Central Composite Design Approach Stability-Indicating RP-HPLC Analysis for Simultaneous Estimation of Monoclonal Antibodies in Bulk and Combined Dosage Form

This study presents the novel quality by design (QbD)-assisted RP-HPLC method for the simultaneous estimation of relatlimab and nivolumab in both bulk and pharmaceutical formulations, utilizing a statistically optimized design via central composite design (CCD). Optimization was performed by CCD by selecting mobile phase ratio of acetonitrile, pH of buffer, and flow rate as factors and evaluating responses, namely retention time and tailing factor, through Waters Alliance-e2695, which occurred using a Kromasil C18 (150 × 4.6 mm, 3.5 µm) column that operated with a mobile phase composed of acetonitrile:ammonium acetate in the ratio of 20:80 v/v. The detector used a photodiode array for absorbance-based reading at 236 nm with an ambient temperature setup. The detection system worked at 1.0 mL/min flow rates. The results obtained from the validation parameters demonstrate that this RP-HPLC method, developed using the QbD approach, is robust and dependable. ICH guidelines served as the basis for conducting the validation of this proposed method. Forced degradation is carried out at various stress conditions. The method offers a rapid, robust, and cost-effective analytical solution with high precision and accuracy, validated as per ICH guidelines, and is well-suited for routine quality control and stability studies.