PDA/FDA Adventitious Agents and Novel Cell Substrates: Emerging Technologies and New Challenges, Nov. 3–4, 2011, Rockville, MD

Meeting Summary

The first day opened with a keynote talk by Johannes Löwer, president of the International Alliance for Biological Standardization (IABS), entitled “Risks Associated with Cell Substrates and Other Biological Materials and Product Safety.” Examples of virus contamination in biological products were presented, such as HIV-1 and torque teno virus (TTV) in blood products and porcine circovirus 1 (PCV-1), simian virus 40 (SV40), endogenous avian retrovirus (EAV), and human rhinovirus in some viral vaccines. In addition, endogenous retrovirus-like particles have been reported in Chinese hamster ovary (CHO) producer cells and cell culture harvests and minute virus of mice (MVM) contamination has been described in CHO cell production systems. Recent occurrences highlight that unexpected virus contamination can occur and any positive signal should be immediately followed-up and intensively studied. In particular, laboratory contamination should be ruled out, and the infectivity of the agent in humans should be evaluated in cell culture. A matrix was presented for adventitious virus risk assessment by scoring the level of risk based upon contaminating virus titer, species barrier, pathogenicity, growth in cell substrate, and inactivation/removal steps in the manufacturing process. The overall message of the talk was that manufacturers should aim for products that are free of all contaminating agents, even if they are not infectious or pathogenic to humans.

The rest of the morning was focused on talks related to Adventitious Agent Testing and Emerging Methods and were moderated by Kathryn King (Center for Drug Evaluation and Research [CDER], U.S. Food and Drug Administration [FDA]) and Houman Dehghani (Amgen). These sessions provided an overview of the currently recommended assays for evaluating safety of biologicals and the need for considering use of new technologies, as well as detailed presentations on some of the more mature and established emerging technologies for broad detection of known and novel adventitious viruses. Michael Wiebe (Quantum Consulting) presented “Conventional Virus Assays and the Need for New Technologies.” He discussed the past virus safety record in biopharmaceutical manufacturing and the need for using a multifaceted approach to virus safety based upon cell bank testing, raw material control, use of closed systems for cell culture, virology knowledge, testing, and viral clearance. Examples of known virus contaminations were listed and the importance of in-process and lot release testing emphasized. The strengths and weaknesses of the currently recommended in vitro culture assays for virus testing were described, with examples of virus contaminations that escaped detection. The potential use of emerging virus detection technologies was introduced with questions for considering their availability, applications in biologics, and if and how they will improve safety over the currently used assays. Concluding remarks put forward a question central to the discussions at the end of day 1: “Are the new virus detection technologies ready for prime time?”

David Munroe (SAIC-Frederick, Frederick National Laboratory for Cancer Research) presented “Different Next-Gen Sequencing Platforms.” His talk included descriptions of established platforms such as 454, Illumina-Solexa, and SOLiD as well as some new sequencing platforms such as PacBio, Ion Torrent, and Nanopore. A comparison of the different platforms indicating strengths and limitations of each was presented that included cost and time to generate results (days to weeks). This analysis also included comparison of the platform characteristics (throughput, read length, accuracy, and ease of operation), application suitability (transcriptome, whole genome, sheared DNA, amplicons, CHiP, miRNA, exome, microbiome, bisulfite, custom capture, and whole genome), and data assembly (de novo and hybrid assembly). The challenges associated with massively parallel sequencing were outlined, with emphasis on analysis, transfer, storage, and management of data and the need for increased resources (experienced personnel and advanced equipment).

Rangarajan Sampath (Abbott Ibis Biosciences) presented “PLEX-ID Technology for Broad Screening of Microbial Contaminants.” A description of the broad-range polymerase chain reaction with electrospray ionization mass spectrometry (PCR/ESI-MS) integrated detection system and its applications for microbial agent detection were presented. Case studies demonstrating the ability of the PLEX-ID to detect the following were presented: mouse parvovirus 1c (including typing), a new Bluetongue virus serotype that infected Vero cell culture due to exposure to contaminated raw material, and different mycoplasma strains. The time course for detection of MVM in CHO cells was as early as day 2, and parainfluenza virus 3 could be detected in supernatant by day 4. The time course was similar to results from quantitative PCR (qPCR). The brief run time of the PLEX-ID, which is 6–8 h, was also noted.

