Mammalian cell culture processes used in the production of monoclonal and recombinant proteins require orthogonal steps in the downstream process to clear virus. Virus filtration is a common, size exclusion-based method in these processes. Initially, tangential flow products were used, but as a result of new-generation virus filtration products, most operations are now constant-pressure, normal-flow operations that can be executed in a single shift operation. The virus filtration step toward the end of the downstream process is now yielding increased protein concentrations…
Tag: <span>viral clearance</span>
Viral clearance studies are required for pharmaceuticals derived from human and/or animal sources such as recombinant proteins produced in eukaryotic cell lines, human blood products and vaccines, and even for some critical class III medical devices. It is mandatory to demonstrate that steps in the manufacturing process are capable of inactivating or removing potential viral contaminants. For this, a laboratory-scale (downscale) of the process step is developed and challenged with different model virus solutions. The viral concentrations are quantitatively determined in the feed material and the relevant product fraction. The ratio of both defines the reduction in virus and specifies the viral inactivation or viral removal capacity of the investigated process step…
The safety of biological products (biologics) derived from in vitro cultured cell lines of animal origin is dependent both on clinical studies to evaluate efficacy, and a matrix of controls throughout the manufacturing process to assure consistency, quality, and safety of the marketed product for human use. One major area of concern is virus safety. Through the combination of: (A) careful selection of raw materials; (B) testing of process intermediates; and (C) virus clearance evaluations of the individual steps in the manufacturing process; biologics manufacturers can demonstrate that their products are free from detectable adventitious and endogenous viruses. Comprehensive regulatory guidance suggests approaches for virus testing of biologics at early and later stages of clinical development and, although some countries have specific requirements, many of these approaches are harmonized worldwide. In general, regulatory authorities expect purification processes to include multiple steps with complementary, or orthogonal, methods for virus reduction including inactivation and separation or removal. Each step in the process may be more or less effective for reducing levels of different test viruses, and therefore selection of the appropriate panel of test viruses for the specific product is critical for viral clearance studies…
Viral clearance validation studies evaluate the efficacy of upstream or downstream process steps for clearing (inactivating or removing) potential viral contaminants from biologics process streams. Inactivation steps are designed to render viruses non-infectious, while removal steps achieve actual physical removal of viruses from the process stream. During validation, the efficacy of viral clearance steps is challenged through evaluation of inactivation and removal capacity, both for viruses known to be capable of infecting the manufacturing process (relevant viruses) as well as for worst-case model viruses (i.e., those believed to be most resistant to removal or inactivation). Worst-case viruses are used to challenge the process steps in order to assure that unknown or novel viruses that may be present in the process stream will be adequately cleared. Historically, the parvoviruses have been used as worst-case models for viral clearance studies due to their small size and lack of a lipid envelope. These characteristics are known to challenge removal by viral filtration and inactivation by a variety of physical and chemical means. In the present paper, we examine the literature on removal of viruses by filtration, and inactivation of viruses by heat, ultraviolet light, and gamma radiation. We conclude that for viral filtration, as well as ultraviolet and gamma irradiation, the use of a parvovirus as a worst-case model virus may not adequately assure that all types of viruses will be cleared using these steps…
Tissue-derived products are a class of biological materials harvested directly from animal or human tissue, in contrast to recombinant DNA materials grown in cell culture bioreactors. Tissue-derived products are often used for structural purposes and are typically regulated as medical devices. However, when used to treat human patients, tissue-derived products are subject to many of the same concerns as recombinant DNA biotherapeutics, with viral safety being one of them. To address this, the tissue source material must undergo a risk analysis and testing regimen for the presence of viral contaminants. In addition, viral clearance studies must be performed to evaluate whether the purification process is robust enough to remove and/or inactivate viruses that may be present in the starting material.
The goals of viral clearance studies are the same for tissue-derived products and biotherapeutics, but the design and performance of these studies can be quite different because of the diverse nature of the materials. In this article, we will present an overview of viral clearance studies for tissue-derived products based on our experience in performing a large number of such studies. Rather than discussing the issues related to viral clearance in general, our focus will be on the unique challenges that tissue-derived products pose.
