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2 V I R U S S A F E T Y OVERVIEW Methodologies for Viral Safety by Cheryl Scott Good manufacturing practices (GMPs), as agreed upon by the World Health Organization and regulatory agencies around the globe, require thorough control of manufacturing processes used to make biological therapeutics and diagnostics. Viral contamination is just one of the reasons why. A process begins with raw material sourcing, selection, and testing. Ingredients for culture media, for example, must be characterized, qualified, and documented to show that a company is taking appropriate steps to ensure that quality and safety begins at the start of the manufacturing process. As one such step, raw materials should, whenever possible, come from sources other than human or animal tissues, cells, or fluids. Experts recommend looking beyond the obvious, too: Just because a protein is labeled recombinant does not mean that it has had no exposure to human/ animal-derived components. The manufacturing process... may include use of constituents such as albumin, transferrin, Tween-80, lipoproteins, and gelatin that are bovine sourced. Similarly, yeast used for the production of yeast extract, which is a common additive in production processes, may be grown in a medium containing animalderived peptones. (1) How far beyond the obvious should companies look? One step removed should be enough. In addition to primarily derived sources, manufacturers should look into secondary sources that is, the vendor s process and ingredients. Transmission electron micrograph of smallpox viruses (colorized). US DEPT. OF HEALTH AND HUMAN SERVICES, CENTERS FOR DISEASE CONTROL AND PREVENTION ( Cell line characterization is a big part of biotech process development, and production cell lines and cell banks can also be considered raw materials. Because cells in culture provide an ideal environment for viral replication, it is essential to establish a master cell bank (MCB) based on the cell clone that has been genetically engineered to produce and express a recombinant product. Some of the methods discussed in this article serve to identify and remove (or at least inactivate) the viruses present in those cultures. Anything truly dangerous to a cell line will kill it in culture, but established cultures are likely to contain endogenous retroviruses and/or nonpathogenic infections. For each production run, cells are derived from a working cell bank (WCB), which is maintained separately from the MCB to provide an insurance level of redundancy. Viruses can find their way into production bioreactors, processing operations, and even cell line storage, despite the presence of specific rules to follow for handling and working with production materials to keep that from happening. Though pretreatment of raw materials upstream of bioreactor production is not required by regulation, it makes good business sense because it can make later processing easier. Possible sources of viral contamination include process materials, personnel, and contamination from improperly maintained equipment as well as the source material itself. Filtration and other methods discussed later in this article can present a barrier to some adventitious agents, and compliance with GMPs for hygiene and environmental control, as well as proper cleaning, provides further protection. Despite all those precautionary measures, GMPs further require biopharmaceutical companies to test for the presence of adventitious viruses and either remove or inactivate them and of course prove that all the methods of doing so will work reproducibly. The term viral clearance is often used to refer to all of these efforts in general. DETECTING VIRUSES The ideal method for detecting viruses in cell culture would be one that could dependably signal the presence of even a single virus of any type and precisely identify it without affecting the cells in any detrimental way. Unfortunately, the ideal method does not exist. So the methods described here are generally used in various combinations. Which ones are chosen depends highly on the specifics of cell line, product, process, and viruses of interest. Infectivity assays are the classic methods of virus detection. In plaque-forming assays, a test sample BioProcess International NOVEMBER 2005 SUPPLEMENT

