Nucleic Acid Amplification Techniques (NAT) to Address Biosafety of Biological Therapies
Over the past 15 years, nucleic acid amplification techniques (NAT), also referred to as polymerase chain reaction (PCR), have played an increasingly important role in the characterization and evaluation of biosafety for human biological therapies. Adventitious agents are of particular interest, as they pose a significant safety risk if present in vaccines or other biological therapeutics. Adventitious agents may contaminate cell lines from a number of sources including: (A) the individual from whom the cell line is derived; (B) media and tissue culture reagents containing products of animal origin; (C) human operators or others who may have contaminated the cells during handling under non-GMP conditions. Therefore, all materials, cell lines, primary cells, and tissue products used for therapeutic purposes require testing for a number of adventitious agents. The viruses to be assessed depend upon origin of the cell line and raw material used in manufacture (1-16).
One of the most advanced NATs available today is real-time TaqMan® quantitative PCR (qPCR). SGS routinely applies this technology in a wide variety of services, including biosafety testing of biological therapeutics. qPCR for virus detection in cell substrates is especially useful in situations for which viruses cannot be readily grown or detected in culture. In fact, qPCR is currently the most effective NAT used to assess contamination with such viruses. Because qPCR is highly specific, multiple assays are typically performed to detect the full range of microbial sequences of concern. Several investigators, as well as the FDA, recommend that qPCR assays with degenerate or consensus primers be used to detect several agents, provided the assay sensitivity is sufficient to assure product safety (6, 12-15). Approaches for validating a qPCR assay are well documented and involve optimizing and specifying the PCR primers and probes; determining the detection limit, linearity, precision, and reproducibility of the assays; determining the accuracy of quantitative data; validating viral extraction; and measuring the precision and robustness (15).
The European Pharmacopoeia (Ph. Eur.) Section 2.6.21, United States Pharmacopeia (USP) Chapter 1237, and the FDA Vaccine Guidance to Industry (2010) all either describe a specific testing approach or highlight adventitious agents of concern NAT/PCR detection (Figure 1).
Detection of Contaminating Viruses
The possibility that conventional infectivity-based methods may miss detection of contaminating viruses is a significant concern in biotechnology safety testing and highly sensitive PCR assays are crucial for detecting these agents. In addition, under certain circumstances it is not always possible to screen for specific viruses by the 28 day in vitro or by in vivo infectivity assays, especially if the sample to be tested is a viral vaccine seed or harvest that cannot be neutralized by antisera in cell culture, or is cytotoxic to infectivity based methods of virus detection (1-16).
Testing for the presence of human viruses is required when cells of human/primate origin, including products obtained from human blood or tissues, are involved in the production of the therapeutic agent. Examples for which human virus PCR testing is advised include cell substrates of human origin, such as MRC-5, HEK 293, A549 and PER.C6 used in the production of vaccines, and adenovirus vectors for gene therapy or vaccine delivery (6,7). In general, human cell lines should be tested for viruses that are associated with severe or oncogenic diseases and are likely to infect the cell type, particularly those that might establish latent or abortive replication in cells. Moreover, in cases for which specific viruses cannot be readily detected using culture methods, PCR is currently the most effective tool to detect contamination with such viruses. Regulatory authorities encourage the manufacturers of certain influenza vaccines derived from cell culture to test for specific respiratory viruses using PCR-based assays. Certain exogenous avian virus PCR assays are recommended if the influenza virus vaccine seed has historically been cultured within eggs or a cell substrate is of avian origin (17). Chinese hamster ovary (CHO) cells are highly susceptible to infection with minute virus of mice (MVM), and this virus has contaminated bulk harvests during the routine manufacture of biological agents. Real-time PCR serves as a rapid, efficient mechanism to exclude viral contamination of cell substrates and bulk harvests.
