Development of Ionic Exchange High Performance Liquid Chromatographic Method for the Separation of Ovalbumin (OVA), Human Serum Albumin (HAS), and Bovine Serum Albumin (BSA) and Quantification of Ovalbumin

Therapeutic proteins, such as monoclonal antibodies (mAb) and antibody-drug conjugates (ADCs), get more and more importance since they were established as potent drugs in anticancer therapy or for the treatment of autoimmune based diseases. These proteins are susceptible to chemical and enzymatic modifications that can occur during manufacture, formulation, and storage. The large size of mAbs and ADCs and the minor structure diversity between the variants make their separation very challenging. As a result, ion-exchange chromatography is considered the most effective technique for characterizing therapeutic mAbs and ADCs and for monitoring the batch-to-batch process consistency and product stability and purity among several chromatographic modes for their separation such as reversed-phase liquid chromatography, size exclusion chromatography, hydrophobic interaction liquid chromatography. Ion-Exchange Chromatographic approach is based on the electrostatic interaction of the ionic portion of the protein with a cation- or anion- stationary phase. This investigation developed two ion-exchange methods using pH- and Salt- gradients ion exchange modes. A novel, simple and robust method was developed to separate a mixture of OVA, BSA, HSA proteins on Agilent Technologies 1260 Infinity series HPLC with Diode array detector and Agilent Bio SAX, NP5, SS, 4.6 x 250mm, 5µm, non-porous column controlled at 50oC. Under gradient elution technique, 25 mM Bis-Tris methane at pH 5.8 were found optimum buffer strength and mobile phase acidity. DryLab® modeling software was used to simulate the optimum conditions of NaCl concentration and gradient time. The optimum separation conditions were found under 1ml/min and gradient profile: 0 to 35 min, % B: 0% to 20%, 35 to 35.1 min, % B: 20% to 100%, 35.1 to 50 minutes, %B: 100%, 50.1 minutes %B back to 0% with solvent B = 25 mM Bis-Tris buffer at pH 5.8 + 1 M NaCl. Under the optimum salt-gradient separation conditions, the developed method was validated for OVA protein in terms of system suitability test, specificity, robustness, linearity and range, precision, accuracy, LOD, and LOQ. 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