Cell and Gene Therapy Lesson 4: Undefined Methodologies, Unstable Technology and Potential Challenges in the GMP Regulatory Environment

Peter Wunderli, Ph.D., research fellow, PPD Laboratories, GMP lab, discusses cell and gene therapies in an GMP environment.

The U.S. Food and Drug Administration (FDA), European Medicines Agency (EMA) and other national regulatory agencies have provided guidance for the development, manufacture and evaluation of gene therapy medicinal products (GTMPs). These guidelines ensure appropriate good manufacturing practice (GMP) systems are in place to satisfy the mandates for safety, identity, strength/potency, purity and consistency (all product quality attributes) of pharmaceutical products. As more products progress to clinical trials and agencies and industry gain experience in developing GTMPs, initial, updated draft and final guidance documents are being issued that reflect the evolving perspectives and expectations agencies have for evaluating these products. As a result, a very good overview, “Regulatory considerations for gene therapy products in the EU, Japan and the US”¹, published in 2017, is already outdated by more recently issued draft and final guidance^2-7.   

The purpose of this review is to identify some areas where regulations on specific methodologies are not defined, technologies are considered less than ideal or where recent developments have added some potential challenges to the characterization of these products. The specific areas discussed here are methods directed at the quality attributes associated with establishing strength/potency, purity and safety of GTMP products, and especially viral vectored products.    

The first of these, strength/potency, is of paramount importance, not only because these are required under 21CFR sections 600.3(s) and 210.3(b)(16), but because gene therapy vectors are directed at therapeutic, prophylactic or diagnostic applications and function by inserting or modifying genes in target cells and tissues. If this results in too much expression, there could be significant risks to patient health from possible toxicities. Too little expression, and the treatment could fail to offer the intended health benefit. There is also potential risk both from how it is delivered and what happens to the genetic material after it enters the cells. Further, potential host responses to the delivery might significantly impact safety, efficacy and durability of the intended disease treatment.

For typical protein based biotherapeutic entities, determination of product potency is already complex, requiring in vivo or in vitro determinations of relative potency (ideally targeting the known or theoretical mechanism of action of the drug) that allow comparison of product activity to a reference material.  By comparison, GTMPs make that requirement seem simple, as there are multiple indicators of product potency, including:

1. Physical titer – representing the number of copies of the gene of interest (GOI) within a quantity of the product;
2. Infectious titer – establishing the concentration of those genomes than can gain entry to the cell and replicate;
3. Gene expression – demonstration that the gene not only enters the cell, but can direct cellular expression of that genetic material in a way that can be measured and is dose-dependent;
4. Functional potency – the expression product of the gene is shown to have the GOI’s targeted activity.    

There are also multiple ways of measuring each indicator of potency. FDA guidance⁸ acknowledges that “the complexity of CGT products can present significant challenge(s) to establishing potency assays.” The guidance goes on to say that meeting all the requirements may not be possible in early phase clinical investigations and that a single test cannot adequately measure the product attributes that predict clinical efficacy. However, data must be available and evidence substantial “to assure the identity, quality, purity and strength … during all phases of clinical study.” Regulatory agencies strongly encourage development and application of expression and functional methods while products are in development to support indication of new drug (IND) applications and recommend discussions with the appropriate agency as potency measurements are designed, evaluated and validated.^2,7,8,9

At a public meeting in December 2018, the FDA’s Blood, Vaccines and Other Biologics advisory committee held a workshop titled “Quantitation of AAV-Based Gene Therapy Products” to review and discuss best practices for physically quantitating the concentration of recombinant adeno-associated viral (rAAV) vectors present in a product. Standard quantitation has been based primarily on methods applying quantitative PCR (qPCR) as it has a long history and is in widespread use. However, droplet digital PCR (ddPCR) and size exclusion chromatography (SEC) methods were reviewed and discussed at length. Denise Gavin, acting gene therapy branch chief, division of cell and gene therapy, CBER, U.S. FDA, provided the agency’s perspective. She made no recommendations for any specific method, but suggested that product specific processes should be considered and that the agency recommends early development and optimization beginning in preclinical product development to ensure that the data provided demonstrate suitability and can support comparative dosing across production lots and clinical testing.

