Abstract
The development of new products for the prevention and treatment of current as well as emerging and re-emerging diseases has led to the introduction of novel cell substrates in biologics. Examples include the demand for large-scale production of pandemic influenza vaccines and for therapeutic proteins, and the use of novel vectors for AIDS vaccines and in cancer gene therapy. A major safety issue regarding the use of novel cell substrates is adventitious agents, including viruses that may have been exogenously introduced due to cell passage history or indigenous viruses that are naturally occurring in the species of cell origin due to infection, or endogenous retroviruses that exist as a normal part of the host cell DNA. Additionally, in the case of genetically engineered cells, there is a concern for recombinant viruses that may be generated de novo involving vector virus sequences. Furthermore, potential oncogenicity of residual cellular DNA remains a theoretical safety concern related to tumorigenic cell substrates. This paper discusses safety issues related to novel cell substrates with a focus on tumorigenic cells and genetically engineered cells and presents the current testing recommendations for general cell substrate safety with details on additional assays for consideration in testing some novel cell substrates including tumorigenic cells.
- Novel cell substrates
- Viral vaccines
- Adventitious agents
- Latent viruses
- Endogenous retroviruses
- Chemical induction
- Tumorigenic cells
- DNA oncogenicity
- Cell banks
- End-of-production cells
Introduction
A variety of animal-based biological products are regulated by different offices in the Center for Biologics Evaluation and Research (CBER): vaccines, toxoids, and allergenic extracts in the Office of Vaccines Research and Review (OVRR); somatic cell and gene therapy products, tissue and tissue-based products, xenotransplantation products, and tumor vaccines and immunotherapy products in the Office of Cell Therapy and Gene Therapy (OCTGT); whole blood, blood components, blood derivatives, blood substitutes, anti-toxins, anti-venoms, immune globlulins, immune Fabs and in vitro diagnostics in the Office of Blood Research and Review (OBRR); and devices regulated by both OBRR and OCTGT. Additionally, CBER offices review monoclonal antibodies, therapeutic cytokines and growth factors, and toxins when these products are solely used as an ex vivo constituent in a manufacturing process or used solely as a reagent in the production of a product that is under CBER's jurisdiction.
An important aspect in the development of a safe biological product is to assure that contaminating agents are not introduced due to biological raw materials used in its production (discussed in reference 1). Cell substrates are a critical raw material in the development and manufacture of CBER products. In the case of viral vaccines, a variety of animal tissues and cell lines of mammalian and avian origin are used for the development of the virus seed or vector virus stock as well as in the manufacture of the final vaccine product (2). Additionally, yeast cells are used for human papillomavirus (HPV) vaccine and hepatitis B vaccine and an insect cell line for the production of a recently licensed HPV vaccine. Mammalian cell lines of human and rodent origin are used in cell and gene therapy for the development of therapeutic cells, for the generation of packaging cell lines, and for the production of gene therapy vectors as well as in the production of therapeutic proteins such as monoclonal antibodies. Animal tissues, such as porcine-derived tissues, are used in xenotransplantation, and most recently transgenic animals are being used to produce drug products (such as goat for antithrombin). Such products are regulated by various guidances issued by the different CBER offices (3–8). The introduction of novel cell substrates has posed a variety of unique regulatory challenges, particularly in addressing safety concerns related to their use in product development and manufacture. This paper focuses on the use of novel cell substrates in viral vaccines with a detailed discussion of issues specifically related to tumorigenic cells.
