Nanofiltration as a robust method contributing to viral safety of plasma-derived therapeutics: 20 years' experience of the plasma protein manufacturers.

BACKGROUND: Nanofiltration entails the filtering of protein solutions through membranes with pores of nanometric sizes that have the capability to effectively retain a wide range of viruses. STUDY DESIGN AND METHODS: Data were collected from 754 virus validation studies (individual data points) by Plasma Protein Therapeutics Association member companies and analyzed for the capacity of a range of nanofilters to remove viruses with different physicochemical properties and sizes. Different plasma product intermediates were spiked with viruses and filtered through nanofilters with different pore sizes using either tangential or dead-end mode under constant pressure or constant flow. Filtration was performed according to validated scaled-down laboratory conditions reflecting manufacturing processes. Effectiveness of viral removal was assessed using cell culture infectivity assays or polymerase chain reaction (PCR). RESULTS: The nanofiltration process demonstrated a high efficacy and robustness for virus removal. The main factors affecting nanofiltration efficacy are nanofilter pore size and virus size. The capacity of nanofilters to remove smaller, nonenveloped viruses was dependent on filter pore size and whether the nanofiltration process was integrated and designed with the intention to provide effective parvovirus retention. Volume filtered, operating pressure, and total protein concentration did not have a significant impact on the effectiveness of virus removal capacity within the investigated ranges. CONCLUSIONS: The largest and most diverse nanofiltration data collection to date substantiates the effectiveness and robustness of nanofiltration in virus removal under manufacturing conditions of different plasma-derived proteins. Nanofiltration can enhance product safety by providing very high removal capacity of viruses including small non-enveloped viruses.

Prion removal capacity of plasma protein manufacturing processes: a data collection from PPTA member companies.

BACKGROUND: The variant Creutzfeldt-Jakob disease incidence peaked a decade ago and has since declined. Based on epidemiologic evidence, the causative agent, pathogenic prion, has not constituted a tangible contamination threat to large-scale manufacturing of human plasma-derived proteins. Nonetheless, manufacturers have studied the prion removal capabilities of various manufacturing steps to better understand product safety. Collectively analyzing the results could reveal experimental reproducibility and detect trends and mechanisms driving prion removal. STUDY DESIGN AND METHODS: Plasma Protein Therapeutics Association member companies collected more than 200 prion removal studies on plasma protein manufacturing steps, including precipitation, adsorption, chromatography, and filtration, as well as combined steps. The studies used a range of model spiking agents and bench-scale process replicas. The results were grouped based on key manufacturing variables to identify factors impacting removal. The log reduction values of a group are presented for comparison. RESULTS: Overall prion removal capacities evaluated by independent groups were in good agreement. The removal capacity evaluated using biochemical assays was consistent with prion infectivity removal measured by animal bioassays. Similar reduction values were observed for a given step using various spiking agents, except highly purified prion protein in some circumstances. Comparison between combined and single-step studies revealed complementary or overlapping removal mechanisms. Steps with high removal capacities represent the conditions where the physiochemical differences between prions and therapeutic proteins are most significant. CONCLUSION: The results support the intrinsic ability of certain plasma protein manufacturing steps to remove prions in case of an unlikely contamination, providing a safeguard to products.

Effective virus inactivation and removal by steps of Biotest Pharmaceuticals IGIV production process.

The virus validation of three steps of Biotest Pharmaceuticals IGIV production process is described here. The steps validated are precipitation and removal of fraction III of the cold ethanol fractionation process, solvent/detergent treatment and 35 nm virus filtration. Virus validation was performed considering combined worst case conditions. By these validated steps sufficient virus inactivation/removal is achieved, resulting in a virus safe product.

Contribution to safety of immunoglobulin and albumin from virus partitioning and inactivation by cold ethanol fractionation: a data collection from Plasma Protein Therapeutics Association member companies.

BACKGROUND: Virus removal by partitioning into different fractions during cold ethanol fractionation has been described by several authors, demonstrating that cold ethanol fractionation can provide significant contribution to virus removal, even in those cases where virus removal is limited and must be supported by additional measures for virus inactivation during further processing. STUDY DESIGN AND METHODS: Plasma Protein Therapeutics Association (PPTA) member companies collected and evaluated 615 studies on virus removal by the steps of the cold ethanol fractionation process. The studies describe the precipitation and separation of Fraction (F)III or FI/III in the immunoglobulin fractionation process and precipitation and separation of FII/III, FI/II/III, and FIV/IV in the albumin fractionation process. RESULTS: The data indicate a significant contribution of cold ethanol fractionation to the overall clearance of a broad spectrum of viruses, at varied process variables such as pH, temperature, and alcohol concentration and demonstrate the robustness of virus removal by the cold ethanol fractionation process. CONCLUSIONS: The data presented here support the importance of the partitioning steps for virus safety for immunoglobulins and albumin. However, virus removal by cold ethanol fractionation alone cannot provide viral safety of human albumin and immunoglobulins and therefore must be completed by other virus inactivation and removal procedures.