Tom Slezak (Lawarence Livermore National Laboratories [LLNL]) presented “Microarrays: Background for Discussion of Possible Use for Adventitious Agent Testing.” While it was indicated that no single molecular technology is the “best” for adventitious agent detection in biologics, it was emphasized that microarrays can fill an important gap between PCR assays and high-throughput sequencing and generate results in about 24 h. The details of the key parameters that differentiate microarray platforms were presented: manufacturing technology, probe length limitation, number of probes, probe targets, probe design strategy, amplification strategy, labeling/detection strategy, and results analysis approach. Consideration of all of these factors was stated to be important in determining the overall suitability of the arrays for a particular need. Different arrays were presented, including ViroChip from Joseph DeRisi's laboratory at University of California, San Francisco (UCSF) for viral pathogen detection; GreeneChip developed in Ian Lipkin's laboratory at Columbia University for detection of different viruses, bacteria, fungi and parasites; PhyloChip developed by Gary Andersen's laboratory at Lawrence Berkeley National Laboratory (LBNL) used for bacterial community analyses; and Respiratory Pathogen Microarray (RPM), which is a customized array for detection of tropical and emerging infectious agents, developed in Dave Stenger's laboratory at the Naval Research Laboratory (NRL) and U.S. Army Medical Research Institute of Infectious Diseases (USAMRID). Details of the Lawrence Livermore Microbial Detection Array (LLMDA) developed at LLNL, which can detect both bacteria and viruses, were presented. Analysis of complex biological samples was presented to highlight the ability of this assay to simultaneously detect different microorganisms.

Marc Salit (National Institute of Standards and Technology [NIST]) presented “Standards and Standardization” and emphasized this important aspect of development for any new assay or technology. This talk described some reference material products that have been developed to provide confidence in Next-Gen sequencing applications (such as a the new reference material NIST SRM 2374–DNA Library for External RNA Controls, which is the first reference of its kind certified for sequencing) and reference materials in development such as reference materials for whole genome sequencing. The characterization of reference material and related quality control aspects were discussed. Challenges of developing a reference material for adventitious agent detection by sequencing were presented.

The talks in the afternoon sessions were moderated by James Gilbert (Biogen Idec) and David Onions (BioReliance). These talks focused on case studies using the emerging technologies and highlighted the strengths and limitations of the methodologies and their utility for evaluating biological samples. John Kolman (BioReliance) presented “Current and Future Roles of MP-Seq™ in Safety Testing of Biologics.” This talk included case studies demonstrating the potential applicability of Roche 454-massively parallel sequencing (MPS) to raw material screening, cell line characterization, contamination incident investigation, and for implementation as a rapid end-point to the traditional in vitro virus assays. An example of screening bovine serum demonstrated the ability of the method to detect a novel bovine parvovirus that can latently infect human cells, and other previously known bovine parvoviruses. The importance of the development of an algorithm for bioinformatics specifically for detection of viruses and challenges of data management were emphasized.

Matt Friedenberg (Gen-Probe Inc.) presented “A First Look at Third Generation Sequencing.” He reported on Gen-Probe's co-exploration with Pacific Biosciences of new integrated clinical diagnostics systems based upon the SMRT (Single Molecule Real Time) platform developed by Pacific Biosciences using hepatitis C virus (HCV) infection as an example. The talk focused on the potential application of this technology for the identification of HCV quasispecies, which are associated with resistance to antiviral drugs, with the hypothesis that the technology eventually be used to define treatment decisions by monitoring the quasispecies evolution over the course of treatment. Presentation of in-progress experiments revealed the SMRT technology can detect transcript variants in the range of 0.5–1% in a background of expected transcripts.

Houman Dehghani (Amgen) presented “State of the Art for Monitoring and Ensuring Biosafety in Manufacturing using PLEX-ID.” After highlighting the advantages of these rapid technologies relative to standard methods, he spoke on development work to explore the use of the PLEX-ID system in a biotherapeutics manufacturing setting. Development work has focused on the characterization of the method for the detection of bacteria, mycoplasma, and viruses, and showed that the PLEX-ID was capable of detecting most components of mixed microbial agent samples. Advantages of the technology in reducing the turn-around time for investigating contamination were emphasized.

