Safety of fibrinogen concentrate: analysis of more than 27 years of pharmacovigilance data.

Fibrinogen concentrate use as a haemostatic agent has been increasingly explored. This study evaluates spontaneous reports of potential adverse drug reactions (ADRs) that occurred during postmarketing pharmacovigilance of Haemocomplettan P/RiaSTAP, and reviews published safety data. This descriptive study analysed postmarketing safety reports recorded in the CSL Behring pharmacovigilance database from January 1986 to December 2013. A literature review of clinical studies published during the same period was performed. Commercial data indicated that 2,611,294 g of fibrinogen concentrate were distributed over the pharmacovigilance period, corresponding to 652,824 standard doses of 4 g each, across a range of clinical settings and indications. A total of 383 ADRs in 106 cases were reported (approximately 1 per 24,600 g or 6,200 standard doses). Events of special interest included possible hypersensitivity reactions in 20 cases (1 per 130,600 g or 32,600 doses), possible thromboembolic events in 28 cases (1 per 93,300 g or 23,300 doses), and suspected virus transmission in 21 cases (1 per 124,300 g or 31,000 doses). One virus transmission case could not be analysed due to insufficient data; for all other cases, a causal relationship was assessed as unlikely due to negative polymerase chain reaction tests and/or alternative explanations. The published literature revealed a similar safety profile. In conclusion, underreporting of ADRs is a known limitation of pharmacovigilance. However, the present assessment indicates that fibrinogen concentrate is administered across a range of indications, with few ADRs and a low thromboembolic event rate. Overall, fibrinogen concentrate showed a promising safety profile.

Defining acceptable epidemiology ranges in donor populations based on the contamination risk of finished plasma-derived products.

BACKGROUND: For a given plasma-derived product, the risk of final product contamination by hepatitis B virus, hepatitis C virus and human immunodeficiency virus depends upon the epidemiology in the donor population, the virus load in a donation, the product yield and the effective virus reduction capacity in manufacturing. STUDY DESIGN AND METHODS: A Monte Carlo simulation model was developed to estimate the risk of virus contamination of a final product resulting from virus contamination of plasma pools for fractionation. The model was run for both source and recovered plasma at various incidence rates for the three viruses to determine virus loads in minipools and fractionation pools resulting from donations with virus levels below test sensitivities. Together with the virus reduction capacity and yield of a theoretical worst case plasma-derived product, the contamination risk in a final vial was calculated. RESULTS: Acceptable upper-bound centre-level incidence rates in the donor population (per donor centre) result in final products with very high margins of virus safety; the largest determinant of these 'Process Limits' is the virus reduction capacity of the manufacturing process. Short donation intervals and long inventory hold periods for source plasma compensates the lower incidence rates typically observed in recovered plasma donors. CONCLUSIONS: The model calculates process limits for epidemiological data at collection centres based on an appropriate margin of virus safety for final products. The model also takes into consideration the impact of different donor/donation management systems for source and recovered plasma on the number of low viraemic donations entering the plasma pool for fractionation.

Peripuberty stress leads to abnormal aggression, altered amygdala and orbitofrontal reactivity and increased prefrontal MAOA gene expression.

Although adverse early life experiences have been found to increase lifetime risk to develop violent behaviors, the neurobiological mechanisms underlying these long-term effects remain unclear. We present a novel animal model for pathological aggression induced by peripubertal exposure to stress with face, construct and predictive validity. We show that male rats submitted to fear-induction experiences during the peripubertal period exhibit high and sustained rates of increased aggression at adulthood, even against unthreatening individuals, and increased testosterone/corticosterone ratio. They also exhibit hyperactivity in the amygdala under both basal conditions (evaluated by 2-deoxy-glucose autoradiography) and after a resident-intruder (RI) test (evaluated by c-Fos immunohistochemistry), and hypoactivation of the medial orbitofrontal (MO) cortex after the social challenge. Alterations in the connectivity between the orbitofrontal cortex and the amygdala were linked to the aggressive phenotype. Increased and sustained expression levels of the monoamine oxidase A (MAOA) gene were found in the prefrontal cortex but not in the amygdala of peripubertally stressed animals. They were accompanied by increased activatory acetylation of histone H3, but not H4, at the promoter of the MAOA gene. Treatment with an MAOA inhibitor during adulthood reversed the peripuberty stress-induced antisocial behaviors. Beyond the characterization and validation of the model, we present novel data highlighting changes in the serotonergic system in the prefrontal cortex-and pointing at epigenetic control of the MAOA gene-in the establishment of the link between peripubertal stress and later pathological aggression. Our data emphasize the impact of biological factors triggered by peripubertal adverse experiences on the emergence of violent behaviors.

