Christopher J. Stenland

Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein

Determining the risk of transmissible spongiform encephalopathy (TSE) transmission by blood or plasma-derived products requires sensitive and specific assays for the detection of either infectivity or a reliable marker for infectivity. To this end, a Western blot assay that is both sensitive and reproducible for the detection of PrP(RES), a marker for TSE infectivity, was developed. Using the 263K strain of TSE as a model system, the Western blot assay proved to be sensitive, specific and quantitative over a 3-4 log dynamic range. Compared to the rodent bioassay, the assay was shown to detect PrP(RES) down to approximately 10(3.4) IU/ml which is approximately 5-10 pg of PrP or approximately 10-20 ng brain equivalents. The Western blot was applied to monitor the partitioning of spiked PrP(Sc) through three plasma fractionation steps, cryoprecipitation, fraction I and fraction III, that are common to the purification of several human plasma-derived therapeutic products including albumin and immunoglobulins. The results from these studies demonstrated 1 log, 1 log and 4 logs of PrP(Sc) partitioning away from the effluent fraction for the cryoprecipitation, fraction I and fraction III steps, respectively.

A direct relationship between the partitioning of the pathogenic prion protein and transmissible spongiform encephalopathy infectivity during the purification of plasma proteins

BACKGROUND: Experimental evidence from rodent models indicates that blood can contain transmissible spongiform encephalopathy (TSE) infectivity, which suggests a potential risk for TSE transmission via proteins isolated from human plasma. Because methods that can reduce TSE infectivity typically are detrimental to protein function, infectivity must be removed to ensure the safety of these therapeutic proteins. Animal bioassays are conventionally used to detect infectivity, but the pathogenic form of the prion protein (PrPSc) can serve as a marker for TSE infectivity.

Evaluation of virus and prion reduction in a new intravenous immunoglobulin manufacturing process.

Background and Objectives Minimizing the transmission risk of infectious diseases is of primary importance in the manufacture of products derived from human plasma. A novel chromatography‐based intravenous immunoglobulin (IGIV) manufacturing process was developed and the reduction of virus and transmissible spongiform encephalopathies (TSE) during the manufacturing process was assessed. Mechanistically distinct steps that could affect virus reduction were identified, and the robustness of virus reduction over the range of process conditions was determined.

Solvent-dependent precipitation of prion protein

The misfolded isoform of the prion protein (PrP(Sc)) possesses many unusual physiochemical properties. Previously, we and others reported on the differential partitioning of PrP(Sc) from plasma derived therapeutic proteins during their purification processes. To understand the driving force behind these partitioning differences, we investigated the effects of various solvent conditions on the precipitation of PrP(Sc). In a physiological buffer, PrP(Sc) remained in the supernatant after low speed centrifugation. At pH 5, PrP(Sc) precipitation was nearly complete regardless of the salt content. PrP(Sc) could also be precipitated at pH 8 by adding ethanol, but this precipitation was salt dependent. Based on these observations, an empirical mathematical model was constructed in which the PrP(Sc) precipitation trends were fully described as a function of solvent pH, salt, and ethanol concentration. This model consistently predicted PrP(Sc) partitioning during cold ethanol precipitation steps used in plasma protein purification processes, as shown by experimentally determined distributions of PrP(Sc) and transmissible spongiform encephalopathy (TSE) infectivity. These results indicate that pH, salt, and ethanol content are the major solvent factors determining the precipitation of the infectious PrP(Sc) in these processes and may provide a useful tool for assessing the differential partitioning of PrP(Sc) in a given solvent environment.