3 Table 1: CHO retrovirus-like particle (RVLP) assay comparisons (11): With increased sensitivity, smaller sample volumes, higher sample throughput, good reproducibility, and robustness, PCR assays are ideally suited for process development and cell line selection. PCR is also a rapid method for detecting the presence of adventitious viruses such as MMV, which could be introduced at the cell bank or culture stages. Assay: Electron Microscopy CHOp Q-RT PCR Q-PERT Type of Method: Particle count Real-time RT-PCR amplification Real-time RT activity detection Target: Virions (virus particles) CHOp RNA RT activity Sample Volume: Milliliters to liters 140 µl 100 µl Time Required: Five days One day One day Specificity: RVLP CHO particles Retroviral RT Sensitivity: 10 6 partcles/ml 10 3 particles/ml 10 6 RT molecules/ml Throughput: Low High High CHOp = Chinese hamster ovary cell particle; Q = quantitative; PCR = polymerase chain reaction; RT = reverse transcriptase; PERT = product-enhanced RT is used to inoculate a small laboratoryscale monolayer cell culture. Plaqueforming units are counted, showing areas where cells have been killed by infection. Tissue culture infectious dose (TCID 50 ) assays are named thus because they show the dilution of virus required to infect 50% of an inoculated cell culture. Multiwell plates are often used for such test cultures. Both types of infectivity assay have been used extensively in the biopharmaceutical industry, so regulatory inspectors are very familiar with them. They are exquisitely sensitive, and each assay performed is virus-specific. So they are almost always a top choice despite their drawbacks. They do take more time (weeks) and trouble (a different assay system for each virus tested) than most other methods. Molecular probe methods are much faster and even more specific than infectivity assays. The most popular are based on the multistep nucleic acid amplification process. Thanks to the Thermus aquaticus (Taq) polymerase enzyme discovered in the 1980s, the polymerase chain reaction (PCR) technique provides a fast and easy method of quantifying precise amounts of DNA (from viral contaminants, for example) present in a sample. A reverse-transcription version of PCR (RT-PCR) is used to detect RNAcontaining viruses. Because the nucleic acid amplification is exponential, users of quantitative PCR (Q-PCR) can determine the amount of nucleic acid present in the original test sample after the chain reaction is complete by using some pretty straightforward math (2). Table 1 illustrates the usefulness of this method, but analysts must be careful. The PCR technique can also lead to false positive results when only viral DNA fragments are present rather than whole viral particles that may be infectious (3). Hybridization assays are special PCR-based assays that involve pairing specific single-stranded labeled DNA probes to their complementary sequences. These are used to identify particular viruses by their unique genetic sequence. Different detection methods may be used, including fluorescence (as in fluorescence in-situ hybridization or FISH), colorimetry, and radiolabeling. Antibody production tests (also known as in vivo infectivity assays) require injecting experimental animals with test samples and then testing their blood for the presence of certain antibodies at specific points over a period of time. Mice, rats, hamsters, and guinea pigs are commonly used, but many tests can be run using embryonated chicken eggs instead. Table 2 lists the types of viruses tested in the four most prevalent in vivo systems. Clearly, embryonated eggs present a versatile alternative to the use of live animals. Generally, test results are considered negative if over 80% of infected individuals survive with no antibodies observed. Both the World Health Organization and European Table 2: Main viruses detected in animal antibody production tests (3) Test Subject Suckling mouse Adult mouse Guinea pig Chicken embryo Viruses Tested Pharmacopoeia (see the For Further Reading box) have described standard methods for these in vivo assays. Microscopy: The morphological means of detecting viruses that is, by visualization using transmission electron microscopy (TEM) are beginning to go out of favor. You can see just about any kind of virus in a sample if you know what to look for but the results can be somewhat random. Virus particles must be present in large numbers (100,000 per ml) for users to have a good chance of catching sight of one. Photomicrographs in this article illustrate the TEM technique. Other Tests and Assays: Several biochemical assay methods (e.g., Western blotting and immunoassays) can be applied to the problem of virus detection. But many are problematic enough (sensitivity too low, specificity too high) that they aren t often applied for it, despite the speed and reliability that make them very good for other uses. Animal safety tests (similar in methodology to the antibody production tests described above) find application in cell bank characterization. The most well known are the suckling mouse safety test (to detect Coxsackie virus) and the rabbit Arbovirus, Colorado tick fever, Coxsackie, Dengue, foot-and-mouth disease, Herpes simplex, Variola virus Arbovirus, EEEV, EMC, HSV, JEV, Lassa, LCMV, rabies, St. Louis encephalitis virus Arbovirus, Ebola, EMC, Junin, Lassa, LCMV, Marburg, Monkey B virus, rabies Arbovirus, EMC, HSV, Influenza, Mumps, Newcastle disease, Ornithosis, Parainfluenza, Rabies, Vaccinia, Variola, VEEV, WEEV SUPPLEMENT NOVEMBER 2005 BioProcess International

