The FDA’s January 2020 guidance, Chemistry, Manufacturing and Control (CMC)^[1] Information for Human Gene Therapy Investigational New Drug Applications (INDs), outlines the analytical methods that define the quality, safety and efficacy of gene therapy therapeutics. The analytical package, consisting of release, stability, and characterization tests, includes data generated throughout the product development and manufacturing process. For example, analytical procedures may be used to test raw materials or starting materials for purity, identity, and safety. For gene therapy vectors, this testing encompasses production cell lines, master and working cell banks (MCB, WCB), and virus banks. Depending on the gene therapy vector, microbial systems may be employed to produce vector DNA (such as in adeno associated virus (AAV)), in which case there may be bacterial cell banks and plasmid banks.

Analytical testing at various stages of the manufacturing process are part of the control of critical steps and intermediates. Safety testing (such as bioburden, mycoplasma, adventitious virus testing) are typically performed on the bulk harvest step while additional safety tests are performed further downstream on the drug substance, depending on the cell line and the product type.

Gene therapy products follow the general model of analytical testing for biologics products in that the release testing should include an identity test, tests for purity and the presence of impurities, tests for the quantitation of active substance and additional safety tests. We will now briefly review the typical portfolio of tests using AAV as an example gene therapy product.

Typical Portfolio of Test Using AAV

Safety

Drug substance (DS) testing will typically include safety tests on the bulk harvest, since the likelihood of detection of adventitious agents is much higher in the bulk harvest vs. the purified DS. Bulk harvest would typically be tested for bioburden, mycoplasma, and viral contaminants based on the species of the mammalian cell line used in production. As an example, HEK-293 cells are typically screened for human viruses whereas testing of Sf9 cells (insect cells used in baculovirus production) may include rhabdovirus and residual baculovirus.

Identity

The purified DS testing should include one or more identity tests. For example, genome titer by qPCR can serve as an ID test for the AAV genome, whereas a serotype specific Western Blot test can detect specific serotypes like AAV9.  A more rigorous identity test may sometimes be required, such as DNA sequencing of the entire AAV genome.

Strength

The strength or quantitation of the number of viral particles, often referred to as genome titer, is performed by ddPCR or qPCR.  The genome titer is a critical quality attribute and is directly connected to dosing in clinical trials. Also, an infectious titer assay is performed because not every AAV virion is infectious. The infectious titer may be orders of magnitude below the genome titer, due to low transduction efficiency of some cell types and AAV serotypes.  The genome titer/infectious titer ratio is also a critical quality attribute.  IND sponsors are expected to provide protocols and reports for the qualification and/or validation of the genome titer test methods.

Purity

The purity of gene therapy products reflects the product’s genome titer and the levels of specific process-related impurities and product-related substances.  Process impurities arise from the cell substrate and manufacturing processes (media, filters, chromatography resins, etc.). Product-related impurities are closely related to the active ingredient (DS), such as sequence variants or partially filled capsids. Refer to Table 1 for a list of test methodologies for typical process impurities and product-related substances.

Table 1: Test Techniques for Process Impurities and Product-Related Substances
Item	Type	Technique(s) to Detect/Measure
Partially filled capsid, empty capsid, overfilled capsid	Product related substance	A260/280 ratio, TEM AUC, qPCR
Sequence Variants	Product related substance	DNA sequencing, next-generation sequencing
Virus aggregates	Product related substance	DLS, SEC-MALS
rcAAV	Product related substance	PCR, co-culture
Protein impurities	Process impurity	SDS-PAGE- Silver stain
Residual Host Cell Protein (HEK-293, Sf9)	Process impurity	ELISA
Residual Host Cell DNA	Process impurity	qPCR
Residual plasmid DNA Residual Baculovirus DNA	Process impurity	ddPCR, qPCR
Residual Triton X100	Process impurity	HPLC
Residual Benzonase	Process impurity	ELISA
Affinity Ligand	Process impurity	ELISA

Glossary:

• AUC: Analytical ultracentrifugation
• rcAAV: Replication competent adeno associated virus
• HEK-293: Human embryonic kidney 293 cells (commonly used in research)
• HPLC: High Performance Liquid Chromatography
• Sf9: Insect cells used in baculovirus production
• A260/280: Ratio of sample absorbance at 260 nm and 280 nm
• TEM: Transmission Electron Microscopy
• ddPCR: Droplet digital polymerase chain reaction
• qPCR: Quantitative polymerase chain reaction
• DLS: Dynamic light scattering
• SEC-MALS: Size exclusion chromatography – matrix assisted light scattering
• SDS-PAGE: Sodium dodecyl sulfate – polyacrylamide gel electrophoresis
• ELISA: Enzyme linked immunosorbent assay

For many viruses, an A260/280 ratio test is performed as each virus has a characteristic ratio that is reflective of the overall virus purity and the quality of the virions. For AAV, as the percent of empty capsid increases, the A260/280 ratio drops. Orthogonal identity tests are typically performed, such as analytical ultracentrifugation (AUC), PCR with specific primer sets and transmission electron microscopy (TEM).

Purity

The purity of the virus is typically measured by gel electrophoresis and staining of the viral proteins (VP1, VP2 and VP3) and determining the percent of contaminant proteins.  Process impurities (host cell DNA or host cell protein [HCP], for example) are tested by qPCR and HCP ELISA.  Other process residues such as residual Benzonase, Triton X-100 and either plasmid DNA or baculovirus DNA are also typically determined using appropriate methods (see Table 1).

Potency

The potency of the gene therapy product may be determined in several ways. In early development it may be sufficient to transduce target cells and measure production of the therapeutic transgene by ELISA, flow cytometry or other detection methods.  However, for some AAV serotypes with low transduction efficiencies, direct measurement of the target protein may not be feasible. In these cases, it may be acceptable to measure the viral mRNA (containing the encoding gene) using reverse transcription polymerase chain reaction (RT-PCR).  As the gene therapy product progresses through clinical development, the health authority expectation will be a potency assay (typically cell-based) that is reflective of the product’s mechanism of action.  Depending on the nature of the product, bioassays can be run as proliferation assays, cytotoxicity assays or signaling assays. The exact design of the bioassay will depend on a multitude of factors, but the goal of a cGMP potency assay is high throughout and suitable precision, accuracy, and sensitivity.

For most potency assays, there will be day to day variation in assay response. Therefore, many potency assays are performed as relative potency assays in which the test article is run against a well-characterized reference standard.  This way, any shift in assay signal will be reflected in both the reference and test article.  Through regular assay trending and a reference standard qualification and stability program, drift of the reference or biases in the assay can be prevented.

Conclusion

Recent FDA Gene Therapy guidances have focused on unique therapeutic targets (retinal disorders, rare diseases, hemophilia, neurodegenerative diseases, for example), as well as unique vector features such as genome editing elements. For these types of specialty targets, FDA may specify unique assay requirements. For example, for vectors with genome editing functionality, the agency requests on-target and off-target editing frequency.

As with other biologics, early interactions to obtain FDA concurrence on the suitability of proposed testing schemes of gene therapy therapeutics are highly valuable.

Premier Consulting CMC experts can assist with phase-appropriate design, development, validation, and documentation of such analytical tests, as well as with interactions with the FDA and other regulatory agencies. Contact us today.

[1] See Table 1 for a glossary of abbreviations.