Analytical Comparability Assessments of 5 Recombinant CRM 197 Proteins From Different Manufacturers and Expression Systems

Abstract

Cross-reacting material 197 (CRM 197 ), a single amino acid mutant of diphtheria toxoid, is a commonly used carrier protein in commercial polysaccharide protein conjugate vaccines. In this study, CRM 197 proteins from 3 different expression systems and 5 different manufacturers were obtained for an analytical comparability assessment using a wide variety of physicochemical and in vitro antigenic binding assays. A comprehensive analysis of the 5 CRM 197 molecules demonstrate that recombinant CRM 197 's expressed in heterologous systems (Escherichia coli and Pseudomonas fluorescens) are overall highly similar (if not better in some cases) to those expressed in the traditional system (Corynebacterium diphtheriae) in terms of primary sequence/post-translational modifications, higher order structural integrity, apparent solubility, physical stability profile (vs. pH and temperature), and in vitro antigenicity. These results are an encouraging step to demonstrate that recombinant CRM 197 expressed in alternative sources have the potential to replace CRM 197 expressed in C diphtheriae as a source of immunogenic carrier protein for lower cost polysaccharide conjugate vaccines. The physicochemical assays established in this work to monitor the key structural attributes of CRM 197 should also prove useful as complementary characterization methods (to routine quality control assays) to support future process and formulation development of lower cost CRM 197 carrier proteins for use in various conjugate vaccines.