4 Table 3: Overall log 10 virus clearance data for purification process steps for virus clearance (4) Process Step CHO Cell Derived MAb Typical Log 10 Clearance safety test (for monkey B virus). Embryonated eggs are used also, to test for arboviruses and myxoviruses. Finally, certain surrogate marker assays can be used as indirect indications of viral infection. For example, certain enzyme levels in animal subjects can indicate cell damage caused by particular viruses. Like TEM, however, these methods are slowly being replaced by the more reliable PCR-based assays in many laboratories. No matter what kind of assay is used, paradoxically, positive results showing the presence of viruses provide more information than negative results suggesting their absence (3). As the adage goes, Absence of evidence cannot be seen as evidence of absence. (The argument works in viral clearance testing even better than it does for discussions of extraterrestrial life!) Because of the inherent limitations in all virus detection methods, most laboratories would rather be able to demonstrate a viral presence upstream and effective clearance of those viruses through downstream processes. REMOVING VIRUSES Viruses that have been detected, identified, and characterized as part of a cell line or other production system must be either removed from the process stream or at least completely inactivated. That is, if for whatever reason removal is not practical, all virions present must be reduced to nonviable particles incapable of causing infection. All is not the best word to use here because just as there is no universally perfect detection method, neither does there exist an ideal removal/inactivation system. The best we can hope for is what s called log clearance: that is, a log 10 reduction of the number of viral particles present or active down to the lowest practical Process Step Human Plasma Derived Proteins Typical Log 10 Clearance Low ph 4 5 Dry heat 5 7 IEX Chromatography 2 5 Solvent detergent treatment 4 7 Nanofiltration 4 5 Nanofiltration 4 5 Overall Overall level. The rule of thumb is usually 4 6 logs, depending on the type of virus and its initial population but experts stress that each case is unique, and thinking in generalities in these matters can be dangerous. Each log reduction is a negative exponent of 10, adding a zero ahead of the previous number to the right of the decimal place. For example, a 4-log reduction of 0.05 mg/l (10 2 ) would be mg/l (10 6 ). Table 3 provides example data for purification of CHO- and plasma-derived protein products (4). It must be noted that even that starting value is a calculated value based on the results of more than one detection method. The final log clearance is calculated by adding together the results of all methods used, which can be misleading and often overstates the success of a purification process. If two clearance methods work by similar mechanisms of action, however, then the combined log clearance must be measured rather than simply added together. Viruses may be removed from a process stream serendipitously that is, unintentionally by nondedicated means. Purification methods intended for other impurities and contaminants (such as host-cell proteins) can remove some viruses too. But viral clearance is too important to leave to chance. So usually one or more unit operations in downstream processing are devoted exclusively to the removal of adventitious viruses. As with virus detection, the combination of viral clearance methods chosen should involve differing mechanisms of action and thus complement each other. Filtration is almost always the first choice as a dedicated viral clearance method; it is ubiquitous in biopharmaceutical manufacturing. Solvent detergent precipitation finds limited use in the industry, but thanks to years of experience in plasma fractionation, viral clearance is one area in which it can be applied. And certain chromatographic methods can also be useful. Chromatography: I ve detailed the various chromatographic techniques used in bioprocessing elsewhere (5). Those applicable for viral clearance are immunoaffinity, hydrophobicinteraction, hydroxyapatite, and