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A thorough cell-bank testing plan is necessary to certify the safety and purity of a resulting biopharmaceutical product.
Whether preparing GMP production or non-production master cell banks (MCB) and working cell banks (WCB), end-of-production (EOP) cell banks, or research and development (R&D) cell banks, mastering the art of cell bank (CB) production requires specialized expertise; optimal equipment and environment; appropriate quality controls; constant monitoring; close communication; and continual troubleshooting. Further, the process for characterizing these cell lines can be extensive, requiring an array of testing options for specific scenarios. From adventitious agent testing to identity and genetic stability testing, it is important to know what options are available, when is the best time to perform these tests, and how testing will impact the overall project schedule and outcome.
Guidelines for the industry
MCB is defined as an aliquot of a single pool of cells that generally has been prepared from a selected clone under defined conditions, dispensed into multiple containers, and stored under defined conditions. A MCB typically originates from a research cell bank that is the “end point” of cell-line development. A WCB is prepared from a homogenous cell suspension obtained from culturing the MCB under defined culture conditions.
Cell banks are typically classified via historical progression: R&D CBs, MCB, WCB, and EOP CBs. Another commonly used terminology reflects potential usage of a bank as well as its GMP status: non-GMP banks (or R&D banks), GMP non-production banks (e.g., bioassay banks), and GMP production banks.
Generally, in GMP facilities, non-GMP (R&D) cell banks are prepared under the same general rules and conditions as GMP banks; however, strict adherence to GMP is not required. GMP non-production banks are typically produced in non-campaign mode, meaning that more than one bank can be prepared simultaneously in the same laboratory. Under these circumstances, appropriate cleaning and changeover procedures must be used to avoid cell line cross-contamination. GMP non-production banks are often used as indicator cells lines in cell-based potency assays.
Recently, there has been an increase in demand for ready-to-use bioassay banks (RTU). The purpose of such banks is to use cells straight from the vial in the bioassay, thereby eliminating the cell expansion step and saving time in the process. RTU banks are usually large in size (400-1000 plus vials) and have a significant number of cells per vial. High cell viability (>80% or even >90%) is a common expectation, therefore, pilot studies are highly advised due to potential viability issues when scaling up from a small research bank to a bank of significant size.
GMP production banks are mostly produced in campaign mode, meaning that only one cell line can be grown in a cell-banking suite at a time. Cells from a GMP production bank are used to make a product. The viability expectation for GMP production banks is typically >70%.
Cell bank biosafety testing: Risk assessment and regulatory expectations
To ensure the purity and safety of the cell bank, it’s necessary to design and implement a comprehensive testing program based on a thorough, accurate risk assessment. As part of this assessment, it’s important to consider the type of cell bank that has been prepared, as the rationale for testing differs depending on the type of cell bank that is produced. For example, the testing program for a non-production cell bank (cells used for analytical assays) or for seed cells that are expanded to create a MCB is typically much more abbreviated than the testing program implemented for a production cell bank.
Testing of all cell banks should include testing for mycoplasma and sterility, which should be performed prior to bringing the cells into a clean facility. Sterility testing ensures that the cells are free from bacteria, fungi, and mold, which can be introduced through contaminated cell lines, the use of contaminated animal- or plant-derived raw materials, operators, or contaminated facilities. Sterility testing is performed as per compendial methods. It’s important that bacteriostasis and fungistasis testing is performed prior to sterility testing to assess the sample matrix for inhibition.
Similarly, mycoplasma are a common cell-culture contaminant, often sourced from the cells themselves, additives, or laboratory personnel. Mycoplasma contamination can not only lead to changes in the metabolism of cells, which ultimately affect product yield and quality but are also associated with human disease. Therefore, it’s important to ensure the cells test negative for mycoplasma. Standard mycoplasma testing is performed as per compendial methods and mycoplasmastasis testing is ideally performed prior to routine testing in order to assess the sample matrix for inhibition. Given that historical mycoplasma testing takes four weeks to complete, rapid methods have been developed that exhibit comparable sensitivities but that take only days to perform. Consequently, many companies are considering these methods as a viable alternative to the compendial 28-day assay.
With respect to non-production seed cells, mycoplasma and sterility are typically the only biosafety tests performed. For non-production cells intended for use in analytical testing, additional characterization testing should focus on the performance of the cells in the particular bioassay for which they are intended.
