Advances in Assay Technologies for CAR T-Cell Therapies

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Extensive testing is required throughout the drug-development process and during manufacturing to ensure the safety and efficacy of marketed medicinal products. Numerous assays for the characterization of biopharmaceuticals and determination of any biologic contaminants have been developed and are highly effective for most biotherapeutics. For many cell-based therapies, such as chimeric antigen receptor (CAR) modified T-cells (also known as CAR-T), however, these conventional methods often take too long and require excessive sample quantities. Consequently, developers of these novel treatments have been faced with a number of challenges. The development of new rapid methods designed to provide comparable results while meeting the need for high-throughput performance show significant promise for addressing these issues.

Vector/cell-testing requirements
CAR T-cell therapies are produced by harvesting blood cells from a patient, selecting and growing the desired T-cell population, and then transducing them with a viral vector (typically lentivirus) carrying the CAR-T gene cassette. Transfecting cell lines with plasmids produces the viral vectors, and special care is taken to ensure that no replication-competent lentiviruses (RCLs) are generated. After CAR T-cell expansion, the cells are reintroduced into the patient.

Microbial and viral testing is performed to determine whether any microorganisms (e.g., bacteria, viruses) are present during the pharmaceutical process, including in intermediates, active ingredients, the manufacturing environment, and formulated drug products. Test methods are employed for detection, screening, enumeration, and identification purposes. Examples include sterility testing for detection, screening for specified microorganisms, determination of the total aerobic microbial count for enumeration, and analysis to identify specific microbes. Other tests to determine the long-term stability (e.g., genomic and epigenetic stability, X-chromosome inactivation) and quality and function (e.g., potency, efficacy, lot-to-lot variability) are also required.

CLICK IMAGE TO ENLARGE Figure 1: Schematic of the manufacturing steps and associated testing regimes for a call therapy production process.

All raw materials must be sourced from approved suppliers and subjected to extensive testing to ensure there is no presence of microbial or viral contamination, as are master and working cell banks (MCBs and WCBs, respectively). Plasmids produced in bacteria are tested for sterility and endotoxin levels, and viral vectors are subjected to identity, purity, adventitious agent, and potency testing before they can be released for the transduction of cells. While no screening of patient blood cells for adventitious agents is required, the transduced cells are tested for sterility, mycoplasmas, RCLs, and endotoxins. The specific tests required for the control of critical raw materials and throughout the production process are determined by regulatory guidelines and are designed to ensure that cell therapy products are well characterized and free of microbial contamination. The required level of testing depends on the phase of the drug-development cycle and the step of the production process; for instance, cell banks and plasmids differ in their testing requirements. Figure 1 presents a schematic of the manufacturing steps and associated testing regimes for a cell therapy production process.

FDA, the European Medicines Agency (EMA), the US Pharmacopeial Convention (USP), and the European Pharmacopoeia (Ph. Eur.) have all published guidance materials related to the production and testing of cell-based therapies. Some examples include:

• EMA guidance on the development and manufacture of lentiviral vectors (1)

• FDA guidance for the manufacture and ex-vivo use of retroviral gene therapy vectors (2, 3)

• USP (4) and Ph. Eur. (5) general guidance on the manufacture and quality control of therapy viral vectors

• FDA supplemental guidance on testing for replication-competent retroviruses in retroviral-vector-based gene therapy products (6)

• FDA guidance on potency tests for cellular- and gene-therapy products (7)

• FDA guidance on preclinical assessment of investigational cellular- and gene-therapy products (8).

Standard methods
A broad range of standard, well-recognized methods that are accepted by regulatory agencies around the world are employed to characterize raw materials, biologic actives, and formulated drug products. For cell-based therapeutics, in addition to the analysis of typical raw materials such as media, assays must also be performed to characterize any cell lines and viral vectors used.

Standard identity tests for cell lines include cytochrome oxidase or short tandem repeat (STR) profiling and DNA fingerprinting, while those for vectors include determination of genetic identity by sequencing of transgenes and restriction enzyme digestion.