Transition in Cell Substrates
Different types of cell substrates such as animal tissues, primary cell cultures (PCCs), diploid cell strains (DCSs) or diploid cell lines, and continuous cell lines (CCLs) are used in U.S.-licensed vaccines (9–11). The history and challenges in the transition of cell substrates for use in biologics has been detailed previously (12, 13). PCCs were used from the 1950s for early viral vaccines (for example, primary monkey kidney cells). Human DCSs were introduced in the 1960s (for example, WI-38 and MRC-5) and later a fetal lung monkey diploid cell line (FRhL-2) was established and characterized for use in viral vaccines. The use of the Namalwa human cancer CCL in the 1970s to produce interferon along with the establishment of biotechnology in the 1980s led to the broader use of animal cells for highly purified therapeutic products such as recombinant proteins in Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells as well as in nonrodent cell lines such as human 293 cells, and monoclonal antibodies in mouse myelomas (NS0 and SP2/0). However, the introduction of CCLs in vaccines was met with greater caution related to the perceived risks associated with immortalized cells. These concerns included the potential transmission of tumorigenic and oncogenic activities and unknown adventitious viruses. The African green monkey Vero cell line was introduced in the 1980s due to its broad susceptibility to a variety of viruses, thereby making it a useful cell substrate for vaccine production as well as for biosafety testing. This was a result of extensive characterization and testing of Vero cells (14, 15) before being accepted by several control authorities for vaccine production. Licensed vaccines produced in Vero cells have been used in large populations in different countries with a demonstrated safety record (10). In the U.S., low-passage, non-tumorigenic Vero cells have been the only continuous animal cell line used in licensed viral vaccines (11). Guidance for cell substrate safety and product quality is provided by various national and international regulatory documents (3–8, 16–20).
Due to the need for novel vaccines against emerging and re-emerging diseases and bioterrorism agents, and the demand for large-scale production in a short timeframe, novel cell substrates are being introduced for the development of vaccines. Since 1998, the U.S. Food and Drug Administration (FDA) has been involved in public discussions related to the use of tumorigenic cells in viral vaccines (11). These scientific discussions helped identify additional safety issues pertaining to use of a novel cell substrate in vaccines and facilitated the use of tumorigenic cells for some investigational vaccines such as Madin-Darby canine kidney (MDCK) cells for influenza vaccines and genetically engineered human 293 and PER.C6 cell lines for defective, adenovirus-vectored vaccines.
Safety Issues Related to Novel Cell Substrates
Novel cell substrates currently being used for some investigational products or under consideration for future products can be broadly grouped into naturally occurring cells and genetically engineered cell lines (Table I). Naturally occurring cell substrates are generally selected based upon their susceptibility to vaccine viruses or vectors and high product yield. Currently, there are a variety of cell substrates for a variety of investigational products including viral vaccines, cancer vaccines, gene therapy vectors, and therapeutics. These include mammalian tumorigenic cell lines and tumor-derived cells, avian embryonic stem cells and cell lines, insects and insect cell lines, plants and plant cell lines, as well as bacteria. Genetically engineered cell lines were developed to meet unique production needs, for example, specific cell lines for complementation or packaging of defective, viral vectors or stable transfectants for high expression of protein products. Additionally, the lack of adequate documentation regarding cell passage history (discussed later) has resulted in the development of new-generation, well-characterized cell lines for high virus particle or protein yield or designed for specific viral vectors.
General Categories and Examples of Novel Cell Substrates
An important safety concern related to cell substrates is the presence of viral contaminants. These (1) may be introduced as exogenous viruses in the cell line during its derivation and passage in vitro; (2) may be present as indigenous viruses that are naturally occurring in the species of cell origin due to infection such as latent DNA viruses (such as adeno-associated viruses, adenoviruses, hepadenaviruses, herpesviruses, papillomaviruses, and polyomaviruses) and some RNA viruses (for example, arenaviruses such as lymphocytic choriomeningitis virus (LCMV) (21); or (3) may occur as endogenous retroviruses that exist as a normal part of the host cell DNA and can be present in a silent or active state. There may be additional safety concerns related to tumorigenic cells, such as tumorigenicity of intact cells and the perceived risk of oncogenicity of residual cellular DNA. Novel vectors such as adenovirus-associated viruses pose potential concerns related to integration into the host genome, and, in the case of genetically engineered cells, the generation of replication-competent viruses (RCVs), such as replication-competent retroviruses, adenoviruses, or lentiviruses, may be an added safety concern. Additionally, co-packaging of “unwanted” RNAs or DNAs may be a concern in case of virus-like particles and packaged virions.
