Viral Safety Research Articles

Viral safety of biological medicinal products

Viral safety of blood donations, plasma products, viral vaccines and gene therapy medicinal products, biotechnical-derived products and tissue and cell therapy products is a particular challenge. These products are manufactured using a variety of human or animal-derived starting materials and reagents; therefore, extensive testing of donors and of cell banks established for production is required. Furthermore, the viral safety of reagents, such as bovine sera, porcine trypsin and human transferrin or albumin needs to be considered. Whenever possible, manufacturing steps for inactivation or removal of viruses should be introduced; however, sometimes it is not possible to introduce such steps for tissues or cell-based medicinal products as the activity and viability of cells will be compromised. It might be possible to implement steps for inactivation or removal of potential contaminating enveloped viruses only for production of small and stable non-enveloped viral gene vectors.

Evaluation of viral inactivation of pseudorabies virus, encephalomyocarditis virus, bovine viral diarrhea virus and porcine parvovirus in pancreatin of porcine origin

Pancreatin is a substance containing enzymes, principally amylase, lipase, and protease. It is obtained from bovine or porcine pancreas and used in the treatment of pancreatic endocrine insufficiency in humans. Regulations and safety concerns mandate viral clearance (virus removal or inactivation) in biopharmaceuticals such as pancreatin. A virus validation study was performed to evaluate virus clearance achieved in the final step of drying under vacuum by testing a panel of four animal viruses: Pseudorabies virus (PRV), Encephalomyocarditis virus (EMCV), Bovine viral diarrhea virus (BVDV), and Porcine parvovirus (PPV). Because of the product's virucidal effect and high cytotoxicity, the starting material was diluted to a ratio of 0.67g of dried pancreatin resuspended in 13.5mL of cell culture medium followed by a 50-fold dilution in cell culture medium before spiking. After heating at 60±1°C for 5h, the samples were diluted about 5-fold in cell culture medium and titered by the plaque assay method. The virus reduction factor ranged from 5.59 (for PPV) to 7.07 (for EMCV) and no viral plaque was observed, indicating that the process step was effective in the reduction and removal of virus contamination. Though no virus contamination events in pancreatin have been reported to date, evaluation of the production process for its ability to inactivate and/or remove virus contamination, particularly from zoonotic viral agents such as hepatitis E virus and Norovirus considered emerging pathogens, is necessary to ensure the viral safety of animal-derived biopharmaceuticals.

New approaches for manufacturing plasma derivatives

Plasma fractionation is the large‐scale production process used to prepare an important class of protein products that are the only potential therapeutic options to treat, manage or prevent life‐threatening conditions resulting from immunological disorders, congenital plasma protein deficiencies, infections and trauma. The main plasma products include immunoglobulin G (IgG), coagulation factors, fibrinogen and albumin. The core plasma fractionation process, based on sequential ethanol precipitation steps, was developed in the 1940s. It has evolved over the years through implementation of chromatographic protein purification steps, viral safety treatments and automation into a complex biotechnological process. Industrial plasma fractionation is nowadays well mastered and scientifically understood in industrialized countries; product quality and safety benefit from the mature regulations overseeing the collection of plasma and licensing of therapeutic biologicals. Several emerging countries, interested in producing plasma products from local plasma resources to increase product supplies, have considered building fractionation plants. However, the complexities of the current plasma fractionation technology and its validation constitute a substantial barrier to entry into the field. Changes in plasma product drivers in industrialized countries have raised interest in novel, easily scalable plasma fractionation technologies capable of enhancing certain aspects, such as IgG recovery. Novel plasma fractionation approaches rely (1) on multiple chromatographic steps to capture target proteins in sequence, (2) membranes and differential electric potentials to partition proteins or (3) aqueous two‐phase system. A ‘mini‐pool’ method of fractionation and viral inactivation using disposable devices is being developed to fill in quality gaps between crude blood components and commercial products.

Use of MMV as a Single Worst-Case Model Virus in Viral Filter Validation Studies.

Demonstrating the virus clearance capability of a purification process is an important aspect of biopharmaceutical process development. A key component of the viral safety of the process is the inclusion of a parvovirus-grade filter as an effective and robust virus removal step. Traditional methodologies for viral clearance studies have been based on a conservative, data-intensive approach, but recent trends in the field of virus clearance and process development show evolution towards streamlined and more efficient study designs that are based on understanding the mechanism of viral clearance by downstream unit operations. The publication of scientific datasets and awareness of the underlying mechanisms involved with these unit operations have fueled this trend. Here, the concept of virus validation on a parvovirus-grade filter using a parvovirus as single, worst-case model virus is presented. Multiple lines of evidence are provided to support this proposal, including a review of published literature and Amgen historical data. The adoption of this approach provides benefits in terms of cost savings for executing viral clearance studies, but it also simplifies the necessary dataset and focuses on only supplying value-added information to demonstrate the viral safety of the process.