Charles Chiu (UCSF) presented “The use of Microarrays and Deep Sequencing for Viral Detection and Discovery.” He spoke on the clinical applications of the ViroChip virus microarray and MPS or deep sequencing for the identification of causative agents in acute infectious disease. Examples were given of the application of both technologies, and challenges associated with data analysis were discussed. The talk concluded with an extended case study on how the ViroChip combined with MPS was used to identify a novel adenovirus, titi monkey adenovirus (TMAdV), which is capable of infecting both monkeys and humans.

Paul Duncan (Merck) presented “Viral Risk Assessment of a Positive Signal from a Random Amplification/Deep Sequencing Strategy.” He presented a case study of a follow-up analysis on positive pestivirus hits from a random amplification/MPS study of a novel human cell line used for production of a novel viral vaccine, highlighting the thought processes involved in decisions, in particular related to what is required to render a hit invalid following a positive match with a viral database.

Arifa Khan (Center for Biologics Evaluation and Research [CBER], FDA) presented “Investigations of New Technologies for Latent Virus Detection.” The talk described some of the novel cell substrates used for the production of investigational viral vaccines and the challenges for the current assays for detection of unknown and latent viruses. Data were presented evaluating three new broad virus detection technologies (PLEX-ID, virus microarrays, and 454-MPS) for the detection of endogenous retroviruses using chemically-treated Vero cells as a model for latent retrovirus induction. Also presented was information on the evaluation of a reverse transcriptase (RT) activity produced from Sf9 insect cells and the results of investigations for endogenous insect retrovirus sequences (errantiviruses) in the RT-containing, pelleted supernatant of these cells. The talk concluded by outlining some considerations for using the new technologies for novel virus detection with emphasis on the development of a strategy to follow-up on a positive result and the importance of continued dialogue and communication, as both the methodology and analyses are complex.

Upon conclusion of the presentations, there was a panel discussion moderated by Kathryn King. The panelists included all of the speakers, session chairs, Vahan Simonyan (CBER, FDA), and Barry Cherney (CDER, FDA). There was agreement that the emerging technologies have impressive capabilities for virus detection and identification and that they have potential for use in the investigation of production contaminants, for characterization of cell banks for new cell substrates or those with missing or uncertain history, and for characterization of raw materials. However, it was accepted that the technologies are complex and there may not be a single one that is appropriate for all situations. Furthermore, results obtained may depend upon the test sample and sample preparation; and interpretation of a positive signal will need a follow-up strategy for confirmation of the results and evaluation of the biological relevance. A gap exists in knowledge and “readiness for validation,” and these methods are not yet ready for use in quality control release or process control assays. Additionally, expertise and resources are needed for bioinformatics and there is a need to re-evaluate the data or update the assays as new sequence information is deposited in the databases. The conclusions were that these technologies will continue to develop and evolve in coming years and that current applicability may be determined on a case-by-case basis in the context of risks posed and mitigations available. Based upon the discussions and data presented, there was general agreement that the new technologies might supplement the current methods for virus detection, but they are not ready to replace them.

The second day was comprised of talks on safety issues associated with development of products in insect and plant cell substrates, with focus on retroelements. The day started with a keynote talk from Marcie McClure of Montana State University entitled “Evolution of Mutualism between Retrotransposons, Retroviruses, and the Cell.” The talk provided an overview of retroid agents and their evolutionary relationships. A phylogenetic tree based upon 65 RT sequences was presented representing the distribution of retroviruses, retrotransposons, pararetroviruses, retroposons, retroplasmids, retrointrons, and retrons among eukaryotes, eubacteria, and arachea. The roles of retroid agents in humans were discussed, and their relationship with humans was categorized as parasitic (including pathogenic viruses such as HIV and HTLV), commensalistic, and beneficial symbiotic. Examples of human retroid agents spanning this mutualism spectrum ranging from the parasitic relationship of deadly disease or genetic disease to the beneficial symbiotic relationship in reproduction and gene regulation were described. Additionally, details of a software for sequence-based identification of all retroids in a genome were provided. The capability of this program to identify and classify new retroid elements was exemplified by the identification of 11 new fish ERVs (FERVs).

