Controls to Minimize Disruption of the Pharmaceutical Supply Chain During the COVID-19 Pandemic.

This article reviews currently available scientific literature related to the epidemiology, infectivity, survival, and susceptibility to disinfectants of Coronaviruses, in the context of the controls established to meet good manufacturing practice (GMP) regulations and guidance, and the public health guidance issued specifically to combat the COVID-19 pandemic. The possible impact of the COVID-19 pandemic on the pharmaceutical supply chain is assessed and recommendations are listed for risk mitigation steps to minimize supply disruption to pharmaceutical drug products. Areas addressed include a brief history of the COVID-19 viral pandemic, a description of the virus, the regulatory response to the pandemic, the screening of employees, the persistence of the virus on inanimate surfaces, cleaning and disinfection of manufacturing facilities, the use of GMP-mandated personal protective equipment to counter the spread of the disease, the role of air changes in viral clearance, and approaches to risk assessment and mitigation. Biological medicinal products have a great record of safety, yet the cell cultures used for production can be susceptible to viruses, and contamination events have occurred. Studies on SARS-CoV-2 for it ability to replicate in various mammalian cell lines used for biopharmaceutical manufacturing suggests that the virus poses a low risk and any contamination would be detected by currently used adventitious virus testing. The consequences of the potential virus exposure of manufacturing processes as well as the effectiveness of mitigation efforts are discussed. The pharmaceutical supply chain is complex, traversing many geographies and companies that range from large multinationals to mid- and small-size operations. This paper recommends practices that can be adopted by all companies, irrespective of their size, geographic location, or position in the supply chain.

Increasing West Nile virus antibody titres in central European plasma donors from 2006 to 2010.

We analysed by neutralisation assay 55 intravenous immunoglobulin preparations produced from human plasma collected in three central European countries, specifically Austria, Germany and the Czech Republic, from 2006 to 2010. The preparations from 2009 and 2010 contained increasing titres of neutralising antibodies against West Nile virus (WNV) in the absence of reported human WNV cases in these countries.

A new liquid intravenous immunoglobulin with three dedicated virus reduction steps: virus and prion reduction capacity.

BACKGROUND AND OBJECTIVES: A new 10% liquid human intravenous immunoglobulin (US trade name: Gammagard Liquid; European trade name: KIOVIG) manufactured by a process with three dedicated pathogen inactivation/removal steps (solvent/detergent treatment, 35-nm nanofiltration and low pH/elevated temperature incubation) was developed. The ability of the manufacturing process to inactivate/remove viruses and prions was investigated. MATERIALS AND METHODS: Virus and prion removal capacities were assessed with down-scale spiking experiments, validated for equivalence to the large-scale process. RESULTS: Lipid-enveloped viruses were completely inactivated/removed by each of the three dedicated virus clearance steps, and for human immunodeficiency virus 1 (HIV-1) and pseudorabies virus (PRV), also by the upstream cold ethanol fractionation step. Relevant non-enveloped viruses [i.e. hepatitis A virus (HAV) and parvovirus B19 (B19V)] were effectively removed by nanofiltration and the cold ethanol fractionation step, and partial inactivation of non-enveloped viruses was achieved by low pH incubation. Overall log reduction factors were > 20.0 for HIV-1, > 18.1 for bovine viral diarrhoea virus, > 16.3 for West Nile virus, > 10.0 for influenza A virus subtype H5N1, > 21.8 for PRV, 12.0 for HAV, > 12.1 for encephalomyocarditis virus, 10.6 for B19V and 10.3 for mice minute virus. Prions (Western blot assay) were completely removed (> or = 3.2 mean log reduction) by a step of the cold ethanol fractionation process. CONCLUSIONS: Introducing three dedicated virus-clearance steps in the manufacturing process of immunoglobulins from human plasma provides high margins of safety.

H5N1 influenza virus and the safety of plasma products.

BACKGROUND: The ever-increasing number of human H5N1 influenza virus infections may enable these viruses to acquire the ability to spread effectively among humans and potentially to cause a pandemic. Recently, more systemic virus dissemination was reported during H5N1 virus infection of humans, resulting in significant virus concentrations also in the blood. The observation has raised concerns about the safety of labile blood products for transfusion and consequentially also for plasma derivatives. To confirm the safety margins of plasma products, dedicated virus inactivation processes used during their production were investigated for their effectiveness in inactivating this virus of recent concern. STUDY DESIGN AND METHODS: Virus inactivation by steps commonly used during the manufacture of plasma derivatives, such as pasteurization for human albumin, solvent/detergent treatment for intravenous immunoglobulin (IVIG), vapor heating for factor VIII inhibitor bypassing activity, and incubation at low pH for IVIG, were investigated with a reassortant strain of H5N1 influenza virus. RESULTS: The results show that H5N1 influenza behaves as expected for lipid-enveloped viruses; that is, the virus is effectively inactivated by all the commonly used virus inactivation procedures tested. CONCLUSION: The safety margins of plasma derivatives against the theoretical transmission of H5N1 influenza virus are very substantial.