The regulatory expectation for the testing of production cell banks requires a much greater level of characterization than non-production banks. MCBs are the starting material for the entire production process. The regulatory expectation is that these are fully characterized for the microbial organisms already discussed, as well as for viral agents, including adventitious agents. This testing is performed one time only, unless changes are made to the bank, in which case the testing should be repeated with the new cells. As WCBs are only a small number of passages beyond the MCB, a reduced package of testing is acceptable. This reduced package typically includes sterility, mycoplasma, and minimal adventitious viral testing. Cells at the limit of in vitro cell age (CAL) or EOP cells are considered to be the worst-case scenario for amplification of any contaminants present. These cells also require one-time full characterization testing. Refer to Table I for a summary of the regulatory testing expectations for each type of cell bank.
Table I: Summary of the regulatory testing expectations for each type of cell bank. MCB=master cell bank, EOP=end-of-production cell bank, WCB=working cell bank.
Like the microbial agents already discussed, viral agents may be introduced through starting materials, such as cells or raw materials, as well as from operators or contaminated facilities. Raw materials such as bovine serum and trypsin continue to be common sources of viral contaminants in the industry. It’s crucial to understand the entire history of the cell bank and perform a thorough risk assessment when designing the viral-testing plan. Has the cell line been exposed to serum or trypsin? If so, was it irradiated? While the animal-based raw materials may no longer be used in the production process, if these reagents were used in the past history of the cells, they are still a concern and something that must be addressed in the testing plan. Was the cell line exposed to other cell species or substances derived from other species? What non-animal additives have the cells been exposed to? What viruses is the cell line susceptible to? These are all important questions that should be considered when performing the risk assessment and developing the testing plan.
Characterization testing for viral agents includes both general, broad-screening assays that are intended to detect unknown viruses as well as tests for specific viruses. The general, broad screening tests are implemented to detect an unknown and wide range of possible contaminants. These tests include in-vitro virus assays, in-vivo virus assays, and electron microscopy. There are also a number of assays available that detect contaminants associated with specific species of concern; the bovine and porcine screening tests, which are performed to identify contaminants from serum or trypsin, as well as human or rodent screening assays based on product-specific concerns. There are also a number of assays to detect the presence of retroviruses, which may be both adventitious and/or endogenous (viruses that are known to integrate into the host-cell genome).
With in-vitro virus assays, multiple indicator cell lines are selected and used to detect viral contaminants. The cells are inoculated with a cell-lysate sample and observed for indications of viral infection (cytopathic effects [CPE], hemadsorption of red blood cells [HAD], hemagglutination of red blood cells [HA]) over the course of several weeks. When selecting indicator cells to use in the testing, it’s important to select them based on their theoretical susceptibility to the viruses of concern. A human diploid cell line (MRC-5) and a primate cell line (Vero) are always used to detect viruses that are infectious for human cells. A third indicator cell line of the same or similar cell type as the cell bank substrate being tested is selected to detect viruses that are infectious to those cells. Additional cell lines may be added to address particular pathogens of concern. For example, A9 or 324K cells may be added to the testing panel to detect murine minute virus (MMV), a common industry contaminant. As with sterility and mycoplasma testing, it’s recommended that matrix interference testing be performed prior to routine testing to assess any sample inhibition or toxicity issues.
In-vivo virus assays are also broad screening assays performed to detect unknown viruses. The rationale is that some viruses that don’t cause CPE or other noticeable effects in a cell-culture system may be detected in an animal system. In-vivo testing involves inoculation of cell lysate into suckling and adult mice, guinea pigs, and embryonated chicken eggs (allantoic fluid and yolk sac). The animals are then observed for clinical signs every working day of the test.
Testing for species-specific panels of viruses is also an important addition to the virus testing plan, and their use is based on the risk assessment. The most common species-specific testing includes testing for bovine and/or porcine agents, most often due to current or past exposure to serum and/or trypsin, respectively. The testing involves inoculation of indicator cells with cell lysate sample and observation of the cells over several weeks for CPE and HAD. The cells are then stained for the presence of a number of specific bovine and/or porcine agents as outlined by the Code of Federal Regulations (9CFR) and European Medicines Agency testing requirements.
For biopharmaceutical products prepared in rodent cells, the presence of rodent viruses is a significant concern, as many rodent viruses are able to replicate human cells. In these instances, antibody production tests can be performed, which are sensitive methods for the detection of adventitious rodent viruses. Viral antibody-free animals are injected with the test article and serologic analyses are performed at the end of the incubation period to determine whether antibodies were produced against specific viruses. Tests may be performed in mice, rats, or hamsters.
