[Show abstract][Hide abstract] ABSTRACT: Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases caused by aberrantly folded cellular proteins (PrP(Sc); prions) that are generally resistant to conventional pathogen-inactivation techniques. To ensure effective decontamination and inactivation of prions that could be present in source material, we investigated critical factors that influence prion inactivation by NaOH.
A decrease in prion infectivity correlates with the disappearance of the protease-resistant core of PrPSc (PrPRES) observed in biochemical assays. To model prion inactivation, hamster scrapie (strain 263K) brain homogenate (SBH) was incubated for specific periods of time in 0.1 m NaOH at 4 or 18 degrees C, with or without detergent. Neutralized samples were subjected to limited digestion with proteinase K (PK) and then analysed using an endpoint dilution western blot assay and antibody 3F4. Structural changes in prions exposed to NaOH were examined using differential immunoprecipitation.
Treatment of SBH with 0.1 m NaOH for 15 min, in the absence of detergent, at 4 and 18 degrees C caused a reduction in the PrP(RES) signal of 3.5 and 4.0 log10 units, respectively, with some residual signal remaining. The presence of the detergent sarkosyl during a 60-min incubation in NaOH further enhanced PrPRES reduction to > or = 4.5 log10 units (i.e. below the limit of detection). NaOH treatment induced conformational changes in PrP that resulted in the exposure of a hidden epitope and enabled prion immunoprecipitation by antibody 3F4.
The use of NaOH can effectively reduce prion levels in an in vitro inactivation assay. After pretreatment of SBH with detergent, NaOH completely eliminates the PrPRES signal. Detergent may liberate lipid membrane-protected PrPSc to improve access to NaOH, which can then inactivate PrPSc by altering its structure. In cases of unidentified exposure to PrPSc during manufacturing, sanitizing procedures combining the use of detergent and NaOH may help to ensure minimal levels of contamination carryover in products.
[Show abstract][Hide abstract] ABSTRACT: Protein products isolated from human plasma are an important class of therapeutics that are used to treat patients afflicted with hereditary deficiencies, trauma, and severe infections. Because of the human origin of the starting material for the production of these biological products, there is a risk of transmitting infectious agents, including viruses and the infectious agents that cause transmissible spongiform encephalopathies (TSEs). The agent that is thought to cause TSEs is a disease-associated, misfolded form of the prion protein or PrP(Sc). Unlike viruses, there are no donor screening tests for TSEs available, and PrP(Sc) is resistant to traditional viral inactivation methods. Therefore, manufacturers of plasma products are faced with special challenges to ensure product safety with respect to TSEs. Fortunately, a growing body of evidence supports the capacity of manufacturing processes to remove infectious prions from the product stream during the purification of plasma products. This can be attributed in part to the unusual physicochemical nature of PrP(Sc), which is distinct from that of soluble therapeutic proteins. Although there is no reported TSE transmission through the use of plasma products to date, many unknowns remain to be addressed through long-term epidemiologic monitoring and further experimental studies.
No preview · Article · Feb 2006 · Transfusion Medicine Reviews
[Show abstract][Hide abstract] ABSTRACT: Specific detection of the pathogenic prion protein, PrP(Sc), is essential for determining the prion clearance capacity of purification processes for therapeutic proteins. Use of a previously described indirect (two-antibody) Western blot assay sometimes resulted in the appearance of non-specific protein bands that interfered with the detection of small amounts of PrP(Sc)-specific signal, limiting the amount of clearance that could be determined for steps so affected. It is shown that these non-specific signals are due to the interaction between immunoglobulin fragments in the sample and the secondary antibody used in the assay. To circumvent this problem, a direct Western blot assay using a prion-specific primary antibody conjugated to the reporter enzyme alkaline phosphatase was developed. Application of the direct Western blot assay resulted in a significant reduction of non-specific signal while retaining the detection sensitivity for PrP(Sc)-specific signal. Therefore, the direct Western blot assay format is an improved tool for determining prion clearance capacity, particularly for immunoglobulin-rich samples.
Full-text · Article · Jun 2005 · Journal of Virological Methods
[Show abstract][Hide abstract] ABSTRACT: 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.
Virus and TSE reduction by processing steps were assessed using a scaled-down version of the IGIV manufacturing process.