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.

Effective poxvirus removal by sterile filtration during manufacture of plasma derivatives.

As a consequence of the September 2001 terrorist events, programs to protect against further such acts including potentially the use of biological warfare agents have been launched in the USA and elsewhere. As part of these initiatives, Vaccinia virus was procured for the pre-emptive vaccination of key personnel against smallpox as well as population-wide protection after an eventual exposure. The introduction of this live virus into a population at a relatively large scale represents a theoretical challenge for the safety of the blood supply, and potentially for plasma for fractionation. To strengthen further the demonstration of safety margins for plasma derived products against Vaccinia virus, the capacity of sterile filtration procedures to remove the virus was investigated. An infectivity assay for the Vaccinia virus strain which represents the majority of smallpox vaccine stocks available currently was used to investigate the potential removal of this virus by sterile filtration processes during the manufacture of plasma derivatives. Vaccinia virus behaves as predicted based on its size, i.e., an artificially added virus load is removed about 10,000-fold by the sterile filtration procedures tested. As the current investigation covered a range of different protein concentrations, filter materials and filters from different manufacturers, the results obtained are considered to be widely applicable. The current investigation supports further the high safety margins of plasma derivatives against any potential Vaccinia virus content of plasma for fractionation. As the large size is a general feature of Orthopox viruses, the results would also provide assurance against poxviruses identified more recently, for example, Monkeypox virus.

West Nile virus: recent experience with the model virus approach.

During recent West Nile Virus (WNV) epidemics in the USA, virus transmissions through transfusion of blood components have been observed, also raising concerns about the safety of plasma derivatives. To verify the safety margins of these products, as initially shown using model viruses very similar to WNV, virus inactivation procedures incorporated into their manufacturing processes were re-investigated using WNV directly. The results show that WNV behaves exactly as had been predicted based on available data for similar ("model") viruses, i.e. it is readily inactivated by e.g. pasteurisation of human albumin, solvent-detergent treatment of intravenous immunoglobulin and factor VIII, vapour heating of factor VIII inhibitor by-passing activity, and incubation of intravenous immunoglobulin at low pH. Our investigation thus verifies the safety margins of plasma derivatives against Flaviviruses in general, and now WNV in particular. Also, use of the model virus concept was confirmed to be well capable of predicting the behaviour of a certain virus based on available data for closely related viruses.

Passive immunization reduces immunity that results from simultaneous active immunization against tick-borne encephalitis virus in a mouse model.

Concomitant administration of an antigen and antibodies of the respective specificity has been shown to result in reduced levels of actively produced antibodies. This has also recently been observed in a clinical trial on simultaneous passive and active immunization against tick-borne encephalitis virus (TBEV). In the current study the influence of simultaneous passive and active immunization on vaccine induced protective immunity against TBEV has been evaluated in a mouse model. Two immunizations with licensed whole-killed TBEV vaccines gave close to complete protection. Administration of human or mouse TBEV antibodies together with the first vaccine dose resulted in a significant reduction of vaccine induced protection against TBEV challenge. This effect was even more pronounced than that observed earlier on the levels of vaccine induced antibody.

Vaccination against tick-borne encephalitis virus, a flavivirus, prevents disease but not infection, although viremia is undetectable.

By adoptive transfer of sera or immunoglobulin preparations, vaccine-induced protection against TBEV has been demonstrated to be mediated by antibodies to the surface protein of TBEV, glycoprotein E. Nevertheless, the mechanism of vaccine-induced protection against TBEV remains unclear. Protection by E antibodies without in vitro neutralization was shown by one group, whereas others found a correlation between protection in vivo and neutralization in vitro. Here, the authors confirm in a mouse model of tick-borne encephalitis (TBE) that immunization with the whole-killed virus vaccine protects mice against a subsequent challenge with a highly lethal dose of virus, i.e. 250 LD50 doses. Vaccine-induced immunity, however, is not completely neutralizing as demonstrated by the development of immune responses to a non-structural virus protein absent from the vaccine, yet expressed in the course of virus replication. Antibodies specific for the non-structural protein 1 (NS1) and cytotoxic T-cells could be detected after, but not prior to, virus challenge of vaccinated animals, establishing that protection by this highly effective vaccine is not equivalent with complete neutralization of the challenge virus.

Neutralizing antibodies protect against lethal flavivirus challenge but allow for the development of active humoral immunity to a nonstructural virus protein.

