Elimination of Ghost Peaks by Optimization of Anion Exchange Chromatography Method for Determination of gamma-Carboxyglutamic acid (Gla)-domainless Impurity in Recombinant Activated Clotting Factor VII Drug Products

Publication date: Available online 22 March 2019Source: Journal of Pharmaceutical and Biomedical AnalysisAuthor(s): Hamed Tahmasbi, Nahid Zarezadeh, Somaie Marjaninia, Behnaz Molavi, Shayan Maleknia, Fereidoun Mahboudi, Hossein BehrouzAbstractSecreted recombinant activated clotting factor VII activated (rFVIIa) in cell culture media missing gamma-carboxyglutamic acid (Gla) domain as a result of failure in gamma-carboxylation or cell lysis is called Gla-domainless impurity which has less negative charge compared to native rFVIIa. Based on risk assessment, this type of impurity is considered as critical drug product quality attribute of rFVIIa and its quantitative analysis in product batches is a critical issue in quality control laboratories.Analysis of Gla-domainless impurity is accomplished by Strong Anion Exchange Chromatography (SAX) in recombinant factor VIIa using Tris and Bis-Tris propane salt buffers as equilibrating buffers and high concentration ammonium acetate as an eluent. Appearance of ghost peaks with notable intensity during elution time of Gla-domainless impurity caused distortion of the related peak and interference with robust and accurate quantification of this impurity. Subsequently, the ghost peak was analyzed by LC-ESI-MS to determine the structure which showed the m/z values at 905.27, 623.53 and 341.60 and 563.73.To find the source of these ghost peaks, quality of water, buffer salts and Chelex-100 together with ionic strength of mobile phase A (additi...
Source: Journal of Pharmaceutical and Biomedical Analysis - Category: Drugs & Pharmacology Source Type: research