Because it is understood that not all vector particles that contain the GOI in a product may be capable of entering cells and activating the genetic material, transfection or transduction assays are applied to determine the specific activity of the delivery vectors as a ratio of the physical number of genomes/particles (established by the methods described above) to those capable of successfully transfecting or transducing cells.  Methods can involve the use of helper virus and have evolved from titration assays with rather subjective cellular cytopathology determinations to those that establish the presence of the vector delivered product (via qPCR or ddPCR). As these methods have inherent variability that may be compounded by variability of vector production and purification processes, other assays are suggested and are in development as more accurate or less variable alternatives to support development of viral vectored products.¹⁰

Regulatory agencies also acknowledge that product complexity may limit the ability to know or demonstrate the mechanism of action of gene therapy products. However, some measure of “the level of transgene expression, associated biological activity, and factors associated with the proposed mechanism of action of the vector/delivery system including maintenance of the therapeutic sequence in the target cell should be analyzed⁷;” and “the potency assay should incorporate both a measure of gene transfer and the biological effect of the transferred gen.”⁸ Both the USFDA and EMA accept that measuring the functional activity might not be possible, and suggest that multiple assays might be necessary.^7,8 They suggest that “immunochemical methods to determine the integrity and quantity of an expressed protein product” can “supplement” functional activity7 and discuss “progressive potency assay implementation” and detailed evaluation and modification of such assays as the product development process continues.⁸

Viral vector products, and rAAV in particular, may be a heterogeneous mixture of empty capsids (i.e. do not contain DNA), uninfectious particles (i.e. contain DNA, but DNA amplification in-vitro is not observed) and infectious particles (enters the cell and transgene expression/DNA amplification is observed in-vitro).⁹ Particles that do not result in expression/amplification are considered product- related impurities that can impact product immunogenicity and need to be quantified.^2,9 Production conditions and purification processes can dramatically impact the levels of these impurities, and there is some debate around the impact of these impurities on product performance,¹¹ but the regulatory perspective of these particles as contaminants suggests attempts should be made to at least reduce, if not eliminate, non-transgene expressing particles.

Residual nucleic acid material from production cells and/or plasmids/helper virus present within or external to capsids or other delivery vehicles is also a concern. Both the size and quantity of these contaminants pose risks of either unintended transfer of a gene with functional expression capabilities or genetic material capable of integrating with the chromosomes of recipient cells and altering cell function. Such events could result in adverse events in patients. Guidance requires selection of cell lines and helper sequences to reduce risk and that product related impurities “be identified and their levels quantified”⁷ and that process related complexed nucleic acids “be addressed with respect to their impact on safety and performance of the complex when administered to the patients.”²  The FDA recommends testing for such impurities, optimization of manufacturing processes “to reduce non-vector DNA contamination” and to “monitor and control the amount of extraneous nucleic acid sequences.”² While the extent of the identification of the impurities required by agencies is still evolving, there are growing indications that agencies have an expectation to at least assess the quantity and the size distribution of non-transgene nucleic acid fragments contained within the product.

Another safety expectation of the agencies is to assess viral vectored gene therapies for the ability to replicate within cells.^2,7 The FDA has issued a specific guidance on these methods for lentiviral vectors,³ but this expectation applies to all viral vectored products and extends to products that are generated using insect cell/virus platforms. It is a requirement for drug substance lot release and, for retro- or lentiviral GTMPs, extends into follow-up assessment in patients. Whether second- and third-generation vectors designed to prevent spurious recombination and potential vector variant replication can sufficiently demonstrate their ability to eliminate that risk and the need for such testing remains to be seen. 

