UV-C Irradiation: Virus Inactivation in Fibrinogen Concentrate Manufacturing Process

Introduction: Ensuring the viral safety of therapeutic proteins is a critical part in the production of plasma-derived medicinal products. Virus inactivation methods, such as heat treatment or low pH treatment, can compromise protein structure and function. The efficacy of these methods is often limited to lipid-enveloped viruses. New viral inactivation methods that ensure viral safety and preserve the integrity of sensitive therapeutic proteins are needed. Ultraviolet light at 254 nm wavelength (UV-C) targets preferentially nucleic acids over proteins and inactivates especially small, non-enveloped viruses without significant damage to therapeutic proteins.This study investigated the effectiveness of a UV-C irradiation system (UVivatec®) to inactivate viruses during the manufacturing process (small- and large-scale) of a newly developed fibrinogen concentrate, while preserving the structural and functional integrity of the therapeutic protein.

Methods: Fibrinogen concentrate is irradiated with UV-C light during the manufacturing process as an additional step. Target and model viruses (lipid-enveloped and non-enveloped) for relevant pathogenic viruses were used in virus clearance studies to evaluate UV-C pathogen reduction in fibrinogen concentrate. Therefore, in-process material from the fibrinogen concentrate manufacturing process were spiked in at laboratory scale with different viruses. Then, the samples were processed using the UVivatec Lab III system at defined standard conditions but with a UV-C dose below manufacturing specification. Virus titers in the pre- and post-irradiation samples were assessed using the respective cell-based virus infectivity assay and virus reduction factors in log₁₀ for this process step were calculated showing the virus inactivation capacities for each model virus. Quality and integrity of the manufactured therapeutic product was evaluated during development through a series of biochemical and biophysical analyses, like the measuring of the specific fibrinogen activity (clottable protein) or the aggregate content by HPLC.

Results: Absorbance analyses showed a favorable benefit-to-risk ratio for the use of UV-C irradiation for therapeutic protein solution. The preferential absorbance at 254 nm by nucleic acids compared to proteins absorbance led to low damage to proteins, but high damage to nucleic acids, a critical component of all viruses. In the virus clearance studies UV-C irradiation achieved effective virus inactivation with more than 4 log reduction observed for small non-enveloped Parvoviridae. For other non-enveloped viruses (e.g. HAV or FCV) or lipid-enveloped viruses UV-C irradiation resulted in a lower virus reduction factor but contributing significantly to the overall virus safety margin of the medicinal product. Importantly, product quality was maintained as indicated by fibrinogen activity and fibrinogen aggregation measurements.

Conclusion: UV-C irradiation is an effective, viable method for virus inactivation of especially small, non-enveloped viruses during the manufacturing process of fibrinogen concentrate. This method complements other virus inactivation technologies (e.g. S/D treatment, heat) where the effectiveness towards small non-enveloped viruses is limited. In addition, it offers a non-thermal, efficient approach to maintain the structural and functional integrity of the fibrinogen concentrate protein. This method would present an alternative for maintaining the integrity of other plasma-derived therapeutic proteins. Further optimization and validation could lead to its widespread adoption in the biopharmaceutical industry, contributing to both safety and quality of other plasma-derived therapeutic proteins.

Zirkel:Biotest: Current Employment. Luelf:Biotest: Current Employment. Asper:Biotest: Current Employment. Poelsler:Biotest: Current Employment.