Method Development and Qualification for Quantitation of Cystine, Cysteine in Monoclonal Antibody using Reversed - Phase High Performance Liquid Chromatography (RP-HPLC)

Abstract

Purified drug material is likely to include trace amounts of process-related contaminants. Therefore, efficient elimination of process-related contaminants or process residuals is crucial for the creation of biopharmaceuticals. The final, purified drug material should be examined for the presence or absence of all crucial reagents applied during the procedure. Since, Cysteine and Cysteine are crucial agents in the refolding process, it is important to identify how to remove them from the drug substance. For the detection and quantitation of Cystine-Cysteine, the RP-HPLC technique was used. Since, Cystine-Cysteine is known to be hydrophobic by nature and can be eluted using the RP-HPLC C18 column. Method Development & Qualification of this process was carried out according to ICH Q2 (R1) guidelines. Method Development process was carried out as function of pH of the Mobile Phase A. Retention Time of Cystine and Cysteine was identified at Rt values 3.5 & 4.5 minutes respectively. For validation of an identity test, the specificity parameter was evaluated using 15 ppm standard. In addition to specificity, accuracy and precision (i.e Repeatability and Intermediate Precision) were carried out. Precision was evaluated as function of concentration (15 ppm, 7.5 ppm and 2.5 ppm). The above parameters preliminarily qualify the performance of the method for its intended purpose. The validation activity would further demonstrate the suitability and reliability of the method by an extensive verification of various parameters on the basis of written protocol and defined acceptance criteria as recommended by ICH Q2 (R1).