Characterization of low‐abundance species in the active pharmaceutical ingredient of CIGB‐300: A clinical‐grade anticancer synthetic peptide

Abstract

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.

Design of a Helical-Stabilized, Cyclic, and Nontoxic Analogue of the Peptide Cm-p5 with Improved Antifungal Activity

“…Surprisingly, in the standard cyclization conditions used for commercial peptides in our laboratory (0.5 mM), the CysCysCm-p5 analogue produced considerable quantities of dimeric byproduct (Figures S17 and S18, Supporting Information), considered as parallel and antiparallel dimers (Figure ). Similar dimers were reported as impurities of CIGB-300 anticancer peptide . Peak 4 (Figure S17, Supporting Information) represents a conformer of peak 3, a dimer, as can be seen in the ESI-HRMS spectrum (Figure S18, Supporting Information) and due to coalescence between peaks 3 and 4 if the sample is previously stabilized for 2 h at room temperature.…”

“…Similar dimers were reported as impurities of CIGB-300 anticancer peptide. 38 Peak 4 (Figure S17, Supporting Information) represents a conformer of peak 3, a dimer, as can be seen in the ESI-HRMS spectrum (Figure S18, Supporting Information) and due to coalescence between peaks 3 and 4 if the sample is previously stabilized for 2 h at room temperature. Only at 0.03 mM the production of the cyclic monomer without the formation of dimeric byproducts (Figure S21, Supporting Information) is guaranteed.…”

Anticancer peptide: Physicochemical property, functional aspect and trend in clinical application (Review)

“…For example, CIGB-300, an amidated disulfide cyclic undecapeptide fused to the TAT cell-penetrating peptide via a β-alanine spacer, inhibits CK-2-mediated phosphorylation leading to cancer cell apoptosis in patients with cervical and non-small cell lung cancer ( 185 - 187 ). Wilms' tumor 1 (WT1) peptide-based vaccination combined with the adjuvant drug OK-432 administered to pediatric patients with a solid tumor has been demonstrated to be safe for these children ( 188 ).…”

Capillary electrophoresis in the analysis of therapeutic peptides—A review

“…CIGB-300 is a synthetic peptide-based drug, a potent inducer of apoptosis [118,119]. It contains an amidated disulfide cyclic undecapeptide fused to the TAT cellpenetrating peptide through a beta-alanine spacer.…”