A large number of animal viruses have recently emerged as agents causing safety concerns. Raw materials that GMP facilities use to manufacture biological agents, including fetal calf serum and porcine trypsin, represent a major risk to biosafety (18). Viral contamination can be introduced into a biological product from raw materials of bovine or porcine origin. Therefore, all new animal reagents used in manufacture should be screened using appropriate techniques. Bovine serum and porcine trypsin are typically screened for viruses using an appropriate Title 9 Code of Federal Regulation (9CFR) method involving a cell culture-based system and assessment of cytopathic effects, haemadsorportion, and immuno-fluorescence-based procedures on the test and control cultures. However, detection of certain viruses using this approach is sometimes not feasible, and in such cases qPCR should be employed for added safety, including for the routine screening of raw materials, as well as cell banks and viral/vector seeds that have been previously exposed to animal material. Some examples of viruses that may contaminate raw materials are Bovine and Porcine enterovirus, swine Hepatitis E, Torque teno virus (TTV), Bocavirus, Bovine polyomavirus, and circoviruses. Porcine circovirus type 1 (PCV1) is highly prevalent in swine and was recently reported in some rotavirus vaccines (19).
The recent cases of vaccine contamination emphasize the need to reduce the risk of introducing viruses, especially those that are difficult to detect, from animal-derived materials used in product manufacture. Circoviruses are amongst the smallest mammalian viruses at around 17 nm and, like parvoviruses, are highly resistant in the environment. Highly sensitive and specific qPCR assays have thus been developed and validated to detect circoviruses. Importantly, the qPCR assay is designed to bind a highly conserved region of the circovirus genome, ensuring maximal coverage and detection. The qPCR assay is fully validated to ICH guidelines and can routinely detect <30 circovirus genome copies per 200,000 non-infected cells.
Detection of Contaminating Mycoplasma
Testing cell cultures for Mycoplasma contamination is critical in the production of reliable biotechnology products. Mycoplasma contamination does not produce turbidity but can have adverse effects on various characteristics, such as cell line growth rates and viral vaccine production. Additionally, certain Mycoplasma species are considered human pathogens and are therefore a serious regulatory concern. As such, the US Code of Federal Regulations (CFR), FDA Points to Consider (PTC), the International Conference on Harmonisation (ICH), European and United States Pharmacopeia (Ph. Eur. & USP) all include technical documentation on the procedures required to detect Mycoplasma during biopharmaceutical production. The Ph. Eur. and USP methods can be harmonized, and are based on culture methods using the agar⁄broth culture test (to detect cultivable Mycoplasma) and the indicator cell culture assay (to detect fastidious Mycoplasma that may only grow in mammalian cell culture). However, the 28-day testing period required for assay completion may not be acceptable for therapeutic products that have short shelf-lives (e.g. cell therapies). This long testing period would also be inappropriate for cytotoxic viral suspensions or in-process samples that require rapid testing. Regulatory authorities have thus become more accepting of alternative rapid test for Mycoplasma, as long as it has been suitably validated.
At least two validated PCR methods for the detection of Mycoplasma DNA are available commercially in off-the-shelf kit format: one made by Roche (MycoTOOL) and the other by Life Technologies (MycoSEQ). The Roche Touchdown method is a conventional PCR approach with gel electrophoresis end-point analysis. The Life Technologies method is advantageous in that it is a rapid, highly controlled, closed PCR well, real-time PCR system. The limit of detection (DL) of the PCR methods is the lowest number of genomic DNA copies (GC) of Mycoplasma that can be detected in the sample. The DL of the culture-based methods is reported as the lowest number of viable cells generating colony forming units (CFU) on the surface of a solid medium. It should be noted that in the past the high ratio between GC and CFU in Mycoplasma samples may have obscured the PCR-based DL data reported. Therefore, the availability and use of Mycoplasma reference samples with low GC⁄CFU ratios enables accurate assessment of NAT-based assay DL, allowing direct comparison to the DL of the cultured-based compendia methods.
Detection of Contaminating Mycobacteria
Cell cultures used in therapeutic product manufacture should be tested for Mycobacteria using three media (two solid media and one liquid medium) and incubated for 56 days, as defined by Ph. Eur Section 2.6.2 and FDA vaccine guidance (6). A rapid, broad spectrum qPCR based assay is validated for the detection of Mycobacterium species and exhibits comparable sensitivity to culture method. This assay is ideally suited for testing materials in which insufficient time exists to perform the classical cultivation method prior to release. Typical examples are cell therapies with a short shelf life and the annual production of influenza vaccine grown in cell substrates and eggs: manufacturers are under severe time pressures to release viral seeds and batches in order to prepare for flu season.
Detection of Contaminating Retroviruses
Retroviruses have a unique replication cycle whereby RNA rather than DNA encodes the genetic information. Retroviruses contain an RNA-dependent DNA polymerase (a reverse transcriptase) that directs the synthesis of viral genome DNA after infection of a host cell. One very important use of qPCR technology is detecting contaminating retroviruses by testing for retroviral reverse transcriptase activity (12).