both anion- and cation-exchange chromatographies. In general, chromatography represents a powerful means of separating components of fluid and fluid solid mixtures. If certain structural properties of a virus or viruses are well understood, they can be effectively removed by exploiting chromatographic chemistries. Process buffer conditions in and of themselves can even inactivate remaining viruses, thus making chromatography a removal and inactivation method. Regulators require that such clearance data be separate for documentation purposes. Because of their expense, process chromatography columns are usually washed, equilibrated, and reused a number of times. That can be risky when it comes to viral clearance efforts. Viruses have been observed to carry over from one use to the next as much as or even more than 25% of the time (3). Although most used resins exhibit virus reduction ability similar to that of new media, lowered effectiveness has been observed with reuse. As a result, regulators want to see caution in practice (6). Limits should be prospectively set on the number of times a purification component e.g., a chromatography column can be reused. Such limits should be based upon actual data obtained by monitoring the components performance over time. (7) Precipitation: The process of partitioning is virus specific (3), and it is not an easy unit operation to control at the level necessary for bioprocess applications. The technique is more common among plasma fractionation companies than elsewhere in the industry. Ethanol, ammonium sulfate, polyethelene glycol, methanol, ethylacridin-lactate, and cationic BioProcess International NOVEMBER 2005 SUPPLEMENT

5 detergents are common precipitation agents. Antibodies present in solution can interfere with the process, as can very small changes in the physicochemical environment. If a partitioning method can be validated (see Process Evaluation below) to work reproducibly, however, regulators will accept it as part of a downstream process. Filtration: As you ll see elsewhere in this supplement, filtration is considered the most effective and economical method, and therefore it is the most used method of removing viruses from bioprocess manufacturing product streams. It is a relatively predictable method based on size exclusion and does not contaminate (no additives) or denature products that are subjected to it. Virus filters cannot be reused. This brief treatment should not imply that filtration is easy. For one thing, filter pore-size and other descriptive ratings are not yet standardized (see the article by Brorson, et al., in this supplement). Also discussed later in this supplement are scale-up issues and stock-quality and prefiltration concerns. Pressures, product characteristics, and membrane features all affect the success of filtration. Filter integrity testing is typically a shared responsibility between filter manufacturers (who correlate data and provide instructions, specifications, and suggestions for testing equipment and methods) and users (who test filters and document results before and after filtration). Integrity-testing methods for virus filters include flow and intrusion tests and particulate and microbial challenging. Bubble-point tests aren t used with viral filters because of the high pressures that would be necessary. They could damage filters and even be a hazard to analytical laboratory personnel. Challenge tests can use certain bacteriophages as model viruses. Filter manufacturers who validate performance provide specifications for their partiular products. It isn t wise to try using the same integrity tests for filters from different vendors. INACTIVATING VIRUSES Because none of the methods above can be expected to remove every single virion present in a process stream and because even a single active virus can lead to a dangerous infection viral inactivation is an essential part of biotech manufacturing. The goal is to destroy either the viruses themselves or their ability to ever infect a cell, while leaving the valuable biotech product intact and functional. Chemicals, heat, or radiation may be FOR FURTHER READING: OFFICIAL GUIDANCE EU 3AB9A: July 1995 Note for Guidance on Validation of Virus Removal and Inactivation Procedures: Choice of Viruses pdfs-en/3ab9aen.pdf EU 3AB4A: July 1995 (in rev. 2005) Production and Quality Control of Monoclonal Antibodies pdfs-en/3ab4aen.pdf EU 3AB8A: August 1996 Note for Guidance on Virus Validation Studies: The Design, Contribution, and Interpretation of Studies Validating the Inactivation and Removal of Viruses pdfs-en/3ab8aen.pdf FDA/CBER February 1997 Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use