Determination of human monoclonal antibodies nivolumab and relatlimab in opdualag by using the RP-UPLC technique: method development and validation

Abstract A fixed combination of Nivolumab and Relatlimab (OPDUALAG) are human monoclonal antibodies (mAb) that are extensively used to treat skin cancer melanoma. The current investigation is to develop a robust, stability-indicating, and rapid method for the simultaneous determination of these two drugs by using the RP-UPLC technique. By utilizing this technique, the final chromatographic conditions were achieved with a Luna C18 column (50 x 2.1 mm, 1.6 μm), and a mobile phase made with acetonitrile and 0.1% formic acid, 6:4 ratio, respectively (Diluted 1.0 mL of Formic acid in 1000 mL of HPLC grade water) by adapting isocratic elution. Both Nivolumab and Relatlimab were detected at 229 nm, and a satisfactory separation was achieved in 3.0 minutes of run time with a flow rate of 0.2 mL/min. The method is linear from 15 - 90 μg/mL for Nivolumab (R2 = 0.9996) and 5 - 30 μg/mL for Relatlimab (R2 = 0.9997). Results of method repeatability for Nivolumab (% RSD=1.3 %) and Relatlimab (% RSD=0.48 %) and intermediate precision Nivolumab (% RSD=1.1 %) and Relatlimab (% RSD=0.48%) were found within acceptable limits. Also, the forced degradation study results show that it is discernible; the degradation products of both drugs were separated from the main peak, which demonstrates the efficiency of the stability-indicating method. So this method works well for routine analysis and stability studies. GRAPHICAL ABSTRACT