The morning sessions, moderated by Pawan Jain (CBER, FDA) and Jonathan Stoye (Medical Research Council, National Institute for Medical Research, UK [MRC, NIMR]), focused on issues related to “Insect, Avian, and Mammalian Cell Substrates.” The session included discussion of potential risks associated with retroelements and RT activity, interactions between baculoviruses and insect cells, and the World Health Organization (WHO) recommendations on use of insect cell substrates for manufacture of biological medicinal products.

Jonathan Stoye (MRC, NIMR) presented “Risks Associated with Retroelements: Lessons from Mammalian Systems.” The talk provided an overview of the retroviral lifecycle and highlighted virus properties such as persistence, spread, and disease association that can pose safety concerns with respect to biological medicinal products. Details of virus replication and integration and effects on cellular RNA transcription were described, followed by discussion of the repercussions of virus infection and long-term persistence in the host genome. The overview included a discussion of endogenous retroviruses and factors affecting virus activation. Consequences of recombination with defective viruses were discussed using the examples of gamma retrovirus gene therapy vectors and the recent discovery that xenotropic murine leukemia retrovirus (XMRV)-related virus is a novel recombinant mouse virus. Potential concerns regarding integration of retroviruses and other retroelments were presented: for viral vaccine vectors, based on a report of retroviral insertional activation in a herpes virus; and in the host genome, based on the development of leukemias due to gene therapy vector-mediated insertional activation of the LMO2 gene. Development of lymphomas in monkeys injected with a replication-competent retrovirus that originated from a gene therapy packaging cell system was shown as one of many examples of the pathogenic effects of retroviruses. Conclusions of the talk were that consideration of sequencing the genome of a novel cell substrate might be prudent and that it is critical to monitor for RT activity bearing in mind that any positive result will need follow-up and a negative result does not assure virus absence since latent viruses may be present that can reactivate later.

Celine Breda (Vivalis) presented “The Characterization and Sanitary Status of Avian Cell Substrate EB66.” The talk highlighted the need for alternative platforms for egg-based vaccine manufacture for reasons including: the need to quickly meet vaccine demand in pandemic crisis; to avoid egg component allergies; and risks of bird-associated disease and contaminations, which could lead to vaccine shortages. The EB66 cell line is a well-qualified duck embryonic stem cell line that was derived without genetic insertions or deletions, and without viral or chemical modifications. The characterization program related to both safety and biological properties of the EB66 cell line was described. Examples of the use of this cell line for the production of vaccines and monoclonal antibodies were provided to demonstrate how this high-density cell line can be applied to biologics manufacturing.

Glyn Stacey (National Institute for Biological Standards and Control [NIBSC], UK) presented “New WHO Guidance on Evaluation of Cell Substrates and Considerations for Insect Cell Substrates.” The new document replaces the Requirements for Cell Substrates for Manufacture of Vaccines WHO TRS 848 (1998) Appendix 1 and includes enhanced guidance on cell substrates for vaccines and biotherapeutic products including recombinant therapeutic proteins produced in novel cell types such as insect, avian, and stem cell substrates. The new elements in the WHO guidance were described, highlighting microbial and tumorigenicity testing and the characterization and cryopreservation of insect cells. Considerations specific to insect cells were discussed with regard to adventitious agents, including selection of indicator cell lines for infectivity assays, relevance of the tumorigenicity assay, and considerations for development of cryopreservation protocols and storage conditions optimized for insect cells. It was emphasized that insect and avian cells have fundamental differences in cell biology that must be taken into account during cell substrate safety evaluation. New adventitious agent issues may require new susceptible cell lines; novel microbial agents may require new isolation conditions and new reference organisms; and there is a need for new genetic stability measures and characterization criteria for stem cells. Given these needs, new molecular technologies for virus detection may aid in characterization of novel cell substrates and assist in identifying adventitious agents that may be difficult to isolate and characterize by traditional methods.