Therapeutic indications and adverse reactions to intravenous gammaglobulin

The first therapeutic indication, and still currently the most applied of gamma globulin, is the one which uses its substitutive capacity in patients with antibodies deficiencies. Its immunomodulatory effectiveness with pro and anti-inflammatory activity holds a second place in their indications, in autoimmune and inflammatory illnesses. The first gamma globulin to be used in patients with agammaglobulinemia in the 50’s, were of intramuscular administration, with significant limitations on the volume and the infusion’s amount, as well as being painful . For over 20 years intravenous immunoglobulin (IVIG) has been widely used in human pathology. The first proteolytic enzyme-treated intravenous immunoglobulins allowed large doses infusions, thereby getting higher levels of IgC circulating in plasma, a subclass profile similar to the normal population, as well as a much higher average life expectancy. The pharmaceutical industry efforts brought about new more purified and viral safer immunoglobulins. The Cohn and cold ethanol methods were quickly supplemented with ulterior procedures that strengthened the viral safety of these products. The application of molecular techniques to the screening study of plasma donors, as well as incorporating new viral inactivation procedures during its manufacture, have made of IVIG a safe product whose consumption has exponentially increased in recent years. IVIG’s high cost, the hypothetical plasma supply problems and risks associated to its administration made essential the writing of usage protocols for official agencies, which, besides, hospitals also adopted.

Efficacy and resistance in de novo combination lamivudine and adefovir dipivoxil therapy versus entecavir monotherapy for the treatment-naive patients with chronic hepatitis B: a meta-analysis

BackgroundCurrently, there is no consensus on the efficacy and resistance of de novo combination therapy versus monotherapy for treatment naive patients of chronic hepatitis B (CHB).ObjectivesThe aim of this study was to evaluate the effectiveness and resistance of de novo combination of lamivudine (LAM) and adefovir dipivoxil (ADV) compared with entecavir (ETV) monotherapy for nucleos(t)ide–naive patients with CHB.Study designPublications on the effectiveness and resistance of LAM plus ADV versus ETV monotherapy for nucleos(t)ide-naive patients with CHB were identified by a search of PubMed, Embase, the Cochrane Library, Web of science, OVID, and CBM (Chinese Biological Medical Literature) until May 1, 2013. Biochemical response, hepatitis B e antigen seroconversion, and viroligic response were extracted and combined to obtain an integrated result. Viral resistance and safety were reviewed.ResultsFive eligible studies (328 patients in total) were included in the analysis. LAM plus ADV combination therapy produced more rapid HBV DNA reduction rate at 12 weeks than that of ETV monotherapy. At 48 weeks, the combination group had superior viroligic response rates compared with ETV group (90.0% vs. 78.9%, P=0.01). The difference in the ALT normalization and HBeAg seroconversion rates was not found. At week 96, LAM + ADV was more effective than ETV in ALT normalization [RR = 1. 11, 95% CI (1.02, 1.21), P =0.01] and HBeAg seroconversion [RR = 2.00, 95% CI (1.26, 3.18, P=0.003)], and no significant difference was found in the virologic response (P =0.23). No viral resistance occurred in combination therapy and six patients in ETV group were experienced with viral breakthrough. Both groups were well tolerated.ConclusionThe de novo LAM plus ADV combination therapy for treatment-naïve patients with CHB was greater than ETV monotherapy in both biochemical response and HBeAg seroconversion rate up to 96 weeks. The rate of emergence of viral resistance in the combination group was less than that in the ETV monotherapy.

Virus clearance methods applied in bioprocessing operations: an overview of selected inactivation and removal methods

Viral safety is an integral component of the process design for products derived from biological sources. While each step may contribute to viral safety, specific steps to remove and or inactivate potential viral contamination should be incorporated. The purpose of this article is to provide a brief overview to some of these specific viral clearance steps and how they may be applied in a manufacturing process. Each of these methodologies have a proven ability to remove or inactivate viruses, however selection and implementation of these methods is dependent on a variety of factors beyond the potential reduction achieved. The application of a selected method should be appropriately investigated and characterized before the implementation into the manufacturing process and subsequent viral safety assessment.

