Abstract

ANALYTICAL METHOD DEVELOPMENT AND FORCE DEGRADATION STUDIES OF ANALYTICAL QUALITY BY DESIGN (AQBD) BASED OPTIMIZATION OFA RP-UPLC METHOD FOR DETERMINATION OF NIVOLUMAB INJECTION

V. Deepika*, M. Hima Bindu, Dr. Achari Kandukuri Triveni, N. Vijaya Lakshmi Reddy

ABSTRACT

Nivolumab is a pioneering fully human IgG4 monoclonal antibody checkpoint inhibitor that has revolutionized oncology. Traditional analytical workflows often lack the systematic robustness required for such complex macromolecular formulations. This study describes the development of a highly robust, sensitive, and rapid stability-indicating reversed-phase ultra-performance liquid chromatography (RP-UPLC) method for Nivolumab Injection optimized via Analytical Quality by Design (AQbD) principles. A five-level, three-factor Central Composite Design (CCD) was implemented using Design-Expert® software to explore the method operable design region. Mathematical models systematically evaluated the effects of flow rate, mobile phase methanol percentage, and column temperature on critical analytical responses, including retention time, theoretical plates, and tailing factor. Optimized separation was achieved on a waters UPLC BEH C18 column using a mobile phase of 0.1% orthophosphoric acid (pH 3.00) and methanol at a flow rate of 0.3 mL/min, 30 °C column temperature, and UV detection at 280 nm. The method yielded an ultrafast retention time of 0.512 minutes. Validation per ICH guidelines demonstrated excellent linearity (10%–200%), high accuracy with recoveries spanning 99.6%–100.2%, and sample stability up to 48 hours. Forced degradation studies confirmed excellent specificity, ensuring complete baseline resolution of stress-induced degradants from the intact active peak.

Keywords: Nivolumab; Analytical Quality by Design; RP-UPLC; Forced Degradation; Method Validation.