George Rohrmann (Oregon State University) reviewed “Baculoviruses, Retroviruses, and Insect Cell Lines” and shared recent experimental results related to their interactions. Data were presented that support genetic exchange between baculoviruses and endogenous insect retroviral sequences (errantiviruses) based upon the envelope fusion protein and the baculovirus late promoter element, diversity of retroviruses in Spodoptera frugiperda and Trichonuplusia ni cells, and changes in the levels of errantivirus RNAs after infection of insect cells with baculovirus. The talk provided details of baculovirus structure and replication with details of the origin and recent acquisition of the virus envelope fusion protein Gp64 in one lineage of Baculoviridae (Group I NPVs) and of the F protein, which is present in most baculovirus lineages and is related to the Env protein of errantiviruses. The capture of envelope and its role in virus evolution from a retrotransposon into a retrovirus by altering the biology or infectivity of a virus was discussed. Possible steps in how an insect retrotransposon could obtain a baculovirus envelope gene were described. An example of an errantivirus in Trichonuplusia ni called transposable element D [TED] that integrated into the baculovirus genome was presented. The resultant virus expresses virus-like particles with RT activity and contains a baculovirus late promoter that is highly expressed late in infection, suggestive of a relationship in which the insect retrovirus exploits the baculovirus late expression system to allow for high levels of RNA production. Investigations of activation of errantiviruses by baculovirus infection performed in Sf9 cells at different times after baculovirus infection were described. While infectious retroviruses have been reported in Drosophila cells, further investigations are needed to determine if the RNAs in the insect Sf9 cell line are associated with infectious particles and if they are transpositionally active in noninsect cells.

The next talk, given by Jane Halpern (Novavax), dealt with the practical application of the Sf9 insect cell line for vaccine production. Her presentation “Characterization of an Insect Cell Line/Baculovirus Expression System for Vaccine Production” gave an overview of the Sf9 cell/baculovirus platform for manufacturing influenza and other vaccines and characterization of this expression system, with a focus on investigations of RT activity known to be associated with this cell line. The overview included details regarding derivation of the Spodoptera frugiperda Sf9 cell line and preparation of the cell banks, construction of the recombinant baculovirus, and characterization testing for GMP manufacturing. Potential safety issues related to Sf9 cells and recombinant baculoviruses were presented, highlighting the novelty of the Sf9 cell substrate with regard to adventitious viruses and RT activity encoded by transposable elements in Lepidoptera. Cell bank qualification included additional characterization of the RT activity by evaluation of sequences encoding RT in Sf9 cell banks, characterization of Sf9 cell bank transcriptome for virus sequences using MPS and analyzing against a curated viral database, investigating the particulate nature of the RT activity and its potential infectivity using various human target cell lines, and transmission electron microscopy [TEM] analysis. It was concluded that the new molecular methods for virus detection can be used to complement existing virus testing methods to provide additional assurance in the safety of a novel cell substrate.

The afternoon talks on “Potential Safety and Quality Issues Related to Plants and Plant-based Products” were moderated by Howard Anderson (CDER, FDA) and Hannelore Willkommen (RBS Consulting). This session explored issues related to potential risks associated with plant substrates, plant viruses, and viral adventitious agents, as well as manufacturing strategies being developed and implemented to minimize risks of contamination.

Rosemarie Hammond (U.S. Department of Agriculture [USDA] Agriculture Research Service) reviewed the “Risks Associated with Plant Viruses and Viroids.” The talk covered the features of plant viruses that should be considered when determining the safety of plant-based biologics. An overview of the families and genera of viruses infecting plants, the key features of plant viruses, and general approach for the use of plant viruses as transient expression vectors for production of therapeutic proteins and virus-like particles as vaccine antigens were presented. Differences in the routes of infection of plant and animal virus infection were noted: While plant viruses are mechanically introduced through the cell wall and they encode proteins that facilitate cell-to-cell and systemic spread, animal viruses require cellular receptors at the animal cell membrane for entry, and exit from the cells is achieved by budding of the virus from the cell surface. Although both encode a RT, there are differences between enveloped retroviruses that infect animals and nonenveloped pararetroviruses that infect plants. Details of viroids, which are the smallest known agents of infectious disease but are only known to infect plants, were discussed, and unique features of other enveloped viruses (Bunyaviridae and Rhabdoviridae) that can infect plants were described. With respect to biologics manufacture, it was noted that plant viruses can be maintained in plant tissue culture and that some plant viruses and viroids are resistant to UV and chemical inactivation. The increasing importance of phytoplasma and spiroplasma in plant disease was mentioned. In addition, plant viruses can replicate in insect vectors and, while they are not known to replicate in animals and humans, exposure can result in an immunogenic response. Plant viruses such as circoviruses and pepper mild mottle virus have been detected in human feces, as they are stable throughout the gut after ingestion of naturally-infected plant material. Several points were raised for further discussion, including (1) whether plant viruses recovered from animal feces are infectious, (2) the possibilities of recombination between animal and plant viruses resulting in a novel virus with altered tropism, and (3) what model-viruses should be chosen for viral clearance studies for plant-derived biotherapeutics.