Robustness of nanofiltration for increasing the viral safety margin of biological products

In this study, the virus-removal capacity of nanofiltration was assessed using validated laboratory scale models on a wide range of viruses (pseudorabies virus; human immunodeficiency virus; bovine viral diarrhea virus; West Nile virus; hepatitis A virus; murine encephalomyocarditis virus; and porcine parvovirus) with sizes from 18 nm to 200 nm and applying the different process conditions existing in a number of Grifols' plasma-derived manufacturing processes (thrombin, α1-proteinase inhibitor, Factor IX, antithrombin, plasmin, intravenous immunoglobulin, and fibrinogen). Spiking experiments (n = 133) were performed in process intermediate products, and removal was subsequently determined by infectivity titration. Reduction Factor (RF) was calculated by comparing the virus load before and after nanofiltration under each product purification condition. In all experiments, the RFs were close to or greater than 4 log10 (>99.99% of virus elimination). RF values were not significantly affected by the process conditions within the limits assayed (pH, ionic strength, temperature, filtration ratio, and protein concentration). The virus-removal capacity of nanofiltration correlated only with the size of the removed agent. In conclusion, nanofiltration, as used in the manufacturing of several Grifols' products, is consistent, robust, and not significantly affected by process conditions.

Role of Risk Assessments in Viral Safety: An FDA Perspective

Available guidance documents, such as the International Conference on Harmonization (ICH) Q5a: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin, delineate principles to consider when assessing the viral safety of biotechnology products ([1][1]). The

Препарати фактора згортання крові VIII та способи їх отримання

To correct factor deficiency or prevent bleeding in patients with hemophilia A, the replacement therapy is carried out. This therapy involves administrating plasma or recombinant preparations of factor VIII. A variety of plasma- and recombinant-derived factor preparations are used for haemophilia treatment in Ukraine. Depending on purification level, the following types of plasma-derived concentrates are defined: intermediate purity products obtained through classic techniques of precipitation/adsorption, and high-purity concentrates purified through ion exchange or affinity chromatography. Since the late 1980s, recombinant factor drugs were brought into use in medical practice. They are synthesised by a genetically engineered Chinese hamster ovary cell line or baby hamster kidney. Recombinant factors are classified in generations according to the additional content of impurity human or animal proteins in a final product. The article provides an analysis of the main plasma-derived and recombinant factor VIII preparations registered in Ukraine. Basic properties, methods of preparation and methods of viral inactivation are described. The advantages and disadvantages of plasma-derived and recombinant products are generalized. Keywords: blood coagulation, plasma and recombinant concentrate of factor VIII, chromatography, viral safety.

Viral Safety Perspective from the Paul-Ehrlich-Institut in Europe

According to European Directive 2001/20/EC, authorization of clinical trials is in the responsibility of national member states ([1][1]). In order to facilitate a harmonized evaluation of viral safety issues, a European guideline on viral safety evaluation of biotechnological investigational

Proceedings of the 2011 Viral Clearance Symposium (South San Francisco, CA)

Freedom from viral contaminants is a paramount concern for recombinant biopharmaceuticals and plasma-derived medicinal products. Viral safety is achieved by a rigorous program of cell bank and source material testing in conjunction with a demonstration that the production process is capable of

No evidence of viral transmission following long-term implantation of agarose encapsulated porcine islets in diabetic dogs.

We have previously described the use of a double coated agarose-agarose porcine islet macrobead for the treatment of type I diabetes mellitus. In the current study, the long-term viral safety of macrobead implantation into pancreatectomized diabetic dogs treated with pravastatin (n = 3) was assessed while 2 dogs served as nonimplanted controls. A more gradual return to preimplant insulin requirements occurred after a 2nd implant procedure (days 148, 189, and >652) when compared to a first macrobead implantation (days 9, 21, and 21) in all macrobead implanted animals. In all three implanted dogs, porcine C-peptide was detected in the blood for at least 10 days following the first implant and for at least 26 days following the second implant. C-peptide was also present in the peritoneal fluid of all three implanted dogs at 6 months after 2nd implant and in 2 of 3 dogs at necropsy. Prescreening results of islet macrobeads and culture media prior to transplantation were negative for 13 viruses. No evidence of PERV or other viral transmission was found throughout the study. This study demonstrates that the long-term (2.4 years) implantation of agarose-agarose encapsulated porcine islets is a safe procedure in a large animal model of type I diabetes mellitus.

Novel alphavirus-based vaccine induces strong immune responses against prostate-specific antigen (PSA) and mediates tumor cell elimination in a mouse model of prostate cancer