The absence of microbial contaminants in cell lines and vector products (bulk harvest [BH] purified) is confirmed through sterility and mycoplasma testing. Due to the large number of different viruses that can potentially contaminate biologic drugs, numerous assays, both in vivo and in vitro, must be conducted on cell lines and vectors to demonstrate the absence of adventitious viral agents. These tests include those that detect ranges of viruses (broad specificity) and those that target specific viruses that have been known to be an issue (e.g., bovine, porcine). Virus-specific testing of cell lines is typically accomplished using polymerase chain reaction (PCR)-based methods and are often required if there is a known risk of contamination associated with the components used in a given process. Cell lines may also be subjected to transmission electron microscopy or product-enhanced reverse transcriptase assays.

For CAR T-cell therapies produced using lentivirus as the viral vector, the absence of replication-competent vectors, particularly RCLs, must also be demonstrated. These assays are performed on vector products (BH, purified vector, and ex-vivo transduced cells).

Purified vectors are subject to further tests, including determination of the viral titer, residual bovine serum and plasmid, osmolality pH, endotoxin, host-cell DNA, and protein assays. The vector titer and endotoxin tests are also conducted for the ex-vivo transduced cells.

Unique testing needs of CAR and other cell-based therapies
Although cell-based therapies have been in development for more than two decades, they still face a number of challenges. Regulatory scrutiny is particularly high. Because these drug products contain live cells, terminal sterilization is not possible; therefore, demonstration of the absence of contaminants is essential. Early failures and questions about the safety of initial treatments have also led to intense interest from regulators.

Testing can be difficult, however. Often, there are limited supplies of the key raw materials required for process, product, and test method development. In addition, drug-substance and drug-product lot sizes are often quite small; thus, sample volumes are typically small, leading to the need to use modified test protocols, which must be validated for the same specificity and sensitivity as the original test.

Further complicating the issue is the limited shelf life of most cell-based therapies. In some cases, the transduced cells can be frozen, allowing for completion of testing prior to product release. It is not possible, however, to freeze most CAR T-cell therapies. The difficulty lies in the lengthy nature of most conventional sterility assays and tests for determination of the absence of bacteria, adventitious viruses, and RCL.

Most assays and tests are cell-culture-based methods with extended incubation times to allow turbidity formation in liquid culture and colony formation on solid media, and require up to two to four weeks to complete. In addition, they involve many manual procedures (e.g., sampling, dilution, dispending, incubation, reading, recording, subculture, and microorganism identification), which all take time. Overall, these tests can take as many as 40 days from start to finish, assuming up to one week for sample delivery to the test lab, two to four weeks for testing, and up to an additional week for delivery of the final report.

Rapid tests (PCR- and rapid-cell-growth-based microbiological and RCL assays) must be performed on cell-based therapies that cannot be frozen. Generally, conventional assays must also be run for confirmation of the results of the rapid tests, even though the results won’t be received until well after the treatment has been administered.

Expectations for rapid methods
The potential benefits of effective rapid-test methods have led to interest in their implementation for many more therapies than those that are just cell-based therapies that cannot be frozen. Not only do these assays enable reduced product-release cycle times, they generally require small sample volumes and provide higher-quality results. In addition, most can be automated and combined with electronic data capture, reducing opportunities for human error and the introduction of contaminants, and further decreasing the overall test time to approximately nine days (one day for sample delivery, seven days for testing, and one day for delivery of the final report).

New microbiological testing methods achieve the same results as corresponding classical methods, but within a shorter time period, for example, less than three days for sterility testing, and 24 hours or less for microbial counts and ID tests. The ultimate rapid tests are completed in real time (e.g., one to three hours). The ultimate goal is to develop methods than can be completed in hours rather than days.

Faster access to test results can also improve the manufacturing process, because potential problems can be investigated/addressed much sooner than is possible when conventional methods are employed. Very rapid methods may also enable in-process and raw material testing.

There are, however, several key requirements that must be met by any rapid-test method that is intended to replace an existing compendial method. Most importantly, a rapid method must meet or exceed the performance of the existing assay in terms of both specificity and sensitivity. Extensive validation is required by regulatory agencies to support the use of a rapid method by demonstrating comparability to the standard method. Parameters that are considered part of such an evaluation can include accuracy, precision, linearity, specificity, the detection limits, operational range/sample volume, robustness, repeatability, and intermediate precision.