Comprehensive Safety Scheme for Cell Substrate Testing
Cell substrate and product safety involves development of a comprehensive safety plan that includes testing for known and novel adventitious viruses and implementing steps and procedures to minimize the risk of virus contamination. General considerations for development of such a plan are two-fold: risk assessment of cell substrate contamination and evaluation of risk reduction. The risk of cell substrate contamination can be assessed by obtaining information regarding (a) the host species of origin (e.g., the donor health and any naturally occurring viruses or immunization with live, viral vaccines, if relevant); (b) the cell passage history (e.g., the potential of adventitious agent introduction due to the length and conditions used in cell line development, handling, raw materials, or use of other cell lines in the facility); and (c) cell characteristics such as genotype (diploidy vs aneuploidy or heteroploidy) and phenotype (nontumorigenic vs tumorigenic). Evaluation for risk reduction can be done by obtaining information related to (a) the susceptibility of the cells to known viruses; b) whether there are any inactivation or clearance steps during the manufacturing process; c) design of genetic modifications in the cells that may reduce the generation of RCVs; and d) the use of qualified biological starting materials (e.g., virus seed or vector virus stock) and reagents (e.g., serum, trypsin).
Cell Banking
The concept of cell banking was introduced with the use of DCSs for controlled production in biologics by establishing well-characterized cells for manufacture during product lifetime (12). This can be applied to DCSs and CCLs, where a two-tiered banking system consisting of a master cell bank (MCB) and a manufacturer's working cell bank (MWCB) is generally used; in some cases additional banks may be generated from parent cells (parent cell bank) or at the end of production (EOP cell bank). Usually, extensive characterization and testing is done on the MCB, with more limited testing on the MWCB and some additional tests on the EOP cell bank. The details of the tests at the different stages of vaccine production are shown in the FDA's 2010 Guidance for Industry on Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications (8, appendix 1; Table I). The inability to establish cell banks for primary cells and tissues necessitates addressing safety concerns based upon donor history, donor testing, use of specific pathogen-free donors (when possible), and by extensive testing of control cells.
Conventional Testing
Conventional safety testing refers to the tests and assays described in the 1993 FDA guidance document for cell line characterization (3). Conventional testing as well as additional testing considerations for novel cell substrates are described in the updated guidance document for characterization and qualification of cell substrates (8). Conventional testing includes determination of karyology, identity, and cell phenotype (tumorigenicity) for cell line characterization, as well as adventitious agent testing for non-viral and viral agents. Non-viral agent testing includes tests for mycoplasma (or spiroplasma in the case of insect cells), bacteria and fungal sterility, and mycobacteria. Adventitious virus testing includes general tests for virus detection: in vivo assays generally in adult mice, suckling mice, and embryonated hens' eggs; in vitro cell culture tests for cytopathic and hemadsorbing/hemagglutinating viruses using three cell lines (same species and tissue type as used in production; human diploid cells and monkey kidney cells); and assays for retroviruses, such as transmission electron microscopy (TEM), polymerase chain reaction (PCR)-based reverse transcriptase assays (e.g., PCR-enhanced reverse transcriptase assay (PERT), and infectivity assays. In certain cases, retrovirus detection assays may include drug treatment to induce latent viruses (discussed below). Assays for species-specific adventitious viruses include antibody production assays such as MAP for mouse viruses, HAP for hamster viruses, and RAP for rat viruses, and challenge assay for LCMV in the case of rodent cell lines or if there is a concern related to potential exposure to rodent materials; and tests for animal viruses, for example, bovine and porcine viruses in the case of serum and trypsin (17). Furthermore, virus-specific assays should also be included based upon donor species and passage history (e.g., PCR and infectivity assays for human pathogens in the case of human cell lines). Cell substrate and vaccine safety can be demonstrated by testing at various stages of production (8, appendix 1; Table I).