Virus and TSE reduction at manufacturing process set points were well within safety standards. Robustness studies verified that the reproducibility of virus reduction was maintained at or beyond operating parameter extremes. Virus reduction across two combined manufacturing steps was lower than the sum of virus-reduction values across the individual steps, indicating mechanistic similarity of the two steps with respect to virus reduction. Only reduction from mechanistically distinct steps was claimed.
This comprehensive approach to pathogen safety provides the new immunoglobulin manufacturing process with a detailed, yet realistic, assessment of the risk of transmission of infectious pathogens.
[Show abstract][Hide abstract] ABSTRACT: Therapeutic proteins derived from human plasma and other biologic sources have demonstrated an excellent safety record relative to the potential threat of transmissible spongiform encephalopathy (TSE) transmission. Previously, hamster-adapted scrapie was used as a model agent to assess TSE clearance in purification steps leading to the isolation of biopharmaceutical proteins. The current study investigated the validity of hamster scrapie as a model for human TSE clearance studies. The partitioning of the pathogenic forms of the prion protein associated with human variant CJD (PrP(vCJD)), human sporadic CJD (PrP(sCJD)) and Gerstmann-Sträussler-Scheinker (PrP(GSS)) syndrome was compared to the partitioning of hamster scrapie (PrP(Sc)) in three plasma protein purification steps. Sheep scrapie (PrP(Sc)) was similarly evaluated.
The starting materials for three plasma protein purification steps, cryoseparation, 3 percent PEG separation, and 11.5 percent PEG separation, were spiked with brain homogenates containing human PrP(vCJD), human PrP(sCJD), human PrP(GSS), sheep PrP(Sc), and hamster 263K PrP(Sc). The partitioning of the pathogenic form of the PrP was analyzed.
Clearance of the pathogenic form of the PrP was measured relative to the effluent fraction. Regardless of the source of the pathogenic prion, clearance was similar to hamster PrP(Sc). A nominal amount of clearance (approx., 1 log), an intermediate amount of clearance (approx., 2 log), and a substantial amount of clearance (> or = 3 log) were observed for the cryoseparation, 3 percent PEG separation, and 11.5 percent PEG separation steps, respectively. In the latter step, no PrP was detected in the effluents.
These data demonstrate that human prions, including vCJD prions, can be removed during the purification of human therapeutic proteins and indicate that partitioning of human prions is similar to that observed in the hamster scrapie model.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jun 2002 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: Solvent-detergent treatment, although used routinely in plasma product processing to inactivate enveloped viruses, substantially reduces product yield from the human plasma resource. To improve yields in plasma product manufacturing, a new viral reduction process has been developed using the fatty acid caprylate. As licensure of plasma products warrants thorough evaluation of pathogen reduction capabilities, the present study examined susceptibility of enveloped viruses to inactivation by caprylate in protein solutions with varied pH and temperature. In the immunoglobin-rich solutions from Cohn Fraction II+III, human immunodeficiency virus, Type-1, bovine viral diarrhea virus (BVDV), and pseudorabies virus were inactivated by caprylate concentrations of >/=9 mM, >/=12 mM, and >/=9 mM, respectively. Compared to solvent-detergent treatment, BVDV inactivation in Fraction II+III solution was significantly faster (20-60 fold) using 16 mM caprylate. Caprylate-mediated inactivation of BVDV was not noticeably affected by temperature within the range chosen manufacturing the immunoglobulin product. In Fraction II+III solutions, IgG solubility was unaffected by </=19 mM caprylate. In albumin solution from Cohn supernatant IV-1, 40 mM caprylate rapidly inactivated BVDV, demonstrating versatility in inactivating enveloped viruses potentially present in other protein solutions. Our data show that caprylate is a robust enveloped virus inactivating agent for immunoglobulins and albumin which may potentially be utilized for other proteins; viral inactivation was not adversely affected by protein content and the buffer composition conditions evaluated. Within the parameters examined, caprylate inactivation of enveloped viruses provided comparable activity or advantages relative to the current, standard solvent-detergent treatment.
[Show abstract][Hide abstract] ABSTRACT: 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.
STUDY DESIGN AND METHODS: Seven plasma protein-purification steps were performed after the plasma intermediates were spiked with TSE-infected material. Resulting fractions were analyzed for PrPSc by using a Western blot assay and for TSE infectivity by using an animal bioassay. Western blots were quantitated by an endpoint dilution analysis, and infectivity titers were calculated by the Spearman-Kärber method.
