Host cell protein profiling and immunogenicity assessment in E. coli-produced interferon beta

Host cell proteins (HCPs) are critical process-related impurities in biotherapeutic products and may impact product safety and efficacy. Although Escherichia coli expression systems are widely used for recombinant protein production, systematic assessments of bacterial HCP immunogenicity risk remain limited compared with CHO-derived platforms. Here, we report an integrated HCP profiling and immunogenicity risk assessment of a commercial recombinant human interferon beta-1b (rhIFNβ-1b) produced in E. coli. HCPs were identified across five product batches using LC-MS/MS, revealing nine residual proteins, predominantly cytoplasmic enzymes with acidic isoelectric points. Three representative HCPs (W7, 42, and E6) were recombinantly expressed and evaluated using orthogonal immunogenicity assays. While W7 and E6 induced pro-inflammatory gene expression in THP-1 monocytes at supraphysiological concentrations, neither protein activated NF-κB signaling in RAW-Blue™ reporter cells nor elicited detectable Th1/Th17 responses in ex vivo human T-cell assays across multiple HLA-DR-typed donors. HCP 42 showed no immunostimulatory activity in any platform tested. Collectively, these results indicate a low immunogenicity risk associated with the evaluated bacterial HCPs at levels relevant to purified drug products. This study provides a comprehensive framework for bacterial HCP identification and risk assessment, and addresses critical knowledge gaps in non-CHO cell expression platforms.