Analysis of Molecular Heterogeneity in Therapeutic IFNα2b from Different Manufacturers by LC/Q-TOF.

Recombinant human IFN alpha 2b (rhIFN alpha 2b), as an important immune-related protein, has been widely used in clinic for decades. It is also at the forefront of the recent emergence of biosimilar medicines, with numerous products now available worldwide. Although with the same amino acid sequence, recombinant proteins are generally heterogeneous due to post-translational modification and chemical reactions during expression, purification, and long-term storage, which could have significant impact on the final product quality. So therapeutic rhIFN alpha 2b must be closely monitored to ensure consistency, safety, and efficacy. In this study, we compared seven rhIFN alpha 2b preparations from six manufacturers in China and one in America, as well as four batches of rhIFN alpha 2b preparations from the same manufacturer, measuring IFN alpha 2b variants and site-specific modifications using a developed LC/Q-TOF approach. Three main forms of N-terminus, cysteine, methionine, and acetylated cysteine were detected in five rhIFN alpha 2b preparations produced inE. coli(1E similar to 5E) and one inPseudomonas(6P), but only the native form with N-terminal cysteine was found in rhIFN alpha 2b preparation produced inSaccharomyces cerevisiae(7Y). Two samples with the lowest purity (4E and 6P), showed the highest level of acetylation at N-terminal cysteine and oxidation at methionine. The level of oxidation and deamidation varied not only between samples from different manufacturers but also between different batches of the same manufacturer. Although variable between samples from different manufacturers, the constitution of N-terminus and disulfide bonds was relatively stable between different batches, which may be a potential indicator for batch consistency. These findings provide a valid reference for the stability evaluation of the production process and final products.