Optimization of SEC-HPLC Method for the Quantitative Estimation of a Process-Related Residual Impurity, Dimethylaminopyridine (DMAP) in the Manufacturing of Pneumococcal Polysaccharide Conjugates Vaccine

The conjugation technology is used for the manufacturing of polysaccharide conjugate vaccines against pathogenic organisms like Streptococcus pneumoniae, Haemophilus influenzae type b, etc. Conjugation using CDAP chemistry is widely utilized because of high yield and short reaction time. The cyano group of CDAP is attached to hydroxyl group of polysaccharides, resulting in activated polysaccharide with generation of process-related residual impurity, dimethyl aminopyridine (DMAP) which should be < 1.5 µg/day as per regulatory guidelines. In the present study, we reported the SEC-HPLC coupled with UV detector for quantitative estimation of residual DMAP content in pneumococcal polysaccharide–protein conjugates. The method was validated as per the ICH guidelines using representative pneumococcal conjugate of serotype 1, 6B and 23F. The method specificity was confirmed using placebo buffers and the identity of DMAP was ensured using orthogonal GC–MS method. The method showed linearity between 0.1 and 15 µg/mL with linear regression of > 0.99. Detection and quantitation limits of 0.03 µg/mL and 0.1 µg/mL were observed respectively. The overall % RSD of DMAP content was found to be between 0.94 and 1.91% suggesting precision of the method. Accuracy was performed by spiking known amount of DMAP at six different concentration levels and % recovery of 87 to 104% was noted. The validated SEC-HPLC–UV method was successfully applied for various stages of pneumococcal polysaccharide conjugate vaccines including crude, in-process and final purified samples. This has suggested that the SEC-HPLC–UV method can be used as routine process analytical tool to monitor residual DMAP.