Utilization of extracorporeal membrane oxygenation in congenital hypertrophic cardiomyopathy caused by maternal diabetes.

Extracorporeal membrane oxygenation (ECMO) is an advanced strategy utilized in many neonatal intensive care units for a specific list of indications. This case illustrates a rare but effective use of this therapy for a newborn infant with severe hypertrophic cardiomyopathy induced by maternal diabetes. Such infants who are unresponsive to conventional therapies may benefit from ECMO support, if it is used in conjunction with management strategies that optimize cardiac output.

Pathogen safety of plasma-derived products - Haemate P/Humate-P.

Plasma-derived factor VIII (FVIII) and von Willebrand Factor (VWF)/FVIII concentrates have been successfully used to treat haemophilia since the late 1960s. These products are derived from pools of plasma donations that may contain viral contaminants - including hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) - and may therefore present a transmission risk to recipients. To ensure the safety of Haemate P/Humate-P, a plasma-derived VWF/FVIII concentrate, donors of plasma are carefully selected and all donations are screened for viral antigens (HBV), virus-specific antibodies (HIV-1/2, HCV) and genomic material [hepatitis A virus, HBV, HCV, HIV-1 and high titres of human parvovirus B19 (B19V)]. As a quality control measure, plasma pools for fractionation are only released for further processing when non-reactivity has been demonstrated in serological and genome amplification assays. The manufacturing process for plasma-derived products, especially the fundamental procedure of pasteurization, is effective in inactivating and/or removing a wide variety of viruses that may potentially be present despite the screening process. This has been demonstrated in virus validation studies using a range of different viruses. New emerging infectious agents, including prions, which potentially pose a threat to recipients of plasma derivatives, are also the subject of safety evaluations. The multiple precautionary measures that are inherent in the overall production process of Haemate P/Humate-P have resulted in an excellent safety record, documented during 25 years of clinical use, and will help to maintain the high safety margin in the future.

Investigations of prion and virus safety of a new liquid IVIG product.

A highly purified, liquid, 10% immunoglobulin product stabilized with proline, referred to as IgPro10 has recently been developed. IgG was purified from human plasma by cold ethanol fractionation, octanoic acid precipitation and anion-exchange chromatography. The manufacturing process includes two distinctly different partitioning steps and virus filtration, which were also assessed for the removal of prions. Prion removal studies used different spike preparations (brain homogenate, microsomes, purified PrP(sc)) and three different detection methods (bioassay, Western blot, conformation-dependent immunoassay). All of the investigated production steps were shown to reduce significantly all different spike preparations, resulting in an overall reduction of >10log(10). Moreover, the biochemical assays proved equally effective to the bioassay for the demonstration of prion elimination. Four of the manufacturing steps cover three different mechanisms of virus clearance. These are: i) virus inactivation; ii) virus filtration; and iii) partitioning. These mechanisms were assessed for their virus reduction capacity. Virus validation studies demonstrated overall reduction factors of >18log(10) for enveloped and >7log(10) for non-enveloped model viruses. In conclusion, the IgPro10 manufacturing process has a very high reduction potential for prions and for a wide variety of viruses resulting in a state-of-the-art product concerning safety towards known and emerging pathogens.

In vitro amplification and detection of variant Creutzfeldt-Jakob disease PrPSc.

Variant Creutzfeldt-Jakob disease (vCJD) poses a serious risk of secondary transmission and the need to detect infectivity in asymptomatic individuals is therefore of major importance. Following infection, it is assumed that minute amounts of disease-associated prion protein (PrP(Sc)) replicate by conversion of the host cellular prion protein (PrP(C)). Therefore, methods of rapidly reproducing this conversion process in vitro would be valuable tools in the development of such tests. We show that one such technique, protein misfolding cyclic amplification (PMCA), can amplify vCJD PrP(Sc) from human brain tissue, and that the degree of amplification is dependent upon the substrate PRNP codon 129 polymorphism. Both human platelets and transgenic mouse brain are shown to be suitable alternative substrate sources, and amplified PrP(Sc) can be detected using a conformation-dependent immunoassay (CDI), allowing the detection of putative proteinase K sensitive forms of PrP(Sc).

Critical factors influencing prion inactivation by sodium hydroxide.

BACKGROUND AND OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Improved conformation-dependent immunoassay: suitability for human prion detection with enhanced sensitivity.