Particular laws, regulations and guidance regarding rapid testing for biopharmaceutical manufacturing include:

• EDQM’s “EP 2.6.1, Sterility”, which discusses approaches to sterility testing (9)

• EDQM’s “EP 2.6.7, Mycoplasma” includes information on nucleic acid detection for mycoplasma in Europe (10)

• 21 Code of Federal Regulations (CFR) 610.12 update (11)

• The Parental Drug Association (PDA) published PDA Technical Report Number 33 (TR33): Evaluation, Validation and Implementation of New Microbiological Testing Methods (12)

• EurPh’s “EP 5.1.6 Alternate methods for control of microbiological quality” (13).

In 2011, FDA’s Center for Biologics Evaluation and Research (CBER) investigated matrix effects through the evaluation of three rapid microbial test systems: Milliflex Detection (Millipore), BacT/ALERT (bioMerieux), and BACTEC (BD) (14).

It should be noted that when rapid test methods are approved, they are approved as part of the filing for a specific drug product. In addition to equivalent performance and significantly faster turnaround times, rapid methods should also be easy to use and be less costly than the corresponding standard methods. They must also be designed to address potential matrix effects.

The technologies on which rapid test methods are based are divided into four categories for convenience:

• Growth-based methods

• Viability-based methods

• Cellular compound- or artifact-based technologies

• Nucleic acid technologies (NATs).

Examples of rapid microbiological assays and rapid methods for the detection of adventitious agents are presented as follows.

Rapid microbial assays
A variety of technologies have been developed for rapid microbiological assays. Methods derived from blood-culture methods employed in clinical microbiology are attractive because they are based on techniques that have been approved by regulatory authorities, albeit for different applications. Examples include methods that rely on carbon dioxide sensors (pH-sensitive fluorescence and colorimetric response) and the use of a pressure-sensitive transducer to measure changes in headspace pressure.

Many methods have also been developed that are based on technologies that have not previously been used in a clinical setting. Examples include determination of the electrical impedence of the media supporting growing microorganisms, solid-phase fluorescence laser scanning microscopy, flow cytometry of fluorescently labeled organisms, and ATP bioluminescence.

ATP bioluminescence is of interest because the sample preparation is similar to that of compendial methods: It is compatible with a wide range of product types, and the results can be read and reported automatically using compliant software. Nonsterile product release is possible within 23 to 48 hours; however, this method does not provide enumeration of contamination levels. Automation of current compendial cell-culture methods is, however, making rapid microbial enumeration possible. Detection of positive cultures is faster with automated interpretation of culture results (via image processing), the general workload is reduced, and computerized data management provides documentation control. Microscopy, solid-phase fluorescence laser scanning, and ATP bioluminescence have also been applied for total aerobic microbial count enumeration.

Nucleic-acid-based methods have also been developed for rapid sterility and mycoplasma testing. One concern with NATs is that nonviable DNA can provide false positive results. Careful design of test systems to ensure sterile environments for samples is crucial. Such methods can be run after immunomagnetic separation to achieve targeted separation using magnetic beads linked to antibodies or lectins that bind specific organisms. Real-time detection is possible with such systems given that within 20 minutes, 36 to 48 nucleic acid amplification cycles can be achieved for a typical bacterium using PCR.

DNA contamination issues can be minimized through the use of NAT methods based on RNA. Several such methods have been developed for microbial identification. Genotypic identification of bacterial organisms can be completed in eight hours to three days depending on the specific technology. However, the equipment required for these tests is expensive, and specialized skills are necessary to perform them. Biochemical methods and methods involving gas chromatography analysis of the fatty-acid content in cell membranes have also been developed for rapid microbial identification.

Rapid methods for adventitious agent detection

As mentioned previously, due to the large number of potential adventitious viral agents that are possible, many different assays are required to ensure detection of all likely viruses. In addition to being lengthy, cell-based methods may give false negatives, because in some cases, replicate viruses may not give any signs of cytopathic effects, or an infectious virus may not replicate in the cell lines chosen for the assay.

PCR-based assays are much simpler and more rapid, with turnaround times of hours compared with weeks. Each PCR test, however, detects the DNA sequence from a specific virus. While accurate, the results include both viable and nonviable DNA, and are not specific to live virus particles. In addition, PCR alone is not applicable for the detection of multiple viruses in a single assay.