Porcine circoviruses (PCVs) are small (17 nm) non-enveloped viruses with a covalently closed, circular, single-stranded DNA genome. PCV type 1 (PCV-1) and PCV type 2 (PCV-2) belong to the circovirus genus within the Circoviridae family. PCV-1 was originally isolated as a contaminant of porcine kidney (PK15) cells, and although it was found to be widely distributed in domestic swine in both North America and Europe, no correlation to any porcine disease or disorder has been established. PCV-2, however, has been found to be associated with several disease syndromes in pigs. For manufacturers of biologics utilizing porcine tissue or porcine tissue-derived materials, PCVs represent a contamination risk. In fact, an independent academic laboratory detected PCV-1 in a live attenuated rotavirus vaccine using metagenomic analysis and a PCV-1-specific polymerase chain reaction (PCR). While this study did not detect PCV-1 or PCV-2 nucleic acid in rotavirus vaccine from a second manufacturer, subsequent testing by the manufacturer revealed low levels of both PCV-1 and PCV-2 DNA. The source of the PCV nucleic acid contaminating both vaccines was determined to be porcine pancreas-derived trypsin used in the manufacture of the vaccines. The manufacturer of the rotavirus vaccine that was initially found to contain PCV sequences determined that their cell banks and virus seeds were contaminated with the viral sequences. The strong safety record of both vaccines and the benefits of vaccination against rotavirus convinced both the United States Food and Drug Administration (US FDA) and the European Medicines Agency (EMA) to permit their continued use…
Biologics are often produced in or derived from matrices that harbor the potential for introduction of adventitious agents to the drug product. This potential is not strictly theoretical, as viruses such as hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), porcine circovirus (PCV), and minute virus of mice (MVM) have been detected in biological products in the past. From a regulatory and safety perspective, assurance that adventitious agents are not present in the drug product is a critical measure of product quality. Guidelines for assuring safety, with respect to adventitious agents in blood-derived products and products produced in mammalian cell culture, are addressed in specific guidances from the Food and Drug Administration (FDA) and the Committee for Proprietary Medicinal Products (CPMP). These guidance documents suggest that safety is best assured through screening donor material or production cell lines, by controlling animal-derived raw materials used during manufacture, incorporating viral removal and inactivation steps in the production process, and protecting the product from the environment during manufacture. Even though Medicago develops products that are produced in plants, a host that does not support the replication of viruses that infect mammals, various regulatory agencies have advised that the production process should contain one or more operations that remove or inactivate adventitious agents. Medicago has investigated multiple methodologies to accomplish this goal, and has found ultraviolet C (UVC) irradiation treatment to be effective for adventitious agent inactivation in the production process used to manufacture their quadrivalent influenza vaccine without detrimental impact to the product…
In this paper, we review the efficacy data for low and high pH inactivation of viruses in solutions (i.e., liquid inactivation) and discuss the mechanisms of action and the impact of temperature and treatment time, as these are the primary determinants of inactivation efficacy, besides pH, for different viruses. Only enveloped viruses were considered for low pH inactivation, as the literature concerning low pH inactivation of non-enveloped virus is not extensive and low pH is not considered to be an effective inactivation approach for most non-enveloped viruses. We conclude that for low pH treatment of enveloped viruses, and high pH treatment of both enveloped and non-enveloped viruses, an enteric flavivirus such as bovine viral diarrhea virus represents a worst-case model virus…
Monoclonal antibodies (mAb) are highly selective molecules, and an unlimited amount of mAbs with equal quality can be produced using mammalian cell cultures and animals. These molecules have remarkable applications in biomedicine, diagnosis and therapy due to the ability to reproduce exactly the same binding properties. The mAbs have been generated against an ostensible set of compounds such as toxins, drugs, blood proteins, cancer cells, viruses, hormones, environmental pollutants, food products, metals and plant materials. In general, mAbs can also be used for creating sensitive tests to detect small amounts of substances, and in therapies, abzymes, and for isolating specific compounds from complex mixtures by immunoaffinity chromatography (IAC)…
Contamination by adventitious agents (bacteria, fungi, mycoplasma, and viruses) represents potential safety risks for biologics produced in mammalian cells. Bacterial and fungal contaminations are usually easy to detect in culture medium due to changes in pH and visual indicators such as color and opacity. Mycoplasma contamination has been detected in 15–35% of cell lines deposited in some cell culture collection. This is because mycoplasma contaminations often cause little changes that can be readily detected by visual inspection. However, bacterial, fungal, and mycoplasma contamination can be more effectively controlled than viral contamination by careful screening of initial parental cell banks, proper environmental monitoring, along with ongoing testing…