Regulatory authorities advise the use of reverse transcriptase activity packaged into extracellular retrovirus particles as a marker for retrovirus contamination of cell banks and/or viral vaccine products originating from mammalian and avian cell substrates (1,3,6,12). Product enhanced reverse transcriptase (PERT) assays, also termed Amp-RT or polymerase chain-reaction-based reverse transcriptase (PBRT) assays have been reported to be up to 106-fold more sensitive than conventional RT assays for detecting the presence of retroviruses (12). These reverse transcriptase (RT) assays are used to detect the conversion of RNA template to cDNA, a process induced by the contaminating retroviral RT enzyme. Importantly, avian cells used to manufacture vaccines contain endogenous retroviral RT activity; appropriate test systems are thus needed to detect infectious avian retroviruses.
Retrovirus testing using PERT assays should be performed on cell-free culture medium. PERT assays can detect all retroviruses, as these viruses encode and contain a functional RT enzyme. Candidate materials for testing include cell substrates, viral seeds, and/or harvests of all viral vaccines produced in mammalian cell substrates, as infection with retroviruses is commonly considered to be restricted to vertebrates. However, the genome of many eukaryotes contains mobile sequences known as retrotransposons with long terminal repeats (LTR retrotransposons) or viral retrotransposons, which have characteristics similar to the integrated proviruses of retroviruses, such as Ty elements in Saccharomyces cerevisiae, copia-like elements in Drosophila, and endogenous proviruses in vertebrates. The gypsy element of Drosophila melanogaster includes LTRs and contains three open reading frames, one of which encodes potential products similar to gag-specific protease, reverse transcriptase, and endonuclease. Evidence suggests that gypsy is an infectious retrovirus and provides evidence that retroviruses also naturally occur in invertebrates (20). TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins potentially capable of forming virus-like particles (VLP) with reverse transcriptase (21). Additionally, the PERT assay is used to detect reverse transcriptase activity in the culture supernatant of the insect cell line Sf9 (12). A co-cultivation or infectivity assay is typically performed in combination with a PERT end point to exclude the presence of infectious retrovirus in the cell substrate (1,6).
A PERT assay can be used in two ways, qualitatively (FPERT) and quantitatively (QPERT). The QPERT version of the assay is utilized when quantitative results are required. In this case the test will contain a highly linear purified RT enzyme standard curve with a known number of molecules over at least a 5-log dynamic range. QPERT is used to determine if there is a significant numerical elevation of RT activity above background in the supernatant at different time-points during certain manufacturing processes. RT activity increasing significantly during manufacturing provides further evidence of infectious retrovirus contamination. For example, QPERT can be used to test products manufactured in primary avian cells tested lot-by-lot, for which endogenous retrovirus levels in bulk harvests versus control cells are assessed.
FPERT is by far the most common assay type and is used when highly sensitive qualitative results are required for the detection of vaccine seed or bulk produced in cell substrates. This method produces either a negative test, or a “suspect” positive test (Figure 2). A suspect positive test requires further investigation using an alternative technology, usually a co-cultivation or transmissibility assay. In our laboratories, the QPERT and FPERT assays are highly specific and sensitive, as they include a cellular DNA polymerase suppressant, activated calf thymus (aCT) DNA. A clarification and an additional ultracentrifugation step are then conducted to concentrate the retroviral particles. Cellular DNA polymerases mimic retroviral RT activity and can cause false positive results; thus, the inclusion of aCT DNA ensures specific detection of retroviral RT activity (Figure 3). A sample of DNA polymerase with a cycle threshold (CT) = 25 in the absence of aCT DNA will have a CT = 40 in the presence of aCT DNA. In contrast, aCT DNA does not suppress CT values in a sample containing retroviral RT enzyme, thus including aCT DNA in FPERT ensures maximal specificity and sensitivity for retroviral detection.
When tumourigenic or novel cell substrates are proposed for use in manufacturing, regulatory authorities may request induction studies with PERT end points. These studies involve pre-treating the cell substrate with chemical agents known to induce reactivation or replication of endogenous or latent viruses (6). Subsequent co-cultivation or transmissibility assays with cells susceptible to a broad range of retroviruses, combined with a relevant detection system (e.g., FPERT assay, qPCR and electron microscopy) may be used to confirm the absence of detectable virus, or to amplify viruses with a specific host cell tropism (e.g., co-cultivation with HEK 293 cells to detect retroviruses capable of infecting human cells).