The presence of pathogenic prion protein (PrP(Sc)) in lymphoid tissues of variant Creutzfeldt-Jakob disease (vCJD) patients raises questions as to whether prions may be present in bodily fluids as well. Currently, transgenic mice are highly sensitive in vivo tools for the study of prions in tissues or fluids containing high levels of normal prion protein (PrP(C)). We report here an in vitro assay with virtually equivalent sensitivity incorporating a capture antibody into a sandwich conformation-dependent immunoassay (CDI), resulting in 30- to 100-fold increased sensitivity compared with the original, direct CDI. Furthermore, spiking plasma with vCJD prions in different preparations demonstrated that sandwich CDI detects prions with different biophysical properties at high sensitivity, even without proteinase K pretreatment of samples. Thus, sandwich CDI represents a powerful tool to study prions in bodily fluids of CJD/vCJD patients, with a turnaround time of less than 24 h.

Purity of spiking agent affects partitioning of prions in plasma protein purification.

Prions are not detectable in the blood or plasma of persons afflicted with classical or variant Creutzfeldt-Jakob disease, and they have never been shown to be transmitted by blood or plasma products. Despite the uncertainty as to the presence and biophysical properties of prions in plasma, prion removal studies have been conducted using brain homogenate or microsomes prepared from prion-infected rodent brains as model prions. In this study, we compare the partitioning of different prion spiking agents, having different biophysical properties, in the processes used for plasma protein purification. We have found that membrane-bound prion spiking agents partition similarly, whereas purified, unbound pathogenic prion proteins can have significantly different partitioning properties depending on the conditions in the production process. We conclude that prion spiking studies for the evaluation of prion reduction in plasma protein purification should employ spiking agents with different biophysical properties to mimic partitioning of the theoretical prion contaminant. This will give greater assurance as to the prion safety margins of the life-saving plasma protein therapeutics and excipients.

High-titer screening PCR: a successful strategy for reducing the parvovirus B19 load in plasma pools for fractionation.

BACKGROUND: Human parvovirus B19 (B19) is regarded as a potential risk factor for certain patient populations receiving plasma components. STUDY DESIGN AND METHODS: The prevalence of B19 was determined in a limited plasma donor population. Conditions for high-titer screening PCR were designed to allow the removal of plasma donations in the acute phase of infection with virus loads >or=10(7) genome equivalents per milliliter before manufacturing. Antithrombin III lots originating from screened plasma were compared to lots originating from untested plasma with respect to their B19 DNA load by a sensitive PCR assay. RESULTS: B19 was shown to have a prevalence of about 1 per 800 plasma donations. Only a minority (1/8000) of occurrences were in the acute phase of infection. Removing plasma units with high virus load as determined by high-titer screening PCR significantly decreased peak virus loads of plasma pools for fractionation. Together with a virus-removal capacity of 10.4 log(10) of the manufacturing process, this screening resulted in a final antithrombin III product that was nonreactive for B19 on PCR. CONCLUSION: Combining the strategy of high-titer screening PCR with the virus reduction capacity of the manufacturing process considerably increased the margin of B19 virus safety of antithrombin III. This strategy should have positive impact on other plasma components as well.

GB virus C/hepatitis G virus and intravenous immunoglobulins.

BACKGROUND AND OBJECTIVES: Different intravenous immunoglobulins (IVIGs) were found to be GB virus C/hepatitis G virus polymerase chain reaction (GBV-C/HGV-PCR)-positive. The potential transmission of this virus to recipients by a PCR-positive IVIG batch was investigated. MATERIALS AND METHODS: Polyclonal IVIGs of different manufacturers and with different virus inactivation procedures were analyzed by GBV-C/HGV-PCR and anti-E2-ELISA. Follow-up sera of 13 participants of a clinical trial performed with a GBV-C/HGV-PCR-positive batch were retrospectively investigated for GBV-C/HGV seroconversion (specific antibodies, viral RNA). RESULTS: Four out of ten IVIGs analysed by GBV-C/HGV-PCR were - at least for some batches - virus genome-positive. Virus inactivation by solvent/detergent treatment resulted in GBV-C/HGV-PCR-negative products. GBV-C/HGV-specific antibodies were detectable in all IVIGs analyzed. There was no transmission of GBV-C/HGV observed when recipients of a large amount of a GBV-C/HGV-PCR-positive batch were analyzed by an antibody test and specific PCR. CONCLUSIONS: Despite PCR positivity of an IVIG preparation no transmission of GBV-C/HGV to recipients was observed. Possible explanations are a sufficient virus inactivation procedure and/or presence of specific antibodies in the final products.