Yoseph Shaaltiel (Protalix Ltd.) presented “Mammalian Virus Free Cell Culture System.” The ProCellEx plant cell manufacturing bioreactor system was described for the manufacture of biological medicinal products in a plant cell substrate. The talk highlighted the benefits of a plant cell substrate with respect to virus safety and how the platform further reduces risk of contamination by being a closed system. The system used large, flexible plastic containers for culturing and harvesting cells in consecutive cycles, with a central unit to provide oxygen and nutrients. Carrot cells transformed using Agrobacterium were used to produce a human recombinant glucocerebrosidase enzyme for enzyme replacement therapy of Gaucher's disease. Details of reducing the risk of mammalian viruses in the manufacturing process were described, and the uniqueness of plant cell cultures as compared to whole plants in reducing the risk of plant viruses was presented. Analysis of 80 different plant viruses showed that the master cell bank was negative.

A second talk on the practical application of plant-based expression systems to biological medicinal product manufacture was presented by Vidadi Yusibov (Fraunhofer USA Center for Molecular Biotechnology, FhCMB) and was entitled “Plant-based Production: Development and Facilities.” Plant-based expression systems have evolved from using transgenic plants, plant viral vectors, agrobacterial vector, to hybrid systems. The key features of FhCMB's expression technology are launch vectors for target expression, vacuum infiltration for delivery of the vectors into plant biomass, and a hydroponic system for contained, controlled plant biomass generation. Construction of the launch vector and rapid response capability with a total time from sequence to purified target of 21 days, and surge capacity for this expression system, were discussed. Quality systems, quality control, and stability were described including results from a viral clearance study with two model viruses. The platform technology is being used for a variety of products, including vaccines and recombinant therapeutic/diagnostic proteins.

A third talk on the application of plant-based expression systems to biotherapeutics was given by Victor Klimyuk (Icon Genetics GmbH) and was entitled “Manufacturing of Personalized Vaccines for Treating Non-Hodgkin's Lymphoma using a Plant-based Transient Expression System.” Disease characteristics, mechanistic basis of active immunotherapy, and an innovative trial design in non-Hodgkin's lymphoma [NHL] were presented, followed by a description of the manufacture of an idiotype vaccine using the magnICON Sytem with details of the idiotype Ig identification, cloning in magnICON vector, and transformation of Agrobacteria and production in Nicotiana benthamiana, as well as the conjugation process. The platform is versatile and can be used for producing small to large size antigens. Quality control and safety studies were presented. A point-by-point solution to the management of potential risks of virus contamination was discussed. The talk concluded by highlighting the advantages of the platform for application to personalized medicine and enhanced safety due to the removal of animal-derived raw materials.

The day concluded with a panel discussion moderated by Arifa Khan that included the session chairs, speakers, Daniel Adams (Proteins Sciences Corp.), and Robin Levis (CBER, FDA). There was general agreement that a major challenge in the development of plant-derived and insect cell–derived biologicals was a lack of knowledge about the viruses and virus-like elements in these novel cell substrates relative to mammalian and avian cells. The principles used successfully to prepare biologicals in mammalian cells can be applied to novel cell substrates; however, details of characterization may differ. For example, with respect to ICH Q5A, it would be expected that different viruses would be present, different indicator cells would be necessary, and incubation would occur at a different temperature. Mammalian viruses (when biophysicochemically equivalent) may be acceptable substitute models for plant and insect viruses in clearance studies to assess general process virus clearance capability. Positive results in RT assays require biophysical and virological follow-up to assess whether the RT activity is associated with particles and viral nucleic acid, and if it is infectious (e.g., as was done following the discovery of RT activity in chicken cell-grown products using the highly sensitive, product-enhanced RT assay). There is a need for better understanding of retroid families, functionalities, interactions, and experience for scientifically based risk assessments for biological products from novel cell substrates.

