Viruses, model

Where the structure of one or more similar viruses is available, low-resolution starting phases can be calculated from the model virus (or summed structures) correctly placed in the cell of the unknown structure (Fry et al., 1993). Cryo-EM reconstructions can provide adequate starting phases and envelope information for the determination of novel structures, for example BTV (Grimes effl/., 1998), Reovirus... 

Independent Assays for Proving Virus Removal. Retroviruses and viruses can also be present in culture fluids of mammalian cell lines (15,24). Certainly the absence of vims can be difficult to prove. Model viruses, eg, NIH Rausher leukemia vims and NZB Xenotropic vims, were spiked into fluids being purified, and their removal subsequendy validated when subjected to the same purification sequence as used for the product. 

The virus reduction factor of an individual purification or removal—inactivation step is defined as the log10 of the ratio of the virus load in the pre-purification material divided by the virus load in the post-purification material. A clearance factor for each stage can be calculated and the overall clearance capacity of the production process assessed. Total virus reduction is calculated as the sum of individual log reduction factors. Individual manufacturing steps must possess fundamentally different mechanisms of virus removal or inactivation in order for values to be considered cumulative. Additionally, because viruses vary greatly with regard to inactivation or removal profiles, only data for the same model virus can be cumulative. 

Validation of viral clearance is a major concern for products derived from mammalian cell culture and transgenic animals, as well as for viral vectors used for gene therapies. As we learn more and more about potential risks from newly found viruses, the requirements for validation increase. The increased concerns may be reflected in the number and types of viruses that are used for viral clearance studies. Both relevant and model viruses are used. A recent review of validation of the purification process for viral clearance evaluation provides further information on selection of viruses and performance of the studies [36],... 

Virus identification can start during cell bank characterization and continues until the final product is obtained. It is essential to demonstrate that the viruses are not co-purified with the product (FDA, 1993a, 1997 ICH, 1997). The efficiency of removal or inactivation procedures should be demonstrated on a small scale through spiking experiments, by the use of the viruses themselves, when identified and possible, or with model viruses which mimic any possible variants large or small, DNA or RNA genome,... 

Terms used for virus classification related to the biological of interest and the manufacturing processing are relevant viruses, specific model viruses, and nonspecific model viruses. The first refers to a virus that is likely to be present in the initial crude starting biological material, the second is a model... 

Arindam Bose (Pfizer Central Research) further discussed the ICH documents and presented a rationale for the recommended combination of test procedures and process clearance validations required to demonstrate that marketed biopharmaceuticals are free of adventitious agents. He showed that testing of Pre-Seed Stock (PSS), the Master Cell Bank (MCB), and the Working Cell Bank (WCB) is required to demonstrate that they are free from contamination by mycoplasma, bacteria, molds, and yeasts. In addition, viral clearance validation studies must be performed on scaled down versions of each chromatographic step and the viral inactivation/removal step employed in the product purification scheme. Finally, clearance studies must be conducted with a panel of relevant and model viruses (typically three to four) to establish that the purification scheme will indeed purge any viruses that may be inadvertently introduced during processing. 

A major issue in performing virus validation studies is determining which viruses should be used. The Committee for Proprietary Medicinal Products (CPMP) has issued guidelines on the selection of viruses to evaluate in validation studies. Processes must be validated for their capacity to inactivate/remove relevant viruses, or viruses that are known to contaminate plasma or other materials in the production process. If relevant viruses cannot be easily propagated in cell culture or assayed, then validation studies should include specific model viruses with characteristics similar to relevant viruses. If relevant viruses do not represent viruses with... 

The virus reduction studies of the three process steps discussed here were performed with HFV-l, Bovine viral diarrhea virus (BVDV), Pseudorabies virus (PRV), Reovirus type 3 (Reo), Hepatitis A virus (HAV), and Porcine parvovirus (PPV). HIV-1 was included as a relevant enveloped virus, while BVDV and PRV were tested as specific model viruses for HCV and HBV, respectively (Table 1). Reo was chosen as a non-specific model non-enveloped virus, HAV was included as a relevant virus and PPV was used as a surrogate for human parvovirus B19. All viruses were propagated using standard cell culture conditions. " The appropriate cell lines were infected, at a low multiplicity of infection, and incubated until 4-1- cytopathic effects were observed. The infected cells were frozen and thawed three times to release virus, centrifuged at low speed to remove cell debris and the clarified supernatants were removed for use as virus spikes. 

A process step with many variables to control is equally difficult to scale down for bench scale studies. However, results from animal studies and human clinical trials have been consistent with the in vitro model virus clearance studies of the terminal freeze dry/dry heat treatment. 

HIV (>31ogio HIV-I reduction), but was less effective for HBV-like viruses (>llogio PRV reduction, 2.41ogio VSV reduction) and for HCV-like viruses (2.91ogio Sindbis reduction). Studies in animals and humans confirmed the model virus results. Chimpanzees inoculated with Hemofil T, that had been spiked with HBV (30,000 chimpanzee infectious doses) and freeze dried/dry heated, later developed hepatitis During clinical trials,... 

The results from the albumin studies demonstrated that virus selection (e.g., HM175/18f and HM175/24a) and preparation (e.g., chloroform extracted and untreated virus) were just as important as the process conditions (e.g., protein concentration and temperature) in determining the outcome of virus reduction during pasteurization. Since laboratory adapted viruses and naturally occurring viruses may differ in their sensitivity to physico-chemical treatments, the results observed with model viruses must be carefully interpreted before they can be extrapolated to relevant viruses of concern. 

