Development and Validation of a Cationic Exchange High Performance Liquid Chromatographic Method for the Separation of Ribonuclease (Ribo), Chymotrypsin (Chymo), and Lysozyme (Lyso), and Quantification of Lysozyme
Location
SU-003
Department
Chemistry
Abstract
Ion-exchange chromatography is considered the most effective technique for characterizing therapeutic mAbs and ADCs and monitoring the batch-to-batch manufacturing process consistency, product stability, and purity among several chromatographic modes for their separation such as reversed-phase liquid chromatography, size exclusion chromatography, and hydrophobic interaction liquid chromatography. This investigation involved two ion-exchange methods using Salt- and pH- gradient ion exchange modes. A novel, simple and robust method was developed to separate a mixture of RIBO, CHEMO, and LYSO proteins on Agilent Technologies 1100 series HPLC with a Diode array detector and Agilent Bio SCX column with 4.6 x 250 mm dimension and 5µm particle size controlled at 50o C. Under gradient elution technique using 25 mM disodium phosphate at pH 6.5 and 1.0 mL/ min flow rate, simulating with the DryLab® modeling software, we identified optimum gradient elution conditions for gradient times of 0.0, 3.0, 11.1, 12.0, and 12.1 minutes. Under the optimum salt-gradient separation conditions, the developed method was validated for Lysozyme protein in terms of system suitability test, specificity, robustness, linearity and range, precision and accuracy along with pH buffer AMPSO and CAPS study. The validation results fulfilled the U.S. Food and Drug Administration guidelines (FDA). Optimization of separation conditions using pH-gradient ion exchange chromatography will conclude this presentation.
Faculty Sponsor
John Albazi, Northeastern Illinois University
Development and Validation of a Cationic Exchange High Performance Liquid Chromatographic Method for the Separation of Ribonuclease (Ribo), Chymotrypsin (Chymo), and Lysozyme (Lyso), and Quantification of Lysozyme
SU-003
Ion-exchange chromatography is considered the most effective technique for characterizing therapeutic mAbs and ADCs and monitoring the batch-to-batch manufacturing process consistency, product stability, and purity among several chromatographic modes for their separation such as reversed-phase liquid chromatography, size exclusion chromatography, and hydrophobic interaction liquid chromatography. This investigation involved two ion-exchange methods using Salt- and pH- gradient ion exchange modes. A novel, simple and robust method was developed to separate a mixture of RIBO, CHEMO, and LYSO proteins on Agilent Technologies 1100 series HPLC with a Diode array detector and Agilent Bio SCX column with 4.6 x 250 mm dimension and 5µm particle size controlled at 50o C. Under gradient elution technique using 25 mM disodium phosphate at pH 6.5 and 1.0 mL/ min flow rate, simulating with the DryLab® modeling software, we identified optimum gradient elution conditions for gradient times of 0.0, 3.0, 11.1, 12.0, and 12.1 minutes. Under the optimum salt-gradient separation conditions, the developed method was validated for Lysozyme protein in terms of system suitability test, specificity, robustness, linearity and range, precision and accuracy along with pH buffer AMPSO and CAPS study. The validation results fulfilled the U.S. Food and Drug Administration guidelines (FDA). Optimization of separation conditions using pH-gradient ion exchange chromatography will conclude this presentation.