The talks on the third day focused on “Adventitious Agents and Raw Materials.” The first session was moderated by Zenobia Taraporewala (CBER, FDA) and Mark Plavsic (Genzyme). The session goals were to discuss the safety issues associated with the use of animal and certain nonanimal-derived raw materials, and ways to mitigate the risk of contamination by adventitious agents with focus on the challenges in adventitious agent screening/testing and reduction strategies for raw materials used in banking and batch production.

Tara Tagmyer (Merck) presented “Evaluating Safety of Vendor Sourced Raw Materials Used in Vaccine Manufacture.” The talk began with a summary of adventitious agent contamination events in biologic manufacturing and the impact of such incidents on public health as it relates to vaccines. Cell banks and virus seeds are at high risk for adventitious agent contamination due to the use of animal-derived raw materials (ADRMs) in their derivation and manufacture. The current methods to reduce adventitious agents risks associated with ADRMs were discussed, indicating advantages and limitations of the current compendial testing for viral agents and the inability to use viral inactivation and clearance methods for all raw materials or all vaccine manufacturing processes. Based upon these limitations, an adventitious agent failure mode and effects analysis (FMEA) was presented for ADRMs. However, this can only assess the known agents based upon current literature and can only identify theoretical risks. Therefore, emerging virus detection methods were discussed to determine the actual potential adventitious agent risks. Steps to integrate the new methods for virus safety evaluation were outlined with a risk assessment and remediation plan for potential adventitious agents in ADRMs. It was noted that these methods are evolving and no one technology is perfect but that appropriate application of well-controlled emerging technologies to evaluating raw materials can aid in reducing adventitious agent risks in the final product.

Ivar Kljavin (Genentech) presented “Identifying Source Materials of Reagents Used in Biopharmaceutical Manufacturing.” The talk focused on raw material sourcing and control with details of what “sourcing” implies and what is involved in control. He emphasized that knowing the risks of the raw materials used is itself a viral barrier. The various animal-derived raw material products and associated risks were discussed. Knowledge of the origin of the raw material, the manufacturing process, and the point at which the raw material is used can help in assessing the risk and take action to address it. Risk mitigation strategies for raw materials such as high temperature–short time (HTST), autoclaving, virus filtration removal of serum from processes, and UV treatment were described. Discussion of the pros and cons of using plant-derived raw materials were presented. Additionally, considerations in sampling and challenges in interpretation of false-negative and false-positive results in cell culture methods used for raw material testing were described. Conclusions emphasized that the primary defense against adventitious agents is careful sourcing of raw materials and that critical factors involved in this process are knowledge of the origin and manufacturing process, assessment of risk, removal of risky materials, testing, and treatment of raw materials.

Rosemary Versteegen (International Serum Industry Association [ISIA]) presented “The Safe Use of Animal Serum: Fetal Bovine Serum” (FBS). The talk introduced the audience to ISIA and its history. Details were provided regarding the collection, production, and quality control testing of FBS, indicating the barriers against adventitious virus contamination and the challenges of meeting large volume demand. Details on selecting the safest serum possible were presented that included sourcing based upon geography, infrastructure, and segregation with emphasis on requesting Type 1 FBS and viral mitigation by gamma irradiation. Details of the effectiveness of gamma irradiation for virus inactivation relative to UV or HTST were mentioned. Quality control testing methods discussed included those described in USP, EP, or 9CFR. ISIA is working with USP to develop an FBS reference standard and would also welcome standardization of adventitious agent testing. Quality assurance systems in place were described, and a clearly defined traceability program is currently being implemented. The talk concluded with an open invitation to end users to interact with the ISIA through scientific discussions to continue to improve safety and mitigate risks.

The second session was moderated by Kurt Brorson (CDER, FDA) and Anthony Lubiniecki (Janssen R&D). The session focus was common control strategies that can mitigate the risk of contamination due to raw materials that are used in the development of the production cell line and in the manufacturing process.

Mark Plavsic (Genzyme) presented “Raw Material Treatment Methods and Analytical Considerations.” He discussed the types of agents that can be found in various types of raw materials used for cell culture production, and the types of physical inactivation methods (gamma irradiation, UV irradiation, heat) that can be utilized to mitigate risks. The analytical considerations important for conducting these studies were also reviewed. The need to apply prudent inactivation methods to biological raw materials in order to ensure consistent levels of viral safety, while also retaining the useful growth promotion value of the raw material was emphasized.