Recent information regarding the presence of replication-competent insect rhabdovirus within some Sf9 cell banks (Sf-RV),¹² including those used for manufacture of viral vectored gene therapies, was discussed at the Spring 2019 ISBioTech meeting. Robin Levis (deputy director, division of viral products from the office of vaccine research and review, CBER, FDA) publicly suggested that this should be considered by the agency and drug product manufacturers. While purification processes for standard biological products are validated for their general ability to remove potential adventitious virus particles, viral vectored GTMPs obviously can’t do so.  This does result in an expectation that testing for residual Sf-RV nucleic acid will be performed in such products, but should product release testing be extended to include methods to detect the presence of replication competent Sf-RV?

GTMPs are generally very complex products that have already shown their potential to dramatically change existing treatment modalities and provide options for rare and genetic diseases that did not previously exist.  Like the evolution of regulatory expectations for what are now considered standard biotherapeutic products and the building upon lessons learned from those products, GTMP regulations will also continue to evolve. How regulations of GTMPs evolve will depend upon safety profiles established from the long-term follow-up of already approved products, responses to issues identified from subsequent authorizations and the results of experimentation reported by developers in their efforts towards characterizing these products for potency, purity and safety. The best recommendation for being aware of these changes is for GTMP drug developers to start communications with regulatory agencies early in the process and to meet regularly to ensure their development plans remain aligned with agency expectations.

1. Regulatory Considerations for Gene Therapy Products in the US, EU and Japan. https://www.researchgate.net/publication/321951805_Regulatory_Considerations_for_Gene_Therapy_Products_in_the_US_EU_and_Japan
2. Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs), Draft Guidance for Industry, FDA,  July 2018 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/chemistry-manufacturing-and-control-cmc-information-human-gene-therapy-investigational-new-drug
3. Testing of Retroviral Vector-Based Human Gene Therapy Products for Replication Competent Retrovirus During Product Manufacture and Patient Follow-Up, Draft Guidance for Industry, FDA,  July 2018  https://www.fda.gov/media/113790/download
4. Human Gene Therapy for Rare Diseases, Draft Guidance for Industry, FDA, July 2018 https://www.fda.gov/media/113807/download
5. Human Gene Therapy for Retinal Disorders, Draft Guidance for Industry, FDA, July 2018 https://www.fda.gov/media/113807/download
6. Human Gene Therapy for Hemophilia, Draft Guidance for Industry, FDA, July 2018 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/human-gene-therapy-hemophilia
7. Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal, EMA/CAT/80183/2014 https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-quality-non-clinical-clinical-aspects-gene-therapy-medicinal-products_en.pdf
8. Potency tests for cellular and gene therapy products, Guidance for Industry, FDA, January 2011 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/potency-tests-cellular-and-gene-therapy-products
9. Reflection paper on quality, non-clinical and clinical issues related to the development of recombinant adeno-associated viral vectors, EMEA/CHMP/GTWP/587488/2007 Rev.1, June 2010 https://www.ema.europa.eu/en/documents/scientific-guideline/reflection-paper-quality-non-clinical-clinical-issues-related-development-recombinant-adeno_en.pdf
10. Accurate titration of infectious AAV particles requires measurement of biologically active vector genomes and suitable controls, Molecular Therapy, Methods and Clinical Development, 2017 https://www.cell.com/molecular-therapy-family/methods/pdfExtended/S2329-0501(18)30067-6
11. AAV empty capsids: for better or for worse? Molecular Therapy, 2014 https://www.researchgate.net/publication/259566547_AAV_Empty_Capsids_For_Better_or_for_Worse
12. Assessing and addressing the risks associated with Sf-Rhabdovirus, an adventitious agent in the baculovirus-insect cell system, Am Pharm Sci, 2014 http://www.americanpharmaceuticalreview.com/Featured-Articles/190742-Assessing-and-Addressing-The-Risks-Associated-With-Sf-Rhabdovirus-An-Adventitious-Agent-In-The-Baculovirus-Insect-Cell-System/