Antibody-mediated neutralization of viruses has been extensively studied in vitro, but the precise mechanisms that account for antibody-mediated protection against viral infection in vivo still remain largely uncharacterized. The two points under discussion are antibodies conferring sterilizing immunity by neutralizing the virus inoculum or protection against the development of disease without complete inhibition of virus replication. For tick-borne encephalitis virus (TBEV), a flavivirus, transfer of neutralizing antibodies specific for envelope glycoprotein E protected mice from subsequent TBEV challenge. Nevertheless, short-term, low-level virus replication was detected in these mice. Furthermore, mice that were exposed to replicating but not to inactivated virus while passively protected developed active immunity to TBEV rechallenge. Despite the priming of TBEV-specific cytotoxic T cells, adoptive transfer of serum but not of T cells conferred immunity upon naive recipient mice. These transferred sera were not neutralizing and were predominantly specific for NS1, a nonstructural TBEV protein which is expressed in and on infected cells and which is also secreted from these cells. Results of these experiments showed that despite passive protection by neutralizing antibodies, limited virus replication occurs, indicating protection from disease rather than sterilizing immunity. The protective immunity induced by replicating virus is surprisingly not T-cell mediated but is due to antibodies against a nonstructural virus protein absent from the virion.

Detection of tick-borne encephalitis virus by sample transfer, plaque assay and strand-specific reverse transcriptase polymerase chain reaction: what do we detect?

Experimental inoculation of mice provides a well characterized model for studying infection with tick-borne encephalitis virus (TBEV), a flavivirus pathogenic for humans. Conflicting data on the kinetics of viremia and the development of virus titers in the brain, however, were only recently shown to have resulted from the use of assay systems with different levels of sensitivity in the titration of TBEV, i.e. plaque assay or sample transfer into naive recipient mice. Theoretically, RT-PCR could extend further the detectability to antibody-neutralized virus and when undertaken strand-specifically discriminate active replication from the mere presence of TBEV. We have compared the conventional methods for detection of TBEV with a newly devised RT-PCR method. As expected, RT-PCR, in contrast to the infectivity assays, detected antibody-neutralized virus. Furthermore, the mere presence or active replication of the virus could be differentiated by strand-specific RT-PCR. Plaque assay and sample transfer, in contrast, both detected only infectious virus. However, whereas sample transfer provides higher sensitivity for detection of TBEV from solid organs, the plaque assay is less costly and considering animals welfare more convenient. Thus, the newly devised method may allow the resolution of unanswered questions, while both the traditional infectivity assays retain their benefits in certain situations.

Antibodies protect mice against challenge with tick-borne encephalitis virus (TBEV)-infected macrophages.

TBEV is a flavivirus highly pathogenic for humans. By transfer of antibodies directed to the TBEV surface glycoprotein E into mice, immune protection against subsequent inoculation with free TBEV particles could be achieved. After natural TBEV infection via the skin, however, cells of the monocyte/macrophage lineage were recently demonstrated to represent an important source of local virus replication before viraemia occurs. Whether antibodies can protect against virus challenge when contracted in the form of infected cells, however, is still unclear. In the current study, TBEV antibodies protected mice against challenge with either free virus or TBEV-infected macrophages equally well. This observation may be of more general significance.

Inhibition of IL-10 protein synthesis induces major histocompatibility complex class II gene expression in class II-deficient patients.

Major histocompatibility complex (MHC) class II deficiency is an inherited autosomal recessive combined immunodeficiency, characterized by a lack of constitutive expression of the human leukocyte antigen (HLA) class II genes. The patients investigated in this study are histoidentical twin brothers with a new phenotype in MHC class II deficiency. Examination of HLA-D locus genes in their fractionated peripheral mononuclear cells (MNCs) revealed an unusual and uncoordinated mRNA pattern. Here we analyzed the distribution of pro- and anti-inflammatory cytokines expressed in these patients' adherent and nonadherent MNCs. We show that gene expression of IL-1 alpha, IL-1 beta, IL-6, granulocyte-colony-stimulating factor, and IL-10 was induced in both cell fractions, whereas increased mRNA levels of interferon-gamma and the inducible nitric oxide synthase were exclusively detected in the patients' nonadherent MNCs. As IL-10 is known to be able to downregulate transcription of MHC class II and expression of IL-10 in the patients' MNCs was increased, we investigated the regulatory function of this cytokine. Interestingly, inhibition of IL-10 protein synthesis with IL-10-specific antisense oligonucleotide DNA (IL-10-AS-ODN) induced HLA-D locus genes in these MHC class II-deficient patients. Exposure of the nonadherent cell fraction to IL-10-AS-ODN resulted in a profound induction of a previously absent DR beta 1 and DP alpha gene expression. HLA-DQ beta mRNA levels, however, were increased in both the adherent and the nonadherent MNC population. Albeit expression of HLA-D locus genes was inducible via inhibition of IL-10 translation, surface expression of HLA class II antigens on the patients' MNCs was essentially negative. The data presented support the concept of a coordinated network of pro- and anti-inflammatory cytokine regulation and this network obviously has a significant role in the cell-type-specific regulation of MHC class II expression.