Analysis of human plasma products: polymerase chain reaction does not discriminate between live and inactivated viruses.

BACKGROUND: The viral safety of human plasma products is based on the careful selection of donors and donations and the removal and inactivation of human pathogenic viruses that could potentially contaminate human plasma. For the analysis of the final products for potential virus contamination, the use of polymerase chain reaction (PCR) has been proposed. To test whether this method can discriminate between infectious and inactivated viruses, the following studies were performed. STUDY DESIGN AND METHODS: Infectious and virus-inactivated preparations were titrated with specific PCR, using viruses such as hepatitis B virus (HBV), hepatitis C virus, bovine viral diarrhea virus, and poliovirus. The inactivation method employed was pasteurization (10 hours, 60 degrees C) or solvent/detergent (SD) treatment; in the case of HBV, there was consecutive treatment by both methods. RESULTS: Pasteurization of HBV and hepatitis C virus as well as SD treatment of HBV or pasteurization of HBV followed by SD treatment did not affect the detectability of these viruses by PCR, whereas an infectivity study in chimpanzees demonstrated that infectious hepatitis C virus was inactivated by pasteurization. Pasteurization also had no effect on the PCR titers of stabilized bovine viral diarrhea virus or poliovirus preparations, but it destroyed the infectivity of these viruses completely after only 4 hours' heat treatment. CONCLUSION: Pasteurization or SD treatment destroys the infectivity of the viruses tested, but neither significantly affects their detectability by specific PCR. Therefore PCR is not a suitable measure for testing the viral safety of finished plasma products that have been subjected to virus inactivation.

[Nanofiltration in production of Beriplex P/N: increasing the capacity of virus elimination while maintaining product quality]. / Nanofiltration bei der Herstellung von Beriplex P/N: Erhöhung der Kapazität zur Viruseliminierung unter Beibehaltung der Produktqualität.

For the manufacture of the PCC Beriplex P/N, nanofiltration was introduced into the production process of Beriplex HS providing an additional means to heat treatment for the clearance/inactivation of viruses. By nanofiltration, large enveloped viruses (HSV-1, HIV-1) were completely eliminated by a factor of more than 7 log10. While medium-sized enveloped viruses (HBV, BVDV) were cleared by a factor of approximately 4 log10, small non-enveloped viruses (poliovirus) were not removed. The product profile remained, no thrombogenic activities were detected.

Synthesis of biologically active influenza virus hemagglutinin in insect larvae.

The hemagglutinin of influenza (fowl plague) virus was expressed in larvae of Heliothis virescens by using recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector. Animals were infected with the recombinant virus either by parenteral injection or by feeding. For oral uptake, recombinant virus occluded in polyhedra obtained from cultured Spodoptera frugiperda cells after coinfection with authentic AcNPV was used. Immunohistological analyses of infected animals revealed that the hemagglutinin was expressed only in those tissues that are also permissive for the replication of authentic AcNPV. These tissues included hypodermis, fat body, and tracheal matrix. After oral infection, hemagglutinin was also detected in individual gut cells. The amount of hemagglutinin synthesized in larvae after parenteral infection was 0.3% of the total protein, compared with 5% obtained in cultured insect cells. The hemagglutinin was transported to the cell surface and expressed in polarized cells only at the apical plasma membrane. It was processed by posttranslational proteolysis into the cleavage products HA1 and HA2. Oligosaccharides were attached by N-glycosidic linkages and were smaller than those found on hemagglutinin obtained from vertebrate cells. Hemagglutinin from larvae expressed receptor binding and cell fusion activities, but quantitation of the hemolytic capacity revealed that it was only about half as active as hemagglutinin from vertebrate or insect cell cultures. Chickens immunized with larval tissues containing hemagglutinin were protected from infection with fowl plague virus. These observations demonstrate that live insects are able to produce a recombinant membrane protein of vertebrate origin in biologically active form.

Interaction of Autographa californica nuclear polyhedrosis virus with two nonpermissive cell lines.

The interaction of Autographa californica nuclear polyhedrosis virus with two nonpermissive cell lines was investigated. The insect cell line, CP 169, and the Chinese hamster cell line, CHO-K1, were able to adsorb and engulf virus particles, but there was no evidence for viral replication in these cells based on virus growth titrations, electron microscopy, dot hybridization, and synthesis of viral induced proteins.