Combining PCR with degenerate probes enables the detection of a broad range of viruses. Information on the virus family, subfamily, and genus is obtained using these methods. However, these degenerate/virus-family PCR assays are limited by the fact that contaminant DNA (nonviable) can influence the results.

PCR is also being combined with mass spectrometry and microarrays for lot-release testing. New cell-based methods in development using engineered cells are designed to detect viral contaminants within 48 hours. Massive parallel sequencing, or deep sequencing, can be used to detect multiple DNA sequences from different viruses. Notably, multiplexing allows the rapid detection of virus families. These next-generation sequencing technologies help to minimize the risks associated with conventional and simple PCR-based methods for adventitious agent detection.

Automation is also playing an important role in advancing rapid methods for the detection of adventitious viruses. For nucleic acid extraction, specially designed software can aid in primer/probe selection, while bioinformatics tools help facilitate multiple sequence alignments. Automated PCR assembly leads to improved design principles and chemistries, and real-time PCR amplification and analysis dramatically reduce assay times, as does the ability to immediately compare results to existing viral sequence records in databases. Furthermore, these databases are continually updated with information on new viruses soon after they are identified, enabling more comprehensive analyses.

Conclusion
The development of CAR-T and other cell-based therapies has created opportunities for patients with diseases that previously had no treatment options. However, extensive safety testing of such drug products is necessary, as they are based on live cells that cannot undergo a final sterilization step.

Not only is comprehensive characterization of the cells necessary, rigorous testing to demonstrate the absence of any microbial or viral contamination is paramount. In addition, testing strategies must be designed to meet the unique requirements of each cell-based therapy. Advances in rapid testing methods for use throughout the entire manufacturing process for CAR-T therapies will not only provide even greater assurance of drug product safety, but also may facilitate the further development of novel, effective treatments for patients with unmet medical needs.

References
1. EMA, Guideline on Development and Manufacture of Lentiviral Vectors (London, May 2005).
2. FDA, Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy (Rockville, MD, Mar. 1998).
3. FDA, Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic Cell Therapy Investigational New Drug Applications (INDs) (Rockville, MD, Apr. 2008).
4. USP, USP General Chapter <1046>, “Cell and Gene Therapy Products” (US Pharmacopeial Convention, Rockville, MD, 2011).
5. EDQM, EurPh, Gene Transfer Medicinal Products for Human Use 5.14 (EDQM, Strasbourg, France, 2010).
6. FDA, Guidance for Industry: Supplemental Guidance on Testing for Replication Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and During Follow-up of Patients in Clinical Trials Using Retroviral Vectors (Rockville, MD, Nov. 2006).
7. FDA, Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products (Rockville, MD, Jan. 2011).
8. FDA, Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy Products (Rockville, MD, Nov. 2012).
9. EDQM, EurPh, Sterility 2.6.1 (EDQM, Strasbourg, France, 20601, 04/2011) 10. EDQM, EurPh, Mycoplasma 2.6.7 (EDQM, Strasbourg, France, 20607, 01/2008), 11. CFR Title 21, Part 610.12 (Government Printing Office, Washington, DC), pp. 70-75.
12. PDA, Technical Report No. 33, Evaluation, Validation and Implementation of Alternative and Rapid Microbiological Methods (Revised 2013).
13. EDQM, EurPh, General Text 5.1.6. (EDQM, Strasbourg, France, 2011).
14. FDA, Center for Biologics Evaluation and Research, “Identifying Faster Sterility Tests for Biological Products: Regulatory Research Seeks to Reduce the Time Needed to Ensure the Safety of Critical Products,” (Rockville, MD, 2011), www.fda.gov/downloads/BiologicsBloodVaccines/ScienceResearch/UCM266975.pdf, accessed Jan. 29, 2016.

About the Author
Alison Armstrong, PhD, is senior director, development services at BioReliance, UK.

Article DetailsBioPharm International
Vol. 29, No. 2
Pages 32–37
Citation: When referring to this article, please cite it as A. Armstrong, "Advances in Assay Technologies for CAR T-Cell Therapies," BioPharm International29 (2) 2016.