Impaired axonal transport in motor neurons correlates with clinical prion disease.

Prion diseases are fatal neurodegenerative disorders causing motor dysfunctions, dementia and neuropathological changes such as spongiosis, astroglyosis and neuronal loss. The chain of events leading to the clinical disease and the role of distinct brain areas are still poorly understood. The role of nervous system integrity and axonal properties in prion pathology are still elusive. There is no evidence of both the functional axonal impairments in vivo and their connection with prion disease. We studied the functional axonal impairments in motor neurons at the onset of clinical prion disease using the combination of tracing as a functional assay for axonal transport with immunohistochemistry experiments. Well-established and novel confocal and ultramicroscopy techniques were used to image and quantify labeled neurons. Despite profound differences in the incubation times, 30% to 45% of neurons in the red nucleus of different mouse lines showed axonal transport impairments at the disease onset bilaterally after intracerebral prion inoculation and unilaterally -- after inoculation into the right sciatic nerve. Up to 94% of motor cortex neurons also demonstrated transport defects upon analysis by alternative imaging methods. Our data connect axonal transport impairments with disease symptoms for different prion strains and inoculation routes and establish further insight on the development of prion pathology in vivo. The alterations in localization of the proteins involved in the retrograde axonal transport allow us to propose a mechanism of transport disruption, which involves Rab7-mediated cargo attachment to the dynein-dynactin pathway. These findings suggest novel targets for therapeutic and diagnostic approaches in the early stages of prion disease.

Ultramicroscopy reveals axonal transport impairments in cortical motor neurons at prion disease.

The functional imaging of neuronal circuits of the central nervous system is crucial for phenotype screenings or investigations of defects in neurodegenerative disorders. Current techniques yield either low penetration depth, yield poor resolution, or are restricted by the age of the animals. Here, we present a novel ultramicroscopy protocol for fluorescence imaging and three-dimensional reconstruction in the central nervous system of adult mice. In combination with tracing as a functional assay for axonal transport, retrogradely labeled descending motor neurons were visualized with >4 mm penetration depth. The analysis of the motor cortex shortly before the onset of clinical prion disease revealed that >80% neurons have functional impairments in axonal transport. Our study provides evidence that prion disease is associated with severe axonal transport defects in the cortical motor neurons and suggests a novel mechanism for prion-mediated neurodegeneration.

Alteration of B-cell subsets enhances neuroinvasion in mouse scrapie infection.

Acquired forms of prion diseases or transmissible spongiform encephalopathies are believed to occur following peripheral exposure. Prions initially accumulate in the lymphoid system before spreading to the nervous system, but the underlying mechanisms for prion transfer between the two systems are still elusive. Here we show that ablation of the B-cell-specific transmembrane protein CD19, a coreceptor of the complement system, results in an acceleration of prion neuroinvasion. This appears to be due to an alteration of the follicular dendritic cell (FDC) network within the lymphoid tissue, thereby reducing the distance between FDCs and adjacent nerve fibers that mediate prion neuroinvasion.

The tyrosine kinase inhibitor imatinib mesylate delays prion neuroinvasion by inhibiting prion propagation in the periphery.

Prion diseases are fatal neurodegenerative disorders with no effective therapy. A hallmark of prion disease is the conversion of the normal cellular form of prion protein PrP(C) into a disease-associated isoform PrP(Sc). The authors recently have shown that a tyrosine kinase inhibitor, imatinib mesylate, induces clearance of PrP(Sc) via specific inhibition of c-Abl in prion-infected cell culture models. In this study, the authors assessed the in vivo effects of imatinib mesylate on prion disease using a scrapie-infected mouse model and further investigated prion infectivity of the drug-treated scrapie-infected neuroblastoma (ScN2a) cells. The authors found that imatinib mesylate abolished prion infectivity to almost undetectable level in ScN2a cells and the level of PrP(Sc) was significantly decreased by the drug in scrapie-infected mouse spleens as well as in ScN2a cells. Moreover, the drug treatment at an early phase of peripheral scrapie infection delayed the appearance of PrP(Sc) in the central nervous system (CNS) and onset of clinical disease in mice. However, neither intraperitoneal nor intracerebroventricular delivery of the drug exerted any PrP(Sc) clearance effect in the CNS.

Immunisation strategies against prion diseases: prime-boost immunisation with a PrP DNA vaccine containing foreign helper T-cell epitopes does not prevent mouse scrapie.