ICH Q5A: March 1997, rev. September 1999 ICH Harmonised Tripartite Guideline: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin FDA/CDER March 2001 Guidance for Industry on Monoclonal Antibodies Used as Reagents in Drug Manufacturing EU 3AB12A, rev 3: January 2001 Plasma-Derived Medicinal Products pdfs-en/3ab12aen.pdf 9 CFR 113: Standard Requirements Detection of Cytopathogenic and/or Hemadsorbing Agents WAISdocID= &WAISaction=retrieve 9 CFR 113: Standard Requirements Detection of Extraneous Viruses by the Fluorescent Antibody Technique WAISdocID= &WAISaction=retrieve EU 3AB7A: July 1995 (in rev.) Use of Transgenic Animals in the Manufacture of Biological Medicinal Products for Human Use pdfs-en/3ab7aen.pdf 21 CFR 1271 A F: January 2001 Human Cells, Tissues, and Cellular and Tissue-Based Products CFRSearch.cfm?CFRPart=1271 Directive 2004/23/EC 31 March 2004 on Setting Standard of Quality and Safety of the Donation, Procurement, Testing, Processing, Preservation, Storage, and Distribution of Human Tissues and Cells l_ en pdf SUPPLEMENT NOVEMBER 2005 BioProcess International

6 used to this end, and stabilizers of one sort or another can be added to keep them from affecting a product. Chemical Methods: Chaotropes (e.g., detergents and fatty acids) can be added to a process stream (then later removed) to inactivate viruses. Surfactin, for example, is a cationic detergent lipopeptide produced by certain strains of Bacillus subtilis. It acts to disrupt the lipid envelopes of some viruses. Buffers can be used to lower the ph of a solution (to ph 3 4), which has been shown to inactivate viruses. In fact, serendipitous inactivation can occur during some chromatographic processes that use low-ph buffers. Solvent/detergent inactivation is another method often used in the plasma fractionation industry. For more information on that, see the article by Dichtelmüller, et al., in this supplement. Care must be taken, however, in the use of chemical agents. Proteins may be denatured or otherwise altered by their presence. And toxic residues can remain after the chemicals are removed. Betapropriolactone (BPL) is an alkylating agent that has been long used in vaccine manufacture. Particular caution is needed when it is applied to biotechnology products. BPL has been shown, for example, to alter the antigenicity of immunoglobulin solutions. Alternative chemical methods have been developed in response to such problems. Panacos Inc. ( is a small-molecule drug company working on antiinfectives. In its previous incarnation as VI Technologies, Inc. (Vitex), the company developed its Inactine brand pathogen inactivation technology. Now in clinical trials as a therapeutic, the compound selectively binds and modifies certain sequences of DNA or RNA, irreversibly destroying viruses ability to replicate through the infection process. It has already been shown effective in bioprocessing. Irradiation: Photochemical inactivation of viruses is achieved using ultraviolet or visible light to activate a photosensitizing chemical such as riboflavin or methylene blue. Uultraviolet light is naturally destructive to nucleic acids on its own, and viruses with relatively large genomes are particularly sensitive to it. However, proteins can be sensitive to such radiation as well, especially those incorporating large numbers of cysteine and/or aromatic amino acids (1). Clearant, Inc. ( was founded in 1999 to develop and commercialize a new pathogen inactivation technology. The process combines patented and trade-secret technology based on gamma irradiation to substantially reduce all types of pathogens including enveloped and nonenveloped viruses. Unlike most other techniques, it can be applied at various stages of manufacturing even in final product Colorized transmission electron micrograph of Dengue fever viruses attacking cells. US DEPT. OF HEALTH AND HUMAN SERVICES, CENTERS FOR DISEASE CONTROL AND PREVENTION ( containers. It has so far been used successfully with enzymes, serum proteins, growth factors, antibodies, tissues, and cell culture supplements. Heat and Pressure: Making simple physical changes to their environment can inactivate viruses. Some viruses are more susceptible than others. For example, the human immunodeficiency and murine leukemia viruses need ideal conditions to remain viable. But the minute virus of mice and porcine parvoviruses are very resistant to many methods of inactivation (8). Long used in food processing and medical applications, pasteurization is a simple matter of heating a solution (e.g., 10 hours at 60 C) and then cooling it down. Pasteurization of albumin is even codified in the US federal regulations, which