Prostate cancer is the second most frequently diagnosed cancer in men worldwide and sixth leading cancer-related cause of death in males. Treatments for recurrent prostate cancer are only rarely curative, and the majority of patients eventually demonstrate progressive disease. Animal and clinical studies suggested that immunological approaches can be useful for prostate cancer treatment and prevention. The development of immunotherapy for prostate cancer based on the induction of autoimmunity to prostate-specific differentiation antigens is an attractive concept. Prostate specific antigen (PSA) is a serine protease of the glandular kallikrein gene family with highly restricted expression on prostate tumor cells and normal prostate. Previous attempts to develop PSA-based vaccines for prostate cancer primarily using poxvirus and adenovirus vectors resulted in promising clinical and immune responses. However, these approaches had limitations due to pre-existing antibody responses to viral vectors and insufficient safety. Previously we have constructed novel non-replicative alphavirus vector platform derived from live attenuated human TC-83 IND vaccine against Venezuelan equine encephalitis (VEE) virus infection. In earlier studies, vaccines based on alphaviruses demonstrated efficiency in targeted gene delivery to DC and induction of protective CTL immunity against several human viral pathogens and breast cancer in a rodent animal model. In this study we tested novel TC-83 based PSA-encoding alphavirus vaccine (vVLP-PSA) against prostate cancer using transgenic mouse model with established neonatal tolerance to human PSA. Preventive vaccination using vVLP-PSA demonstrated induction of strong PSA-specific CD8+ T-cell response as detected by IFNγ ELISPOT assay and intracellular cytokine staining. Potent expansion of PSA-specific CD8+ effector T-cells was found in peripheral blood and spleens of vaccinated mice using PSA peptide-loaded MHC I dextramers. vVLP-PSA vaccination also induced PSA-specific antibody production in immunized mice. Injection of TRAMP tumor cells expressing PSA into vaccinated mice resulted in elevated infiltration of tumor site with CD8+ T-cells and rapid elimination of PSA-expressing tumor cells during first several weeks after tumor challenge. Tumor survival analysis revealed tendency for delayed tumor growth in vVLP-PSA immunized mice. Altogether, our data demonstrate that TC83-based vaccine encoding PSA induces strong PSA-specific CTL response and mediates efficient elimination of antigen-expressing prostate tumor cells promptly after tumor challenge.

Report identifies no need for pancreatin viral safety warning

Report identifies no need for pancreatin viral safety warning

Quality by design approach for viral clearance by protein a chromatography.

Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus-like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X-MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design-of-experiment (DoE)—type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product.

Viral Safety of Human Plasma–Derived Medicinal Products: Impact of Regulation Requirements

The viral safety of plasma-derived medicinal products is of paramount importance. This article aims to provide insight into the relative impact of different safety measures on achieving viral safety of finished products, derived from human plasma. Virus removal and/or inactivation during the production process is the most important safety measure, and model-based risk estimates show that with current safety measures, the risk of transmission of known blood-borne pathogens to plasma product recipients is extremely low. However, because the residual risk of virus transmissions is also influenced by the incidence rate of infection in the donor population, it makes sense to control these incidence rates, as well. The current measures are aiming in the right direction, but integration of guidelines is required to adequately address their common goal: controlling the risk of infectious disease transmission by plasma-derived medicinal products. By integration of guidelines, the combination of various types of safety measures to prevent virus transmission-donor selection, donation screening, quarantining, and virus removal and/or inactivation during production-may be consistently interpreted and adequately assessed.

INFORMATION SYSTEM IN BLOOD CENTER: FIRST STEP FOR THE VIRAL SAFETY

In the last decade a special focus has been based on blood donation system. This is due to two key factors: decline in the number of donors and rising amount of infectious diseases incidence, which increase cost of donated blood procurement. According to WHO recommendations, one of the factors that will improve viral safety of donated blood is information space which includes medical facilities and blood centers. We have analyzed information flows in Pavlodar Region Blood Center in order to formalize them for creation of an information system model.

Safety of snake antivenom immunoglobulins: efficacy of viral inactivation in a complete downstream process.

Viral safety remains a challenge when processing a plasma‐derived product. A variety of pathogens might be present in the starting material, which requires a downstream process capable of broad viral reduction. In this article, we used a wide panel of viruses to assess viral removal/inactivation of our downstream process for Snake Antivenom Immunoglobulin (SAI). First, we screened and excluded equine plasma that cross‐reacted with any model virus, a procedure not published before for antivenoms. In addition, we evaluated for the first time the virucidal capacity of phenol applied to SAI products. Among the steps analyzed in the process, phenol addition was the most effective one, followed by heat, caprylic acid, and pepsin. All viruses were fully inactivated only by phenol treatment; heat, the second most effective step, did not inactivate the rotavirus and the adenovirus used. We therefore present a SAI downstream method that is cost‐effective and eliminates viruses to the extent required by WHO for a safe product. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:972–979, 2013

Blood Clotting Factor VIII: From Evolution to Therapy

Recombinant blood clotting factor VIII is one of the most complex proteins for industrial manufacturing due to the low efficiency of its gene transcription, massive intracellular loss of its proprotein during post-translational processing, and the instability of the secreted protein. Improvement in hemophilia A therapy requires a steady increase in the production of factor VIII drugs despite tightening standards of product quality and viral safety. More efficient systems for heterologous expression of factor VIII can be created on the basis of the discovered properties of its gene transcription, post-translational processing, and behavior in the bloodstream. The present review describes the deletion variants of factor VIII protein with increased secretion efficiency and the prospects for the pharmaceutical development of longer acting variants and derivatives of factor VIII.