Method Development and Validation of Reverse-Phase HPLC for Purity Analysis and Size-Exclusion HPLC for Aggregate Analysis of Monoclonal Antibody, SUNmAb

Abstract

Development of analytical method for therapeutic protein as compared to small molecules is a challenging task because of their high molecular weight, storage conditions, environmental conditions. During analytical method development of therapeutic protein, they come in contact with different solvents, parameters like pH, temperature may change which can lead to instability of protein molecule and so method development for protein requires expertise. Here, we are presenting our research work which includes the development of Reverse Phase-HPLC (RP-HPLC) method for purity analysis of therapeutic protein using Zorbax 300SB C-18 (3.5 μm 150x4.6 mm, Agilent) column with solvents like water, acetonitrile, and propanol. Different combination of mobile phases were tried and the results were observed and interpreted. A gradient method with combination of 1-propanol and acetonitrile at 220 nm was found to provide satisfactory results. The method was qualified and was found to be accurate, precise, specific and provided linear responses. Similarly, we have developed a SEC-HPLC method for aggregate analysis of therapeutic protein using TSKgel G2000 SWxL 5μm, 125 Å (300 x 7.8mm, Tosoh) and Biosep SEC s2000 5 μm, 150 Å (300 x 7.8mm, Phenoemenex) column with Sodium and Potassium based buffers as mobile phase. A method with Biosep SEC s2000 column and Potassium based buffer as a mobile phase at 215 nm was found to provide satisfactory results. The method was qualified and was found to be accurate, precise, specific and provided linear responses.