Host Cell Identity
Confirmation of cell identity is critical to the characterisation of master or working cell banks. Manipulation of multiple mammalian cell lines in the same facility introduces the possibility that cell culture cross-contamination can occur. Therefore, confirmation of the purity and identity of biopharmaceutical production cell lines are required for the regulatory acceptance of drug product. Isoenzyme analysis is a suitable cost-effective method in most cases, and NAT-based identification is generally suitable when there is risk of cross-contamination with cell lines of the same species (e.g., HEK293 and PerC6). The European Pharmacopeia requires that both DNA fingerprinting and isoenzyme methods be used to characterize and identify cell substrates used for manufacturing gene therapy and vaccine products (1). Identity tests may also be used to detect cross-contamination of one cell line by another. Therefore, these tests must be able to discriminate between closely related species. This ability is particularly critical when more than one cell line is present in an area used for cell banking procedures, including expansion, pooling, or aliquoting of a cell line.
Random amplification of polymorphic (RAPD) (pronounced "rapid") is a type of PCR in which segments of DNA are amplified at random. In general, several arbitrary short primers 8-12 nucleotides in length are used to amplify template of cell line genomic DNA. The fragments are subsequently resolved by gel electrophoresis. The resulting patterns produce a semi-unique profile (or DNA fingerprint) for the cell line being tested and can be compared to the validated pattern of the reference cell line. No knowledge of the DNA sequence for the targeted gene is required, as the primers bind randomly in the cell genome sequence.
More recently, short tandem repeat (STR) analysis has been more widely used. STR involves the amplification of repetitive DNA elements (e.g., tetranucleotide repeat ACGTn) found in the genome of mammalian cells and creates a unique fingerprint (made up of the number of repeats for each region) for cell substrates. Fluorescently labelled oligonucleotide primers that flank regions of repeats are used to amplify the regions or loci. A multiplex PCR reaction for 8 or more of these core loci is performed with subsequent analysis on a genetic analyzer using laser detection of amplified signals. Commercial kits and dedicated software programs are now available to screen and identify the repeat region targets. The use of an allelic ladder within the typing kit enables the conversion of measured amplicon size into an STR repeat number. This type of testing may be used to confirm cell line identity, establish the values of the STR allele set of the cell line being studied, or study genetic drift of cells kept in culture.
Ensuring Removal of Host Cell DNA Impurities
Vaccines and other biological final products derived from continuous mammalian cell lines (or non-mammalian cell lines such as yeast, bacterial, or insect cells) must not contain greater acceptable levels of residual host cell DNA. The presence of host cell DNA in the final product is of significant concern due to the potential transfer of activated cellular and/or viral oncogenes (particularly if the cell substrate is tumourogenic), the production of infectious viruses from viral DNA, and aberrant gene expression by insertion of sequences into sensitive control regions of genes. Consequently, whether host cell- and vector-derived DNA is present, this should be assessed using a suitably sensitive analytical technique such as qPCR, thus obtaining quantitative data.
Stringent guidelines stipulate the maximum amount of DNA that can be present in the clinical lot. For example, a vaccine produced using a continuous cell line such as low passage Vero should be limited to a maximum level of 10 ng of cellular DNA per dose (1). There may be instances where continuous cell line DNA is considered to pose a greater risk, such as if the cell contains retroviral proviral sequences (22). Therefore, in many cases the maximum amount of residual DNA per dose and the size of the residual DNA should be established on a case-by-case basis dependant on the product.
In summary, biosafety is of utmost concern in bio-therapeutic and vaccine manufacturing, especially because of the increasing rate of discovery of previously unrecognized viruses. Viral contamination from animal products and raw materials represents a significant–and growing– threat to biotechnology products. Importantly, additional steps to detect viral contamination need to be incorporated to ensure safety. Now that streamlined assay validation procedures and GMP-validated NAT tests are readily available, these methods provide manufacturers with the confidence required in production and batch release of products. The high reproducibility and fast throughput of cutting-edge NATs reduces the amount of time needed for development, safety testing, and final marketing of novel biotechnology products.
Archie Lovatt, Ph.D.
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