Evaluation of depth filtration to remove prion challenge from an immune globulin preparation

Plasma-derived therapeutic proteins have the potential to contain transmissible spongiform encephalopathy (TSE) infectivity. This study evaluated the effectiveness and characterized the mechanism of abnormal prion protein removal during a depth-filtration step used in the manufacture of an immunoglobulin preparation.

Scrapie brain homogenate was treated with lysolecithin, sonicated and sequentially filtered through 0.45-, 0.22- and 0.1-microm membrane filters. The scrapie brain homogenate was then added (at a 1:51 dilution) to the Supernatant III fraction used in the manufacture of Rho(D) immune globulin (human). The spiked immunoglobulin preparation was then filtered through a depth filter under the same conditions used in full-scale production. After filtration, the depth filter was washed with hypertonic NaCl solutions to elute the abnormal prion protein (PrPSc) from the filter. A Western blot assay for PrPSc was used to quantify removal from the filtrate and recovery from the filter washes. A second run was performed whereby the PrPSc-spiked Supernatant III was filtered through a 0.22-microm membrane filter prior to depth filtration. A third run evaluated depth filtration of PrPSc in Tris-buffered saline (TBS).

The depth filter removed greater than four logs of PrPSc from the Supernatant III filtrate. A significant portion of the PrPSc could be recovered from the depth filter by elution with high-molarity NaCl solutions. Prefiltration (through a 0.22-microm membrane filter) of the spiked Supernatant III prior to depth filtration removed all detectable PrPSc. Depth filtration removed less than one log of PrPSc from TBS.

Depth filtration appears to remove PrPSc from the immunoglobulin preparation by mechanical straining rather than by adsorption to the filter matrix. The immunoglobulin preparation caused the PrPSc to aggregate from particles <0.1 microm in size to particles of >0.22 microm, probably as a result of the presence of methanol in the preparation. The depth filter failed to remove PrPSc from a purely aqueous environment.