facilitates validation of albumin processing. Lyophilized powders can be heated to 80 C for 72 hours or to 100 C for half an hour in a dry-heat process. Moisture levels and timing are important factors here, as they are in freeze-drying itself. Additives (e.g., tryptophan and caprylate) can also be used to protect bioproducts from the heat effects. Certain stabilizers may also have a protective effect on viruses, however. Other stabilizers include amino acids, sugars, magnesium cations, mannitol, glycine, and potassium citrate. Microwave heating is a new twist on the old method, producing very high temperatures with brief exposure. However, the necessary equipment may cost more than some companies are willing to invest. (They can t just go to a home store and pick up any old microwave oven!) Pressure cycling is another physical method of viral activation. Repetitive increases (up to bar) and decreases of pressures disrupt viral structures by destroying the bonds between proteins that make up their capsids. However, large amounts of protein in a solution (such as in human plasma) can get in the way and lower the effectiveness of this method by running interference, protecting the viruses from the very conditions intended to harm them. One pressure treatment is showing promise for blood preparations (9). PROCESS EVALUATION In the above section on detection methods, I mention that the choice of assay depends on several criteria. Different assays will also be used at different points in a process: Infectivity assays are most commonly used in process validation... whereas for routine monitoring and in-process bulk testing PCR assays appear to be favored (1). Process evaluation is essential to GMP compliance. Spiking studies use scaleddown devices and process conditions to validate the clearance capability of a virus clearance operation. Results are submitted to regulators in marketing authorization applications such as the FDA biologics license application or ICH common technical document. 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7 Proper design of the validation study is critical to ensuring success of the study. Scale-down validity, choice of virus, feedstock quality, virus stock quality and inclusion of proper study controls are examples of the topics that need to be addressed in the study design. To effectively design the study, it may be necessary to perform preliminary testing aimed at characterizing the interactions among the virus, prefilter, virus filter, and feedstock. (10) The goal of virus validation is to provide evidence that a production process will effectively inactivate/ remove viruses that may contaminate or have been shown to be present in the starting materials. In addition, the validation will provide indirect evidence that the production process might inactivate and/or remove novel or unpredictable virus contamination (4). These studies contribute to the overall safety profile of a biotherapeutic. Because of the many variables, it is difficult to exactly duplicate process conditions at the laboratory scale. So process evaluation identifies those production steps that are most effective in reducing the numbers or infectivity of viruses and estimate the ability of the whole process to do so. Worst-case conditions are used to illustrate the extremes of those abilities. In general, at least two distinct process steps are evaluated for virus clearance. At least one of these steps should be effective against nonenveloped viruses.... Validity of the scaled-down process should be demonstrated by comparing process parameters such as ph, temperature, protein concentration, protein transmission, reaction time, column bed height, linear flow rate, elution profile, and step efficiency. (4) Usually, two separate viral clearance studies are performed. In preparation for manufacturing phase 1 clinical trial materials, the first validates the effective and adequate clearance of relevant and known viruses. Before phase 2 3 materials are made, another round of viral clearance testing shows that same clearance as well as removal of novel and unpredictable viruses. The SUPPLEMENT rest of this supplement focuses mainly on the details of process evaluation. CONTRACT LABORATORIES Most viral clearance testing in the biopharmaceutical industry is performed by contract testing laboratories. One reason for this is that GMP requirements will not allow virus stocks to be brought into the same site where product manufacturing occurs. Virus testing and associated process evaluation must be done at a different location from the manufacturing site. Most companies have only one facility so they have no choice but to outsource. Smaller