RESULTS: PrPSc partitioning paralleled TSE infectivity partitioning, regardless of the nature of the protein-purification step. The detection ranges for PrPSc and infectivity were 0 to 5.3 log and 1.1 to 8.9 log median infectious dose per unit, respectively. Clearance of PrPSc and infectivity ranged from 1.0 to 6.0 log.
CONCLUSION: Purification steps for isolating therapeutic proteins from human plasma showed the removal of both PrPSc and TSE infectivity. PrPSc partitioning coincided with infectivity partitioning, which showed a close relationship between PrPSc and TSE infectivity. By exploiting this association, the in vitro Western blot assay for PrPSc was valuable for estimating the partitioning of TSE infectivity during plasma protein purification.
[Show abstract][Hide abstract] ABSTRACT: Manufacturers of human therapeutic proteins derived from biological sources continuously strive to improve the pathogen safety profiles of these products. Efforts to improve pathogen safety margins for these biological products are directed towards several areas within the manufacturing processes including: (a) sourcing and screening of raw materials (b) determining the potential for manufacturing processes to reduce pathogen titres, and (c) incorporating methods designed specifically to remove or inactivate contaminating pathogens. Methods that could potentially reduce pathogen titres are a major focus for many manufacturers. In general, these methods are grouped into two categories, pathogen clearance and pathogen inactivation. Assessments are performed on small-scale, laboratory simulations of the manufacturing process of interest that are spiked with a known amount of a selected pathogen. These studies provide estimates of the potential for a process step to remove or inactivate a particular pathogen. There are several pathogen clearance/inactivation methods that are inherent in manufacturing processes, however, some methods are intentionally incorporated into manufacturing for the sole purpose of reducing putative pathogen titres. Not only are well-known pathogens such as viruses targeted, but also suspected pathogens such as those associated with the transmissible spongiform encephalopathies (TSEs). The production processes for the isolation of several biological products, including recombinant KOGENATE Bayer (Kogenate FS), have been evaluated for the ability to reduce pathogen titres and/or have been designed to incorporate methods for reducing potential pathogen safety risks. Several processing steps with the potential to reduce pathogen titres have been identified.
[Show abstract][Hide abstract] ABSTRACT: Countless patients and clinicians rely on therapeutic proteins, such as intravenous immunoglobulins (IVIGs), isolated from human blood plasma. Since plasma is predisposed to contamination by a variety of blood-borne pathogens, ascertaining and ensuring the pathogen safety of plasma-derived therapeutics is a priority among manufacturers. Even though the pathogen safety records for IVIG and other plasma proteins are excellent, the industry remains active in research programs aimed at improving the margin of safety. Industry initiatives designed to increase the safety of plasma-derived products range from donor screening and testing to implementing methods into the manufacturing processes that can inactivate or remove pathogens from product streams. In general, the industry's comprehensive strategy is designed to provide patients and caregivers with the safest plasma products possible.
No preview · Article · Nov 2001 · Journal of Allergy and Clinical Immunology
[Show abstract][Hide abstract] ABSTRACT: 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 (PrP(Sc)) can serve as a marker for TSE infectivity.
Seven plasma protein-purification steps were performed after the plasma intermediates were spiked with TSE-infected material. Resulting fractions were analyzed for PrP(Sc) by using a Western blot assay and for TSE infectivity by using an animal bioassay. Western blots were quantitated by an endpoint dilution analysis, and infectivity titers were calculated by the Spearman-Kärber method.
PrP(Sc) partitioning paralleled TSE infectivity partitioning, regardless of the nature of the protein-purification step. The detection ranges for PrP(Sc) and infectivity were 0 to 5.3 log and 1.1 to 8.9 log median infectious dose per unit, respectively. Clearance of PrP(Sc) and infectivity ranged from 1.0 to 6.0 log.
Purification steps for isolating therapeutic proteins from human plasma showed the removal of both PrP(Sc) and TSE infectivity. PrP(Sc) partitioning coincided with infectivity partitioning, which showed a close relationship between PrP(Sc) and TSE infectivity. By exploiting this association, the in vitro Western blot assay for PrP(Sc) was valuable for estimating the partitioning of TSE infectivity during plasma protein purification.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Feb 2000 · Journal of Virological Methods