Solvent-Detergent Treatment and Ion-Exchange Chromatography Have No Impact on Prion Removal by 15 nm Nanofiltration in the Manufacture of Factane®.

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The transmission risk for new variant Creutzfeldt-Jakob Disease by medicinal plasma-derived products (MPP) is considered as very low, even theoretical, as to date no cases associated with these products have been reported. As a precautionary measure, European Regulatory authorities require that manufacturers of MPP perform an assessment of their manufacturing processes capacity to remove transmissible spongiform encephalopathy (TSE) agents. In the absence of scientific data, manufacturers are required to perform specific studies demonstrating their processes efficacy towards prion removal. We investigated the efficacy of 3 manufacturing steps in the Factane® process: step 1: precipitation combined with alumina gel adsorption and filtration through positively charged membranes; step 2: ion-exchange chromatography in the presence of Solvent-Detergent (SD) and step 3: combined 35 nm + 15 nm nanofiltration. Removal of TSE infectivity was assessed either by independent experiments, to determine the reduction factor associated with each individual step, or by a study combining several steps in order to investigate the impact of upstream process steps (SD treatment and Chromatography) on the efficacy of nanofiltration. Intermediate products collected from industrial batches were spiked with a microsomal fraction purified from a hamster brain homogenate (scrapie 263K strain). Detection and titration of the infectious agent were performed by Western Blot (WB) and/or Bioassay. When each step was studied individually, step 1 led to a reduction factor of 1.5 log10 (WB, clearance factor: 3 log10) and step 2 to a reduction factor of 1.7 log10 (WB). Step 3 led to reduction factors higher than or equal to 3.3 log10 by WB as well as by Bioassay. In a study combining steps 2 and 3, the total reduction factor was higher than or equal to 5.1 log10 (WB). The bioassay corresponding to this combined study is ongoing. Results from investigational studies of the Factane® manufacturing process highlight the contribution of the 3 identified steps towards prion removal which is in line with previously reported data. Nanofiltration shows a very significant contribution to the removal of TSE agents as evidenced by WB and bioassay experiments. This observation is in agreement with the estimated size of TSE agents (15-45 nm) as well as with their biophysical properties (agregability, insolubility and hydrophobicity). Data from the combined study (reduction factor ≥ 5.1 log10) are compatible with the cumulative effects of the chromatography and the nanofiltration steps. This observation shows that nanofiltration's efficiency in removing prions is unchanged by the upstream treatments, namely SD treatment, and supports the hypothesis that the overal global reduction factor for the manufacture process is equal to the sum of the individual steps. These data confirm the interest of nanofiltration as an additional safety step in increasing the safety margin of plasma products towards the theoretical TSE transmission risk.