spacer. CIGB-300 inhibits the CK2-mediated phosphorylation leading to apoptosis of tumor cells in vitro, and in vivo in cancer patients. Despite the clinical development of CIGB-300, the characterization of peptide-related impurities present in the active pharmaceutical ingredient has not been reported earlier. In the decision tree of ICHQ3A(R2) guidelines, the daily doses intake, the abundance, and the identity of the peptide-related species are pivotal nodes that define actions to be taken (reporting, identification, and qualification). For this, purity was first assessed by reverse-phase chromatography (>97%) and low-abundance impurities (≤0.27%) were collected and identified by mass spectrometry. Most of the impurities were generated during peptide synthesis, the spontaneous air oxidation of the reduced peptide, and the lyophilization step. The most abundant impurity, with no biological activity, was the full-length peptide containing Met transformed into a sulfoxide residue. Interestingly, parallel and antiparallel dimers of CIGB-300 linked by 2 intermolecular disulfide bonds exhibited a higher antiproliferative activity than the CIGB-300 monomer. Likewise, very low abundance trimers and tetramers of CIGB-300 linked by disulfide bonds (≤0.01%) were also detected. Here we describe for the first time the presence of active dimeric species whose feasibility as novel CIGB-300 derived entities merits further investigation.
CIGB-300 是一种首创的基于 25 个氨基酸的合成肽类药物，目前正在癌症患者中进行临床试验。它包含一个酰胺化的二硫键环十一肽，通过β-丙氨酸间隔物融合到 TAT 细胞穿透肽上。CIGB-300 抑制 CK2 介导的磷酸化，导致体外肿瘤细胞凋亡，并在癌症患者体内。尽管 CIGB-300 进行了临床开发，但目前尚未报道其活性药物成分中存在的肽相关杂质的特征。在 ICHQ3A(R2)指南的决策树中，每日剂量摄入、肽相关物质的丰度和身份是定义应采取行动（报告、鉴定和资格）的关键节点。为此，首先通过反相色谱法（>97%）评估纯度，并通过质谱法收集和鉴定低丰度杂质（≤0.27%）。大多数杂质是在肽合成过程中产生的，还原肽的自发空气氧化和冻干步骤也是产生杂质的原因。最丰富的杂质是没有生物活性的全长肽，其中的甲硫氨酸转化为亚砜残基。有趣的是，通过 2 个分子内二硫键连接的 CIGB-300 平行和反平行二聚体表现出比 CIGB-300 单体更高的抗增殖活性。同样，也检测到非常低丰度的 CIGB-300 三聚体和四聚体（≤0.01%），它们也是通过二硫键连接的。在这里，我们首次描述了存在具有活性的二聚体物质，它们作为新型 CIGB-300 衍生实体的可行性值得进一步研究。