A. Positive-Stranded RNA Viruses - The positive-stranded RNA viruses are best represented by the picornaviruses of which poliovirus is a model virus. Poliovirus contains a single-stranded genome RNA, which has a MW of 2.5x10 (7700 nucleotides), and four viral structural proteins. Upon entrance into a cell, the genome RNA is translated into one large polypeptide which is cleaved proteolytically to produce all the viral proteins (see below). The 5 ends of picornavirus genome RNAs are unique as they contain a covalently linked protein. The protein (VPg) has a MW of 6-12,000, and has both a basic and hydrophobic nature. It is linked... 

The phenomenon of viral adsorption to various surfaces was extensively studied from an environmental standpoint as reviewed by Daniels (14) and Gerba (15) for prevention of various waterborne viral transmissions. The problem of virus removal from complex protein solutions is very different from that of sewage and drinking water treatment processes because most protein molecules compete for the active sites of the adsorbents. Hence, both the adsorption rate and capacity diminish in the presence of protein molecules (16). It is the intention of this paper to demonstrate and to compare the antiviral activity of a surface-bonded QAC in aqueous solutions against 2 model viruses with and without the presence of proteins. The efficacy of the accepted antiviral thermo-inactivation was compared with the viral inactivation method by the surface-bonded QAC treatment. Beta-lactamase was used as a thermolabile model protein (17), and bacteriophage T2 and herpes simplex virus type 1 (HSV-1, an enveloped animal virus) were used as model hydrophilic and hydrophobic viruses to test these chemical inactivation methods. 

The models can be scaled to various sizes to fit the needs of the experiment. For example, a very small-scale nanofiltration system, such as a Planova P-15 hollow-fiber cartridge with O.OOl-m surface area (Asahi Kasei Corporation, Japan), can be used to study virus retention capabilities of a virus reduction step in a biological manufacturing process, whereas a scaled-up version of the same system with a surface area of 0.01 m provides an excellent way to study the nanofiltration process variables. In a nanofiltration validation study, a feed sample is typically spiked with a known quantity of a model virus. The mixture is filtered under the expected process... 

Table 3.4 Model virus reduction for rAHF-PFM processing [8]...

A complementary approach to virus safety is the design of virus kill and removal steps of the protein recovery process. These include the physical and chemical principles of separating (theoretical) viral contaminants from the product, or inactivating them. Again, appropriate testing procedures and the demonstration of inactivation and removal of model viruses, as discussed by Wiebe et al. [139] is considered a major provision for the safety of recombinant products from hamster cells. 

The capacity of a pnrification process to clear viruses is demonstrated at a representative small scale nsing model viruses. The most common model viruses used in this validation study are xenotropic murine leukemia virus (x-MuLV), mouse minute virus (MMV), and reovirus (Reo). The viral clearance capacity of the chromatographic steps, inactivation steps, and the viral filnation step is demonstrated by spiking a known amount of a model virus into the load of each of these unit operations and calculating the efhciency of removal by measuring the remaining viral titer in the product containing fractions. 

Viral selection is based on (1) relevant viruses that are actual viruses (or of the same species as actual viruses and relevant to the host cell) that have been identified as contaminants (or potential contaminants) of the process, (2) specific model viruses that are closely related to actual viruses (e.g., same genus or family) and have similar physico-chemical properties, and (3) nonspecific model viruses believed to be representative of the spectrum of different virus physio-chemical characteristics [5, 50]. Nonspecific model viruses are used to show inactivation/ removal of viruses in general and to characterize purification robustness [50]. Virus clearance studies should cover emerging viruses and viruses currently believed to be absent in raw materials. These concerns are not addressed when relying on direct testing to ensure safety, specifically consideration of future virus removal requirements in anticipation of future regulatory changes [5, 41]. 

Model viruses are selected that cover the range of physico-chemical properties of different virus species such as size (i.e., diameter and geometry), enveloped or nonenveloped, RNA or DNA, and single- or double-stranded genome [47,51,53]. 

Examples of model viruses selected for various characteristics are medium-to-large DNA/enveloped (Herpes Simplex I, pseudorabies), small/DNA/nonenveloped (Simian virus SV-40), small/RNA/nonenveloped (Sabin Type I Polio, animal parvovirus), medium-to-large RNA/enveloped (Influenza Type A, parainfluenza virus, Sinbis virus 1), and retrovirus/RNA/enveloped for murine hybridomas (Moloney murine leukemia) [3, 51], Preference in the selection of speciflc model viruses is given to those viruses with signiflcant resistance to physical removal/ chemical agents [47, 49, 51]. 

I, virus validation must be conducted. Typical viruses studied are the murine leukemia virus as a model virus for retrovirus-like particles produced by murine cell expression systems (if applicable to the process) and parvovirus [50]. Before phase... 

At the preclinical product phase, critical and noncritical classification of process input parameters should be initiated [32]. Critical components of facility subsystem validation need to be essentially complete before phase I product manufacture [15]. For phase I, it is necessary to validate aspects of the process related to product safety (e.g., sterility, mycoplasma, viral clearance, impurity removal, and stability) [14]. Abbreviated viral clearance studies for model viruses/retroviruses and impurity clearance studies for host cell DNA often are acceptable, resulting in fewer downstream steps validated at this product stage [3, 5]. If viral clearance results are available in sufficient time, the results can be applied to developing the phase I process steps. All assays do not have to be validated at this stage, but some (especially product-specific ones) should be at least qualified [14]. 

Tangential-flow filtration could also be used to validate virus clearance. Today most virus clearance filters are operated in normal flow mode. However, when the size of the model virus particle for which clearance is being validated, and the desired product are within an order of magnimde of each other, tangential-flow filtration may be the preferred mode of operation. 

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