UV-C Treatment Of Intermediates Of Biologicals To Inactivate Viruses Without Denaturing Desired Protein

Abstract

Dedicated virus reduction steps implemented in the manufacturing process of biologicals, either isolated from human plasma or produced as recombinant proteins, are essential safety measures to assure that a potential virus contamination of the source material will not be transmitted to patients requiring these therapeutic proteins. Currently applied virus reduction steps as solvent/detergent treatment and virus filtration are very effective virus reduction methods with inherent method-dependent gaps regarding the reduction capacity for a very wide range of viruses of diverse physico-chemical characteristics: solvent/detergent treatment does not inactivate non-enveloped viruses and, depending on the pore size of the virus filter, small viruses are not removed when the desired protein is large and has to pass the filter. Therefore, another virus inactivation method was studied which is considered especially effective for small viruses: UV-C treatment using the UVivatec system provided by Sartorius Stedim Biotech GmbH, Göttingen, Germany. Experiments were performed to study the impact of UV-C treatment on the integrity of proteins employing fibrinogen as an example for a large protein and on the inactivation capacity for poliovirus (a small non-enveloped virus). The integrity of fibrinogen was assessed by comparing the untreated fibrinogen with the UV-C treated fibrinogen using HPLC, Clauss assay and thromboelastometry. Virus inactivation was studied in a bioassay using a sensitive cell culture infectivity assay employing a cynomolgus cell line. The results show that UV-C treatment inactivates viruses and modifies fibrinogen in a dose dependent manner; the monomer, dimer and polymer peak in the fibrinogen preparation studied changed from approx. 75% to 60%, 17% to 25% and 8% to 13%, respectively, at a UV-C intensity of 400 J/m² demonstrated by HPLC measurement. In order to protect fibrinogen from modifications, the antioxidant glutathione was added to the fibrinogen preparation. At an UV-C intensity of approx. 300 J/m², sufficient to effectively inactivate viruses studied, a modification of fibrinogen was not any longer detectable. Disclosures: Groener: CSL Behring: Employment.