Reversed-Phase Liquid Chromatographic Method for Separation of Ovalbumin and Bovine Serum Albumin and Quantification of Bovine Serum Albumin

The goal of this research is to develop a reversed-phase high-performance liquid chromatographic method to separate ovalbumin (OVA) and bovine serum albumin (BSA) proteins and to quantify BSA protein in its raw material. This technique is considered one of the most promising analytical techniques for the quantification of peptides and intact proteins, including monoclonal antibodies (mAbs). Two stationary phases, C4 and C3 with 300 Å pore size columns, were tested in this investigation. The C4 column is in 250 × 4.6 mm dimension, while the C3 column is in 75 × 2.1 mm dimension. A 0.08 % TFA was added to the mobile phase as an ion-pairing reagent to control its acidity and column efficiency. Proteins standard solutions of BSA and OVA were prepared in 6 M urea as a denaturing agent to improve chromatographic efficiency. The optimum separation conditions were found under a flow rate 1.0 mL/min, injection volume 20 µL, column temperature 50°C, and a gradient profile: 0 to 10 min, % B: 25% to 80%, 10 to 20 min, % B: 80% to 100% with solvent B = 100% acetonitrile + 0.08% trifluoroacetic acid. Under these separation conditions, the percentage recovery of BSA protein from the stationary phase was found 100%, while only 90- 95% of OVA was recovered. A short alkyl chain column (30 × 4.6 mm) helped improve the recovery of OVA protein and reduced the separation run time of the method. Forced degradation studies were conducted under the optimum separation conditions to determine the stability of these two proteins under sodium hydroxide, hydrochloric acid, hydrogen peroxide, heat, and light. In addition, the developed method was validated to verify its specificity and robustness and confirm its high accuracy and precision. The validation results fulfilled the U.S. Food and Drug Administration guidelines (FDA)