Steven Lang (Janssen Research & Development) presented “Raw Materials and Reagents for Cell Selection.” He discussed the use of biological raw materials during cell line selection and presented a case study in which a candidate cell line was exposed to FBS and a rabbit IgG preparation in order to select high-producer clones. This necessitated examination of the raw material and the resulting cell bank for the potential presence of rabbit adventitious agents in addition to the more typical bovine agents. Eventually, improved screening methodology for the high-producer clones was developed using recombinant DNA sources of reagents, which eliminated the use of rabbit IgG and the need to test for rabbit adventitious agents. The talk highlighted the need to tailor screening programs to the risks and exposures of the cell line during development.

Qi Chen (Genentech) talked about “Viral Clearance Considerations and Overall Process Control Strategy.” She discussed her company's approach of applying multiple overlapping barriers to virus entry into the cell culture process, and multiple virus clearance steps in the purification process. Barriers to virus entry included screening of cell banks, using virus-retentive filters on compressed gases, using process water exposed to high temperature, treating cell culture medium by HTST, and conducting in-process testing employing both general cell culture methods as well as qPCR methods for specific agents like MVM. Virus clearance was consistently achieved by process steps such as anion exchange chromatography and small virus filtration. Together these procedures gave consistently effective protection from virus contamination during the past 4000 large-scale cell culture harvests. In addition, removal of retrovirus-like particles (RVLP) produced from CHO cells was evaluated using samples from full-scale manufacturing processes. Data from five monoclonal antibody (mAb) processes demonstrated that RVLP are removed step-wise to below or near the qPCR assay limit of quantification after the second chromatography step. Based on the data, an alternative approach to retroviral clearance was proposed. Retroviral clearance can be demonstrated by a combination of RVLP removal using full-scale samples from upstream chromatography steps such as affinity and cation exchange chromatography, and by using murine leukemia virus (MLV) as a model for clearance by steps such as low pH inactivation, small virus filtration, and anion exchange chromatography.

The session talks indicated that “zero risk” from a raw material is not achievable, but an integrated approach can reduce risk to lowest practicable levels. This approach should include evaluating raw materials in terms of literature review, risk assessment principles, practicality/utility of testing, mitigation approaches (before use of raw materials in cell culture), and business continuity considerations for each product. Also, replacement/reduction/refinement strategies for ADRMs seem to be prudent and useful.

The final session was an expert panel discussion moderated by Arifa Khan, Anthony Lubiniecki, and Kathryn King on the potential safety concerns for using novel cell substrates and the role of emerging virus detection technologies to fill the gaps for addressing biological product safety. The participants were: Qi Chen, Philip Krause (CBER, FDA), Johannes Löwer, Marcie McClure, David Munroe, David Onions, Mark Plavsic, George Rohrmann, Amy Rosenberg (CDER, FDA), Marc Salit, Ranga Sampath Tom Slezak, Tara Tagmyer, Rosemary Versteegen, Michael Wiebe, and Vidadi Yusibov. The conference concluded with a summary of the meeting highlights by Anthony Lubiniecki. General conclusions from the meeting follow.

Meeting Conclusions

• There was general agreement that the emerging technologies need some further development as potential tools that could complement the existing virus detection methods for cell substrate characterization and safety evaluation, and for support of ongoing investigations of possible viral contamination events.

• Efforts should continue to standardize new virus detection technologies and studies should be done to determine their potential applications in product manufacture, including the potential for assay validation in such applications.

• Establishing consortia/databases will help support knowledge sharing to fill the gaps for advancing the use of the new technologies.

• There should be a follow-up meeting for providing updates on the progress for applications of these emerging technologies for assurance of safety of biological products.

• Studies should be done to better understand retroelements in novel cell substrates and detection of RT activity should be evaluated by thorough follow-up to assess risk to humans.

• There is limited knowledge related to the biology of viruses in plant and insect cell substrates relative to mammalian cells. The principles applied to mitigate the risk of adventitious agent contamination in mammalian cells can be applied, with appropriate adjustments, to these novel cell substrates. Mammalian model viruses, where biophysicochemically equivalent, may serve as acceptable substitutes for viral clearance studies for these cell substrates.

• “Zero risk” from raw materials is not achievable, but an integrated approach to risk reduction can reduce risk to the lowest possible level.