companies especially lack the necessary laboratory facilities and expertise. Even large biotech companies with multiple facilities often will outsource to a contract laboratory to avoid having to work with virus stocks themselves or to access particularly strong expertise in viral clearance and good laboratory practices (GLPs). If your company doesn t have its own staff scientists who are well versed in virology, your best bet is probably to outsource. The disadvantages of the cost and scheduling of test lab availability are very often outweighed by the benefits. Although experts advise placing cost further down on the list of priorities when choosing to outsource, it often plays a more important role in decision making than it should. Choosing the lowest bidder may be more expensive than it first appears. Several considerations must be borne in mind when evaluating a potential vendor: contract lab s technical capabilities, scheduling-related issues, customer service, and cost (1). Auditing a laboratory requires reviewing its sample storage and handling, test procedures, technician training, documentation procedures, and internal auditing program. Outsourcing viral clearance work does not mean outsourcing the responsibility for getting it right. The sponsor company of a product in development is ultimately accountable for all aspects of the development and manufacturing process including the methods, protocols, and results of studies that are not performed in-house. Outsourcing viral clearance work DOES NOT mean outsourcing the responsibility for getting it right. Regulatory agencies expect manufacturers to audit the laboratories they contract with, as well as to review and verify data those contractors produce. A good contract test laboratory will provide sound advice on study design and data interpretation, but in the end it is the sponsor who is responsible for the results. The articles that follow in this special issue should provide a good starting point for approaching the subject of viral safety, and there is additional guidance to be found elsewhere (see For Further Reading ). But viral clearance is an essential aspect of demonstrating product safety, so it is not to be taken lightly. Seek out the advice of experts, study what has been done before, and wherever possible learn from the mistakes of others. Much more than money is at stake. Ultimately, lives could depend on biotech manufacturers getting it right the first time: Although to our knowledge no biotechnological product has ever caused transmission of an infectious virus, viral contamination of processes and even final product have occurred. Examples of contamination include the murine minute virus (MMV) in CHO cell cultures and reovirus in the production of monoclonal antibodies. Viral clearance studies can provide a high degree of confidence in the ability of the manufacturing process to produce a safe product. (11) NOVEMBER 2005 BioProcess International

8 REFERENCES 1 Aranha H. A Guide to Viral Clearance: Strategies for Biopharmaceutical Safety (Guide #9143). D&MD Publications: Westborough, MA, April Pfaffl M. Quantification Strategies in Real-Time PCR: (updated 19 August 2005, accessed 23 August 2005). 3 Aranha H, Wilkommen H. Viral Clearance Strategies for Biopharmaceutical Safety (short course), June 2005, Seattle, WA. IBC Life Sciences: Westborough, MA. 4 Immelmann A, et al. Validation and Quality Procedures for Virus and Prion Removal in Biopharmaceuticals. BioProcess International 3(8, supplement) September 2005: Scott C. Chromatographic Chemistries for Biotechnology Development. BioProcess International 3(5, supplement) 2005: Sofer G. Establishing Resin Lifetime: Key Issues and Regulatory Positions. BioProcess International 1(1) 2003: ICH. Q5A: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin. Federal Register 63(185) 24 September 1998: 51074; 8 Otake T. High Pressure Viral Inactivation and Its Application for Blood Preparations. Foods Food Ingredients J. Jpn. 210(1) Farshid M. Evaluation of Viral Clearance Studies (presentation). Food and Drug Administration, Center for Biologics Evaluation and Research Division of Hematology. 10 Application Note. Ensuring Regulatory Compliance: Validation of Virus Filtration Virus Spiking Study Design. Millipore Corporation: Beford, MA, 2002; publications.nsf/docs/an1650en Chang A, Sofer G, Dusing S. Focus on Compliance: Expediting Compliance for Clinical Biotherapeutics. BioProcess International 2(2) 2004: Cheryl Scott is senior technical editor of BioProcess International, 1200 Executive Parkway #255, Eugene, OR 97401; , fax ; cscott@bioprocessintl.com. BioProcess International NOVEMBER 2005 SUPPLEMENT