Cell based assay identifies TLR2 and TLR4 stimulating impurities in Interferon beta

Area of Science:

• Biopharmaceutical manufacturing
• Immunology
• Drug safety

Background:

• Immunogenicity poses risks to therapeutic protein safety and efficacy.
• Evaluating and mitigating immunogenicity is crucial for biopharmaceutical development.
• Residual innate immune response modulating impurities (IIRMIs) can trigger adverse effects.

Purpose of the Study:

• To investigate the presence and impact of IIRMIs in different interferon-beta (IFNβ) products.
• To determine if IIRMIs contribute to the differential immunogenicity observed in clinical settings.
• To assess the utility of cell-based assays in detecting clinically relevant differences in IIRMIs.

Main Methods:

• Analysis of Betaseron, Extavia, Rebif, and Avonex for IIRMIs.
• NF-κB activation assays in HEK-293 cells expressing TLR2 and TLR4.
• In vivo studies using interferon receptor (IFNAR) knockout mice.
• Measurement of inflammatory mediator expression (IL-6, IL-1β, ccl5).

Main Results:

• Betaseron and Extavia contain IIRMIs that activate NF-κB and induce inflammatory mediators, while Rebif and Avonex do not.
• IIRMIs activate immune pathways in a MyD88-dependent manner, independent of IFNAR signaling.
• Subcutaneous administration of Betaseron-derived IIRMIs increases inflammatory markers in mice.

Conclusions:

• Trace levels of IIRMIs in Betaseron may contribute to its higher clinical immunogenicity.
• Cell-based assays can detect critical differences in IIRMIs between biopharmaceutical products.
• Understanding IIRMIs is essential for accurate immunogenicity risk assessment and improved drug safety.