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A Multi-Peptide Hybrid LC-MS/MS Assay for the Determination of CTI-1601 in Monkey Tissues Provides Insight into its Disposition and Processing

Friedreich’s Ataxia (FA) is a rare genetic disease caused by a deficiency of the mitochondrial protein Frataxin (FXN). CTI-1601, a 24.9 kDa fusion protein currently under investigation as a protein replacement therapy to restore functional levels of FXN in the mitochondria of FA patients, consists of the cell-penetrating peptide TAT linked to the N-terminus of the full-length human FXN protein. CTI-1601 mechanism of action relies on the cell-penetrating ability of the TAT peptide and the subsequent processing into mature FXN after translocation into the mitochondria. To understand the disposition and processing of CTI-1601, a hybrid LC-MS/MS assay was developed to quantify CTI-1601 in cynomolgus monkey buccal cells, skin biopsies, and platelets. After tissue homogenization and cell lysis, CTI-1601 was immunopurified using an anti-FXN antibody followed by trypsin digestion and LC-MRM analysis. Three peptides were monitored, one from the N-Terminal TAT domain and two from the FXN protein.  A full-length SILAC labeled CTI-1601 was used as the internal standard. The assay was qualified for a range of 0.250 to 25.0 ng/mL with accuracy between 80-120% and precision ≤ 20%. The data suggest that, following repeated administration of CTI-1601 for 14 days, CTI-1601 not only accumulates in the tissues outside of the systemic circulation but is also predominantly present as the mature FXN protein. In conclusion, this study exemplifies how hybrid LC-MS/MS assays can be used to simultaneously gain insight into the disposition and processing of biotherapeutics.

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