Vaccination against prion diseases constitutes a promising approach for the treatment and prevention of the disease. Passive immunisation with antibodies binding to the cellular prion protein (PrP(C)) can protect against prion disease. However, immunotherapeutic strategies with active immunisation are limited due to the immune tolerance against the self-antigen. In order to develop an anti-prion vaccine, we designed a novel DNA fusion vaccine composed of mouse PrP and immune stimulatory helper T-cell epitopes of the tetanus toxin that have previously been reported to break tolerance to other self-antigens. This approach provoked a strong PrP(C)-specific humoral and cellular immune response in PrP null mice, but only low antibody titres were found in vaccinated wild-type mice. Furthermore, prime-boost immunisation with the DNA vaccine and recombinant PrP protein increased antibody titres in PrP null mice, but failed to protect wild-type mice from mouse scrapie.

An enzyme-detergent method for effective prion decontamination of surgical steel.

Prions, transmissible agents that cause Creutzfeldt-Jakob disease (CJD) and other prion diseases, are known to resist conventional sterilization procedures. Iatrogenic transmission of classical CJD via neurosurgical instruments is well documented and the involvement of lymphoreticular tissues in variant CJD (vCJD), together with the unknown population prevalence of asymptomatic vCJD infection, has led to concerns about transmission from a wide range of surgical procedures. To address this problem, conditions were sought that destroy PrP(Sc) from vCJD-infected human tissue and eradicate RML prion infectivity adsorbed onto surgical steel. Seven proteolytic enzymes were evaluated individually and in pairs at a range of temperatures and pH values and the additional effects of detergents, lipases and metal ions were assessed. A combination of proteinase K and Pronase, in conjunction with SDS, was shown to degrade PrP(Sc) material from highly concentrated vCJD-infected brain preparations to a level below detection. When RML prion-infected wires were exposed to the same enzymic treatment, intracerebral bioassay in highly susceptible hosts showed virtually no infectivity. The prion-degrading reagents identified in this study are readily available, inexpensive, non-corrosive to instruments, non-hazardous to staff and compatible with current equipment and procedures used in hospital sterilization units.

The tyrosine kinase inhibitor STI571 induces cellular clearance of PrPSc in prion-infected cells.

The conversion of the cellular prion protein (PrP(c)) into pathologic PrP(Sc) and the accumulation of aggregated PrP(Sc) are hallmarks of prion diseases. A variety of experimental approaches to interfere with prion conversion have been reported. Our interest was whether interference with intracellular signaling events has an impact on this conversion process. We screened approximately 50 prototype inhibitors of specific signaling pathways in prion-infected cells for their capacity to affect prion conversion. The tyrosine kinase inhibitor STI571 was highly effective against PrP(Sc) propagation, with an IC(50) of < or =1 microM. STI571 cleared prion-infected cells in a time- and dose-dependent manner from PrP(Sc) without influencing biogenesis, localization, or biochemical features of PrP(c). Interestingly, this compound did not interfere with the de novo formation of PrP(Sc) but activated the lysosomal degradation of pre-existing PrP(Sc), lowering the half-life of PrP(Sc) from > or =24 h to <9 h. Our data indicate that among the kinases known to be inhibited by STI571, c-Abl is likely responsible for the observed anti-prion effect. Taken together, we demonstrate that treatment with STI571 strongly activates the lysosomal degradation of PrP(Sc) and that substances specifically interfering with cellular signaling pathways might represent a novel class of anti-prion compounds.

Expression of truncated PrP targeted to Purkinje cells of PrP knockout mice causes Purkinje cell death and ataxia.

PrP knockout mice with disruption of only the PrP-encoding region (Zürich I-type) remain healthy, whereas mice with deletions extending upstream of the PrP-encoding exon (Nagasaki-type) suffer Purkinje cell loss and ataxia, associated with ectopic expression of Doppel in brain, particularly in Purkinje cells. The phenotype is abrogated by co-expression of full-length PrP. Doppel is 25% similar to PrP, has the same globular fold, but lacks the flexible N-terminal tail. We now show that in Zürich I-type PrP-null mice, expression of N-terminally truncated PrP targeted to Purkinje cells also leads to Purkinje cell loss and ataxia, which are reversed by PrP. Doppel and truncated PrP probably cause Purkinje cell degeneration by the same mechanism.

Molecular biology of prions.

The "protein only" hypothesis holds that the infectious agent causing transmissible spongiform encephalopathies is a conformational isomer of PrP, a host protein predominantly expressed in brain and is strongly supported by many lines of evidence. Prion diseases are so far unique among conformational diseases in that they are transmissible, not only experimentally but also by natural routes, mainly by ingestion. The pathway of prions to the brain has been elucidated in outline. A striking feature of prions is their extraordinary resistance to conventional sterilisation procedures, and their capacity to bind to surfaces of metal and plastic without losing infectivity. This property, first observed in a clinical setting, is now being investigated in experimental settings, both in animals and in cell culture.