Critical quality attributes (CQA) are chemical, physical, biological and microbiological attributes that are defined, measured and monitored during the manufacturing process to guarantee that final product features do not exceed allowable quality limits. They are identified in the first stage of Process Validation, namely Process Design. For autologous CAR T-cell therapies, it is required that each individualised ‘lot’ is tested to guarantee the right quality of the product. It is crucial to consider timeliness in this process in order to avoid product degradation before infusion or cryopreservation. Regenerative medicine CQAs include identity, potency, purity and safety.
Acute lymphoblastic leukemia (ALL) is a type of the blood and bone marrow cancer which is characterized by the development of large numbers of immature lymphocytes. The subgroup of people with relapse/refractory ALL is treated with CAR-T cell therapy (chimeric antigen receptor-modified T-cells). CTL019 is the first CAR-T approved by US FDA which is widely used for relapse/refractory ALL. The target product profile of CTL019 was described as target specific, with a high level of potency T-cells which are capable of rapid expansion and stable persistence in vivo.
Based on this product profile, it was discovered that the CQA of CTL019 can be described as following: cell number, efficiency of transduction, growth rate, phenotype of cells and functional analysis. Cellular phenotype can be described as T-cell subset distribution (whether a cell is a helper cell or a cytotoxic T-cell, etc) and functionality (cell killing, cytokine release, etc).
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Potency
According to CGMP, potency is a crucial attribute that measures quality, consistency and stability of the product. It is also used a quantitative measure of the actual dose administrated to a patient. Potency correlates with clinical efficacy. Traditionally, potency can be measured by an in vivo and/or in vitro quantitative bioassays. However, qualitative assays can be used as part of multiple potency assays. It measures the activity of the product regarding its relevant mechanism of action (MoA), therefore, a proper understanding of the product parameters that predict clinical effects is required.
MoA of CAR T-cells product is antitumor activity. Therefore, potency tests can be based upon the following characteristics of a product: composition of the product, transgene expression, cell function (in vitro cytotoxicity, degranulation and cytokine production), in vivo persistence and activity in the tumour microenvironment. In order to measure the potency of the product, such techniques as flow cytometry or PCR (polymerase chain reaction) can be used.
Flow cytometry is a technique that analyses the expression of extracellular and intracellular molecules, identifies different cell types in a heterogeneous population, evaluates the purity of isolated subpopulations and measures cell volume and size.
In order to detect the function of CAR T-cells, the killing ability of CAR T-cells to target antigen-positive tumour cells in vitro is evaluated. It can be performed by doing 51Cr release assay.
Safety
Safety attribute confirms that the product is not contaminated with microbes or adventitious agents and does not have tumorogenic potential. Therefore, it is important to control such parameters as levels of endotoxin, mycoplasma, superfluous ancillary components and CD3 negative impurities that can be carried over from the apheresis. Sterility testing is thoroughly described elsewhere. The major concern for the manufacturers is if a sterility batch comes from cells which are grown in an open system process as they are more likely to be contaminated with microbes and mycoplasma.
Therefore, closed system processing should be used instead. In addition, reducing serum in the culture protocol also improves the safety of the product as products of animal origin increase the risk of mycoplasma contamination in the final product.
In particular, for CAR T-cell therapies microbial safety is a significant challenge as conditions in the manufacturing processes are much less defined compared with parenteral drugs. In addition, standard methods of sterility testing might be less sensitive for CAR T-cell therapies. For instance, it may refer to microbiological growth media inoculation as it might not identify all present contaminants. As viral vectors are used to engineer CAR T-cells, it is required that master cell banks, end of production cells, vector concentrates and ex vivo transduced T-cells are examined for replicative virus. It is also required to provide information about integrated vector copies per genome, integration profile and integration sites.
Identity
Identity attribute confirms that product contains the intended cellular and non-cellular components. Identity assays are used to demonstrate the identity of the product in the master cell bank, working cell bank and final product as well as distinguish the product from the other products manufactured at the same faculty. It is essential to do these tests because such common process development activities as serum elimination, extending culture duration or scaling up culture vessels can cause change in morphological phenotype in the final cell product. The first measure of identity can be morphology of the cells. However, quantitative assays that can provide qualifiable and validatable measures are more common.
For example, FACS (fluorescent-activated cell sorting) analysis can be used to verify identity of the product. It is a special type of flow cytometry which sorts a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light
scattering and fluorescent characteristics of each cell. Live (or fixed) cells are stained with fluorescently labelled antibodies that recognise cell surface or intracellular expressed proteins. This method is based on the assumption that each different cell population expresses relatively unique protein profile which facilitates identification. Identity specification for a specific cell surface marker can be the requirement that the product must be at least 80% identical.
However, the limitation of this analysis is that it is limited to a quite small number of parameters that are chosen based on previous knowledge and reagent availability. Therefore, it is crucial to establish reference standards for each marker used and the subjective, dependant on operator nature gating strategies.
Another advanced multiplexing technologies, such as mass cytometry, can be used to identify additional phenotypic markers. Mass cytometry is a technique used to determine cell properties and it is based on inductively coupled plasma mass spectrometry and time of flight mass spectrometry. Genome expression profiling also can be used to as a test for the identity of the product. For instance, single-cell RNA sequencing (scRNA-sec) identifies transcriptome signatures correlative with the cell’s proliferation, safety, anti-tumour effector function and persistency.
Purity
Purity attribute confirms that the product is free from undesired components, such as unwanted cell types, endotoxin, residual proteins, peptides or other agents used in manufacturing such as animal serum. It is required to explicitly characterise the contents of a dose of formulated finished product which describes all cell types present and identifies any irrelevant compounds if present.
Cell therapy products must be free from endotoxin and it is required that serum albumin levels are determined for any cell product which was processed in the presence of animal serum. Typical tests used to measure animal serum levels (and other residual proteins) are ELISA-based tests. The requirement for animal serum levels is below 1ppm in the final population of the product. Their advantage is that they are quite easy to qualify to a reference standard and validate. Trypsin levels should also be reduced before final formulation, although there are no guidance levels identified for it. It is also important to show that there are no cytokines, antibodies, growth factors or other components used during the manufacturing process in the final formulation.
One special issue correlated with cell therapy products is elimination of any unwanted cell type in the final product, meaning that it does not exceed a defined and consistent percentage of nontarget cells. However, in case with autologous CAR T-cell therapies a risk of undesired cells types exists due to the targeted transformation in the manufacturing process. Therefore, in case with potency manufactures seek to demonstrate consistency with the previous batches which were proven to be safe and efficacious. Currently the best way to have allowable levels of purity is the use of multitargeted FACS and gene arrays.