Pre- and postexposure protection by passive immunoglobulin but no enhancement of infection with a flavivirus in a mouse model.

Antibody-dependent enhancement of flavivirus infection, which except for dengue virus is without clear proof in vivo, is still under debate. Recently, postexposure immunoglobulin prophylaxis against tick-borne encephalitis virus, a flavivirus, was claimed to possibly have worsened the outcome of infection due to antibody-dependent enhancement. In the present study, antibody-dependent enhancement and pre- or postexposure protection by passive administration of tick-borne encephalitis virus immunoglobulin were evaluated in a mouse model. Preexposure treatment with homologous murine or heterologous human immunoglobulin provided complete protection against lethal challenge with tick-borne encephalitis virus. For postexposure treatment with antibody, the degree of protection correlated with the amount of immunoglobulin administered and was inversely related to the time interval between infection and treatment. Indications of enhancement of infection would have been increased lethality or reduced mean survival time, but neither was observed under the conditions used in our experiments despite the broad range of immunoglobulin and virus challenge doses applied. In contrast to these in vivo results, antibody-dependent enhancement of tick-borne encephalitis virus infection of murine peritoneal macrophages was readily demonstrable in vitro. Thus, antibody-dependent enhancement of viral infection in vitro does not necessarily predict enhancement in vivo.

Nitric oxide and viral infection: NO antiviral activity against a flavivirus in vitro, and evidence for contribution to pathogenesis in experimental infection in vivo.

Upon stimulation murine macrophages produce high levels of nitric oxide (NO), a potent microbicidal and tumoricidal agent recently also implicated as a mediator of antiviral defense. As dysregulated production of NO may lead to extensive tissue damage, the production of this powerful mediator is tightly regulated. Viral infection, however, may alter the regulation of certain macrophage functions, and recent work from our group demonstrated that viral infection--via induction of interferon-alpha beta synthesis -- may either prime for or down-modulate NO production. In light of antiviral activities of NO, down-modulation of NO production in viral infection would seem contradictory to antiviral defense. As others, however, have provided evidence that NO production may contribute to pathogenesis of infection with several neurotropic viruses, the role of NO production was investigated in vitro and in vivo in murine macrophages and in BALB/c mice infected with tick-borne encephalitis virus (TBE-V), a flavivirus. Macrophages from TBE-V-infected mice, but not from control mice, spontaneously produced NO upon culture in vitro. In contrast to the inhibitory effect of NO on replication of several poxviruses and herpes simplex virus, high levels of NO production did not display an inhibitory influence on TBE-V replication in vitro. And finally, in vivo administration of a competitive inhibitor of NO production, aminoguanidine, to TBE-V-infected mice significantly increased their mean survival time. Our results thus demonstrate that the antiviral activity of NO in vitro may be confined to certain viruses, whereas others remain unaffected. Furthermore, we provide evidence that NO production may even contribute to pathogenesis of viral infection in vivo.

Viral infection of macrophages profoundly alters requirements for induction of nitric oxide synthesis.

Activated mouse macrophages can produce high levels of nitric oxide, an antimicrobial effector molecule recently also implicated in antiviral defense. As viral infection may alter macrophage functions, nitric oxide production was investigated in murine macrophages infected with a Flavivirus, tick-borne encephalitis virus. Infected macrophages produced high levels of nitric oxide upon stimulation with lipopolysaccharide without priming, while in control macrophages induction of nitric oxide production by lipopolysaccharide required priming with interferon-gamma. Addition of interferon-gamma to infected macrophages further increased lipopolysaccharide-stimulated nitric oxide production. In contrast, nitric oxide production upon stimulation with interferon-gamma plus tumor necrosis factor-alpha was markedly reduced in infected macrophages. Downregulation of interferon-gamma plus tumor necrosis factor-alpha-induced nitric oxide synthesis by viral infection could be attributed to endogenous interferon-alpha beta produced by infected macrophages. Addition of interferon-alpha beta to uninfected macrophages inhibited interferon-gamma plus tumor necrosis factor-alpha-induced nitric oxide production, and addition of interferon-alpha beta antibodies to infected macrophages increased identically stimulated nitric oxide production to normal levels. Thus, interferon-alpha beta mimicked the effect of viral infection on macrophage nitric oxide production. These findings indicate that viral infection profoundly alters requirements of mouse macrophages for induction of nitric oxide synthesis, depending on the activating signal applied. The described effects of viral infection of macrophages on the regulation of nitric oxide production and new complexity to the role of nitric oxide in host defense against viruses.