Additional Testing
The introduction of novel cell substrates has posed a regulatory challenge for safety testing related to the potential presence of latent or occult viruses that may not be detected by conventional assays. Tumorigenic cell substrates have additional safety concerns related to whole-cell tumorigenicity and potential DNA oncogenicity. These safety issues were identified based upon extensive discussions and meetings involving scientific and regulatory experts (11) and resulted in development of the FDA's 2010 Guidance for Industry that provides a regulatory pathway for introducing a novel cell substrate for product development (8). The overall features of the document are indicated in Table II. It includes the conventional testing described in the 1993 Points to Consider and discussions and details of the additional assays recommended for testing novel cell substrates. The latter include chemical induction assays for detection of latent viruses such as endogenous retroviruses and occult RNA and DNA viruses, an extended tumorigenicity assay for characterization of the tumorigenic phenotype of neoplastic cells, and oncogenicity assays to evaluate cell lysate and DNA for oncogenic activities. These assays may be applied as needed with CBER guidance.
Salient Features of the 2010 Guidance for Industry on Characterization and Qualification of Cell Substrates Used in Viral Vaccines
While concerns regarding tumorigenicity associated with intact cells and DNA oncogenicity may be addressed by removal of whole cells and reduction in DNA size and amount, the demonstration of the absence of latent and occult viruses is challenging and needs to be addressed for cell substrate and product safety. One approach is by using a recently developed algorithm for chemical induction and detection of latent viruses (21). The overall strategy is to treat cells with four chemical inducers that have different mechanisms of activating endogenous retroviruses and latent DNA viruses followed by detection of the induced virus by assays designed to detect known and novel viruses. The additional tests for novel cell substrates are generally recommended on the EOP cell bank.
In the case of some cell types (e.g., neuronal cells), there may be a need to evaluate for the presence of PrPsc, the abnormal form of the host prion protein PrPc (22). There are specific issues related to genetically engineered cells. In the case of transgene- and vector-modified cell lines, evaluation of transgene or vector stability (copy number, intactness, expression) and stability of the cell phenotype needs to be done, as does testing for tumorigenicity of the cells and oncogenicity of residual cellular DNA and cell lysate, and copackaging of “unwanted” RNA or DNAs (in the case of virus-like particles), as well as demonstration of the absence of RCVs.
General Considerations for Product Safety
The development of a comprehensive testing regimen and implementation of a safety plan is critical for assuring product quality and safety. This may be achieved by developing comprehensive testing regimens for detection of known and unknown adventitious viruses to minimize the risk of virus contamination and by including steps in the manufacturing process to inactivate and/or remove contaminating viruses to maximize virus clearance. The salient features of designing safety in biologics include characterization of the cell substrate (e.g., the cell phenotype with regard to tumorigencity, which may be associated with oncogenic viruses or DNA), qualification of cell banks, virus seed/vectored virus and biological raw materials (by extensive testing using general and specific virus detection assays and using raw materials that are certified or tested to be free of detectable virus), in-process testing (based upon a comprehensive testing plan to evaluate bulk/production lots for known and novel virus contaminants), and process validation (efficiently designed to avoid risk of contamination, to eliminate or reduce potential adventitious viral load, and to inactivate potentially contaminating viruses). Additionally, steps need to be present in the manufacturing to reduce the residual host cell material in the final product such as removal of whole cells and removal/reduction of cellular DNA and proteins. The successful implementation of such a strategy will document the absence of contaminating viruses in the raw materials, or their introduction during the manufacture.
Acknowledgments
I thank my colleagues for providing helpful information for my presentation: Robin Levis, Marion Gruber, and Keith Peden in OVRR; Daniel Takefman and Xiaobin Victor Lu in OCTGT; Christine Anderson and Nancy Eller in OBRR; and Patricia Holobaugh in Office of Compliance and Biologics Quality. I further thank Robin Levis and Andrew Lewis for critical review of the manuscript.
- © PDA, Inc. 2010
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