It may also be important to consider the addition of virus-specific real-time polymerase chain reaction (qPCR) assays to the testing panel to detect specific pathogens that aren’t detected in the assays described previously. PCR panels specific for detection of simian, human, canine, avian, or insect viruses are also available.
The last area of concern for cell characterization risk assessment is retrovirus testing. While these agents can be adventitious, many are endogenous to rodent cell lines. Because retroviruses are associated with serious immunosuppressive and degenerative conditions, they are a major safety concern. Therefore, it’s important to demonstrate the absence of infectious retrovirus. Retroviruses have the ability to remain latent within cells and can infect and replicate in the absence of obvious cytopathology, HA, or HAD. Specific co-cultivation procedures are necessary to detect infectious retroviruses. In all of these assays, susceptible target cells are co-cultivated with the cell line of interest. The target cells are subpassaged to allow for amplification of any retrovirus present, followed by detection of infectious virus in a secondary plaque assay for infectious virus or a PCR test to detect viral reverse transcriptase enzyme.
Testing for adventitious agents is crucial for ensuring the safety and purity of a cell bank and resulting biopharmaceutical product. It’s important to perform a thorough and accurate risk assessment to prepare a testing plan and to allow for sufficient time to complete the testing, as many of the tests take weeks/months to complete. Table II shows a summary of standard industry timelines for the various tests. When considering the testing results, it’s also important to consider the implications of the assay results as well. For example, cell-based and animal assays detect infectious virus. In addition, many of these include amplification processes to increase the sensitivity of the assay. Molecular assays, however, detect gene sequences rather than infectious virus and may lead to a “false-positive” result by detecting a virus fragment that may not lead to infection. While it’s important to know the results of both cell-based assays and molecular assays, it’s also important to keep the results of the tests in context.
Table II: Summary of standard industry timelines for various cell-banking tests. qPCR=real-time polymerase chain reaction, StandardTAT=standard turnaround time.
Genetic stability and identity testing
Genetic stability testing is a key component of production cell bank characterization and is a regulatory requirement. Typical mammalian production cell lines are created by stable transfection of the expression vector into the host cell line. During subsequent cell culture, genomic events such as deletion, rearrangement, and point mutation may occur and result in an altered cell phenotype and/or gene expression profile. The instability of the cell line is of great concern because it may negatively affect product integrity, thus posing a risk to the patients. Even when product integrity is not affected, possible reduction of productivity and elevated risk of future events are still of great concern from an operational perspective.
Genetic stability testing typically includes an array of assays performed on the manufacturer’s MCB, EOP, and often WCB. Typical assays include, but are not limited to, those intended to confirm the integrity of the product transcript (mRNA/cDNA sequencing and Northern analysis), the genome structure at integration site (restriction digestion map via Southern analysis), and the ratio of insert gene copy number relative to host genome (via qPCR or Southern analysis). More recently, whole-genome sequencing using next-generation sequencing technology has been discussed within the industry as a potential method to confirm genetic stability and to further enhance cell line characterization.
In addition to genetic stability testing, another important cell bank characterization test is the identity test. Until recently, identity testing was most often performed using isoenzyme analysis, which offered general speciation based on the migration pattern of a selected set of enzymes in the cell lysate when run on an agarose gel. The methodology had several known limitations, such as low sensitivity and difficulty with data interpretation. In addition to the technical limitations, a current shortage of the single-sourced test kit has highlighted the urgent need for alternative identity test methods. Among the possible alternatives, the cytochrome oxidase I barcoding method has become the front runner, due to high interspecies specificity and the availability of an extensive sequence database. For further authentication of human cell lines, short tandem repeat analysis is the preferred method and has been promoted by the American Type Culture Collection.
Both genetic stability testing and identity testing should be performed on each MCB and on one representative lot of EOP cells. Genetic stability testing is optional for the WCB but may be performed at the manufacturer’s discretion. Genetic stability testing results from the EOPC and WCB are compared with those of MCB to allow the detection of any changes that may be indicative of instability. When changes in the manufacturing process are introduced, genetic stability testing may need to be performed on an additional lot of EOP cells.
Article DetailsBioPharm International
Vol. 28, No. 8
Citation: When referring to this article, please cite it as L. Mogilyanskiy, H. Byer, and W. Wang , "Mastering Cell Bank Production," BioPharm International 28 (8) 2015.